Katherine Ferrara

All Publications

• Sonogenetic control of multiplexed genome regulation and base editing. Nature communications Liu, P., Foiret, J., Situ, Y., Zhang, N., Kare, A. J., Wu, B., Raie, M. N., Ferrara, K. W., Qi, L. S. 2023; 14 (1): 6575

  Abstract

  Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.

  View details for DOI 10.1038/s41467-023-42249-8

  View details for PubMedID 37852951

  View details for PubMedCentralID PMC10584809

• PET imaging of focused-ultrasound enhanced delivery of AAVs into the murine brain. Theranostics Ajenjo, J., Seo, J. W., Foiret, J., Wu, B., Raie, M. N., Wang, J., Fite, B. Z., Zhang, N., Malek, R., Beinat, C., Malik, N., Anders, D. A., Ferrara, K. W. 2023; 13 (15): 5151-5169

  Abstract

  Rationale: Despite recent advances in the use of adeno-associated viruses (AAVs) as potential vehicles for genetic intervention of central and peripheral nervous system-associated disorders, gene therapy for the treatment of neuropathology in adults has not been approved to date. The currently FDA-approved AAV-vector based gene therapies rely on naturally occurring serotypes, such as AAV2 or AAV9, which display limited or no transport across the blood-brain barrier (BBB) if systemically administered. Recently developed engineered AAV variants have shown broad brain transduction and reduced off-target liver toxicity in non-human primates (NHPs). However, these vectors lack spatial selectivity for targeted gene delivery, a potentially critical limitation for delivering therapeutic doses in defined areas of the brain. The use of microbubbles, in conjunction with focused ultrasound (FUS), can enhance regional brain AAV transduction, but methods to assess transduction in vivo are needed. Methods: In a murine model, we combined positron emission tomography (PET) and optical imaging of reporter gene payloads to non-invasively assess the spatial distribution and transduction efficiency of systemically administered AAV9 after FUS and microbubble treatment. Capsid and reporter probe accumulation are reported as percent injected dose per cubic centimeter (%ID/cc) for in vivo PET quantification, whereas results for ex vivo assays are reported as percent injected dose per gram (%ID/g). Results: In a study spanning accumulation and transduction, mean AAV9 accumulation within the brain was 0.29 %ID/cc without FUS, whereas in the insonified region of interest of FUS-treated mice, the spatial mean and maximum reached ~2.3 %ID/cc and 4.3 %ID/cc, respectively. Transgene expression assessed in vivo by PET reporter gene imaging employing the pyruvate kinase M2 (PKM2)/[18F]DASA-10 reporter system increased up to 10-fold in the FUS-treated regions, as compared to mice receiving AAVs without FUS. Systemic injection of AAV9 packaging the EF1A-PKM2 transgene followed by FUS in one hemisphere resulted in 1) an average 102-fold increase in PKM2 mRNA concentration compared to mice treated with AAVs only and 2) a 12.5-fold increase in the insonified compared to the contralateral hemisphere of FUS-treated mice. Conclusion: Combining microbubbles with US-guided treatment facilitated a multi-hour BBB disruption and stable AAV transduction in targeted areas of the murine brain. This unique platform has the potential to provide insight and aid in the translation of AAV-based therapies for the treatment of neuropathologies.

  View details for DOI 10.7150/thno.85549

  View details for PubMedID 37908737

  View details for PubMedCentralID PMC10614693

• Combined near infrared photoacoustic imaging and ultrasound detects vulnerable atherosclerotic plaque. Biomaterials Schneider, M. K., Wang, J., Kare, A., Adkar, S. S., Salmi, D., Bell, C. F., Alsaigh, T., Wagh, D., Coller, J., Mayer, A., Snyder, S. J., Borowsky, A. D., Long, S. R., Lansberg, M. G., Steinberg, G. K., Heit, J. J., Leeper, N. J., Ferrara, K. W. 2023; 302: 122314

  Abstract

  Atherosclerosis is an inflammatory process resulting in the deposition of cholesterol and cellular debris, narrowing of the vessel lumen and clot formation. Characterization of the morphology and vulnerability of the lesion is essential for effective clinical management. Here, near-infrared auto-photoacoustic (NIRAPA) imaging is shown to detect plaque components and, when combined with ultrasound imaging, to differentiate stable and vulnerable plaque. In an ex vivo study of photoacoustic imaging of excised plaque from 25 patients, 88.2% sensitivity and 71.4% specificity were achieved using a clinically-relevant protocol. In order to determine the origin of the NIRAPA signal, immunohistochemistry, spatial transcriptomics and spatial proteomics were co-registered with imaging and applied to adjacent plaque sections. The highest NIRAPA signal was spatially correlated with bilirubin and associated blood-based residue and with the cytoplasmic contents of inflammatory macrophages bearing CD74, HLA-DR, CD14 and CD163 markers. In summary, we establish the potential to apply the NIRAPA-ultrasound imaging combination to detect vulnerable carotid plaque and a methodology for fusing molecular imaging with spatial transcriptomic and proteomic methods.

  View details for DOI 10.1016/j.biomaterials.2023.122314

  View details for PubMedID 37776766

• Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression. Nature methods Zengel, J., Wang, Y. X., Seo, J. W., Ning, K., Hamilton, J. N., Wu, B., Raie, M., Holbrook, C., Su, S., Clements, D. R., Pillay, S., Puschnik, A. S., Winslow, M. M., Idoyaga, J., Nagamine, C. M., Sun, Y., Mahajan, V. B., Ferrara, K. W., Blau, H. M., Carette, J. E. 2023

  Abstract

  The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

  View details for DOI 10.1038/s41592-023-01896-x

  View details for PubMedID 37291262

  View details for PubMedCentralID 3337962

• Fast volumetric ultrasound facilitates high-resolution 3D mapping of tissue compartments. Science advances Park, E. Y., Cai, X., Foiret, J., Bendjador, H., Hyun, D., Fite, B. Z., Wodnicki, R., Dahl, J. J., Boutin, R. D., Ferrara, K. W. 2023; 9 (22): eadg8176

  Abstract

  Volumetric ultrasound imaging has the potential for operator-independent acquisition and enhanced field of view. Panoramic acquisition has many applications across ultrasound; spanning musculoskeletal, liver, breast, and pediatric imaging; and image-guided therapy. Challenges in high-resolution human imaging, such as subtle motion and the presence of bone or gas, have limited such acquisition. These issues can be addressed with a large transducer aperture and fast acquisition and processing. Programmable, ultrafast ultrasound scanners with a high channel count provide an unprecedented opportunity to optimize volumetric acquisition. In this work, we implement nonlinear processing and develop distributed beamformation to achieve fast acquisition over a 47-centimeter aperture. As a result, we achieve a 50-micrometer -6-decibel point spread function at 5 megahertz and resolve in-plane targets. A large volume scan of a human limb is completed in a few seconds, and in a 2-millimeter dorsal vein, the image intensity difference between the vessel center and surrounding tissue was ~50 decibels, facilitating three-dimensional reconstruction of the vasculature.

  View details for DOI 10.1126/sciadv.adg8176

  View details for PubMedID 37256942

• Highly Excretable Gold Supraclusters for Translatable In Vivo Raman Imaging of Tumors. ACS nano Yu, J. H., Jeong, M. S., Cruz, E. O., Alam, I. S., Tumbale, S. K., Zlitni, A., Lee, S. Y., Park, Y. I., Ferrara, K., Kwon, S., Gambhir, S. S., Rao, J. 2023

  Abstract

  Raman spectroscopy provides excellent specificity for in vivo preclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity for in vivo Raman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performed in vivo Raman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen over 4 months, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted during the same period. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications of in vivo Raman imaging.

  View details for DOI 10.1021/acsnano.2c10378

  View details for PubMedID 36688431

• Multimodal imaging of capsid and cargo reveals differential brain targeting and liver detargeting of systemically-administered AAVs. Biomaterials Seo, J. W., Ajenjo, J., Wu, B., Robinson, E., Raie, M. N., Wang, J., Tumbale, S. K., Buccino, P., Anders, D. A., Shen, B., Habte, F. G., Beinat, C., James, M. L., Reyes, S. T., Ravindra Kumar, S., Miles, T. F., Lee, J. T., Gradinaru, V., Ferrara, K. W. 2022: 121701

  Abstract

  The development of gene delivery vehicles with high organ specificity when administered systemically is a critical goal for gene therapy. We combine optical and positron emission tomography (PET) imaging of 1) reporter genes and 2) capsid tags to assess the temporal and spatial distribution and transduction of adeno-associated viruses (AAVs). AAV9 and two engineered AAV vectors (PHP.eB and CAP-B10) that are noteworthy for maximizing blood-brain barrier transport were compared. CAP-B10 shares a modification in the 588 loop with PHP.eB, but also has a modification in the 455 loop, added with the goal of reducing off-target transduction. PET and optical imaging revealed that the additional modifications retained brain receptor affinity. In the liver, the accumulation of AAV9 and the engineered AAV capsids was similar (15% of the injected dose per cc and not significantly different between capsids at 21h). However, the engineered capsids were primarily internalized by Kupffer cells rather than hepatocytes, and liver transduction was greatly reduced. PET reporter gene imaging after engineered AAV systemic injection provided a non-invasive method to monitor AAV-mediated protein expression over time. Through comparison with capsid tagging, differences between brain localization and transduction were revealed. In summary, AAV capsids bearing imaging tags and reporter gene payloads create a unique and powerful platform to assay the pharmacokinetics, cellular specificity and protein expression kinetics of AAV vectors in vivo, a key enabler for the field of gene therapy.

  View details for DOI 10.1016/j.biomaterials.2022.121701

  View details for PubMedID 35985893

• A theranostic 3D ultrasound imaging system for high resolution image-guided therapy. Theranostics Bendjador, H., Foiret, J., Wodnicki, R., Stephens, D. N., Krut, Z., Park, E., Gazit, Z., Gazit, D., Pelled, G., Ferrara, K. W. 2022; 12 (11): 4949-4964

  Abstract

  Microbubble contrast agents are a diagnostic tool with broad clinical impact and an increasing number of indications. Many therapeutic applications have also been identified. Yet, technologies for ultrasound guidance of microbubble-mediated therapy are limited. In particular, arrays that are capable of implementing and imaging microbubble-based therapy in three dimensions in real-time are lacking. We propose a system to perform and monitor microbubble-based therapy, capable of volumetric imaging over a large field-of-view. To propel the promise of the theranostic treatment strategies forward, we have designed and tested a unique array and system for 3D ultrasound guidance of microbubble-based therapeutic protocols based on the frequency, temporal and spatial requirements. Methods: Four 256-channel plane wave scanners (Verasonics, Inc, WA, USA) were combined to control a 1024-element planar array with 1.3 and 2.5 MHz therapeutic and imaging transmissions, respectively. A transducer aperture of ~40*15 mm was selected and Field II was applied to evaluate the point spread function. In vitro experiments were performed on commercial and custom phantoms to assess the spatial resolution, image contrast and microbubble-enhanced imaging capabilities. Results: We found that a 2D array configuration with 64 elements separated by lambda-pitch in azimuth and 16 elements separated by 1.5lambda-pitch in elevation ensured the required flexibility. This design, of 41.6 mm * 16 mm, thus provided both an extended field-of-view, up to 11 cm x 6 cm at 10 cm depth and steering of ±18° in azimuth and ±12° in elevation. At a depth of 16 cm, we achieved a volume imaging rate of 60 Hz, with a contrast ratio and resolution, respectively, of 19 dB, 0.8 mm at 3 cm and 20 dB and 2.1 mm at 12.5 cm. Conclusion: A single 2D array for both imaging and therapeutics, integrated with a 1024 channel scanner can guide microbubble-based therapy in volumetric regions of interest.

  View details for DOI 10.7150/thno.71221

  View details for PubMedID 35836805

• Gold-Nanostar-Chitosan-Mediated Delivery of SARS-CoV-2 DNA Vaccine for Respiratory Mucosal Immunization: Development and Proof-of-Principle. ACS nano Kumar, U. S., Afjei, R., Ferrara, K., Massoud, T. F., Paulmurugan, R. 2021

  Abstract

  The COVID-19 pandemic is caused by the coronavirus SARS-CoV-2 (SC2). A variety of anti-SC2 vaccines have been approved for human applications, including those using messenger RNA (mRNA), adenoviruses expressing SC2 spike (S) protein, and inactivated virus. The protective periods of immunization afforded by these intramuscularly administered vaccines are currently unknown. An alternative self-administrable vaccine capable of mounting long-lasting immunity via sterilizing neutralizing antibodies would be hugely advantageous in tackling emerging mutant SC2 variants. This could also diminish the possibility of vaccinated individuals acting as passive carriers of COVID-19. Here, we investigate the potential of an intranasal (IN)-delivered DNA vaccine encoding the S protein of SC2 in BALB/c and C57BL/6J immunocompetent mouse models. The immune response to IN delivery of this SC2-spike DNA vaccine transported on a modified gold-chitosan nanocarrier shows a strong and consistent surge in antibodies (IgG, IgA, and IgM) and effective neutralization of pseudoviruses expressing S proteins of different SC2 variants (Wuhan, beta, and D614G). Immunophenotyping and histological analyses reveal chronological events involved in the recognition of SC2 S antigen by resident dendritic cells and alveolar macrophages, which prime the draining lymph nodes and spleen for peak SC2-specific cellular and humoral immune responses. The attainable high levels of anti-SC2 IgA in lung mucosa and tissue-resident memory T cells can efficiently inhibit SC2 and its variants at the site of entry and also provide long-lasting immunity.

  View details for DOI 10.1021/acsnano.1c05002

  View details for PubMedID 34705425

• In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift. Biomaterials Kheirolomoom, A., Kare, A. J., Ingham, E. S., Paulmurugan, R., Robinson, E. R., Baikoghli, M., Inayathullah, M., Seo, J. W., Wang, J., Fite, B. Z., Wu, B., Tumbale, S. K., Raie, M. N., Cheng, R. H., Nichols, L., Borowsky, A. D., Ferrara, K. W. 2021; 281: 121339

  Abstract

  Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

  View details for DOI 10.1016/j.biomaterials.2021.121339

  View details for PubMedID 35078042

• Two-way magnetic resonance tuning and enhanced subtraction imaging for non-invasive and quantitative biological imaging. Nature nanotechnology Wang, Z., Xue, X., Lu, H., He, Y., Lu, Z., Chen, Z., Yuan, Y., Tang, N., Dreyer, C. A., Quigley, L., Curro, N., Lam, K. S., Walton, J. H., Lin, T. Y., Louie, A. Y., Gilbert, D. A., Liu, K., Ferrara, K. W., Li, Y. 2020

  Abstract

  Distance-dependent magnetic resonance tuning (MRET) technology enables the sensing and quantitative imaging of biological targets in vivo, with the advantage of deep tissue penetration and fewer interactions with the surroundings as compared with those of fluorescence-based Förster resonance energy transfer. However, applications of MRET technology in vivo are currently limited by the moderate contrast enhancement and stability of T1-based MRET probes. Here we report a new two-way magnetic resonance tuning (TMRET) nanoprobe with dually activatable T1 and T2 magnetic resonance signals that is coupled with dual-contrast enhanced subtraction imaging. This integrated platform achieves a substantially improved contrast enhancement with minimal background signal and can be used to quantitatively image molecular targets in tumours and to sensitively detect very small intracranial brain tumours in patient-derived xenograft models. The high tumour-to-normal tissue ratio offered by TMRET in combination with dual-contrast enhanced subtraction imaging provides new opportunities for molecular diagnostics and image-guided biomedical applications.

  View details for DOI 10.1038/s41565-020-0678-5

  View details for PubMedID 32451501

• Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation. Nature communications Seo, J. W., Ingham, E. S., Mahakian, L. n., Tumbale, S. n., Wu, B. n., Aghevlian, S. n., Shams, S. n., Baikoghli, M. n., Jain, P. n., Ding, X. n., Goeden, N. n., Dobreva, T. n., Flytzanis, N. C., Chavez, M. n., Singhal, K. n., Leib, R. n., James, M. L., Segal, D. J., Cheng, R. H., Silva, E. A., Gradinaru, V. n., Ferrara, K. W. 2020; 11 (1): 2102

  Abstract

  Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.

  View details for DOI 10.1038/s41467-020-15818-4

  View details for PubMedID 32355221

• Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites. Proceedings of the National Academy of Sciences of the United States of America Ilovitsh, T. n., Feng, Y. n., Foiret, J. n., Kheirolomoom, A. n., Zhang, H. n., Ingham, E. S., Ilovitsh, A. n., Tumbale, S. K., Fite, B. Z., Wu, B. n., Raie, M. N., Zhang, N. n., Kare, A. J., Chavez, M. n., Qi, L. S., Pelled, G. n., Gazit, D. n., Vermesh, O. n., Steinberg, I. n., Gambhir, S. S., Ferrara, K. W. 2020

  Abstract

  Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

  View details for DOI 10.1073/pnas.1914906117

  View details for PubMedID 32430322

• Immune-mediated ECM depletion improves tumour perfusion and payload delivery. EMBO molecular medicine Yeow, Y. L., Kotamraju, V. R., Wang, X., Chopra, M., Azme, N., Wu, J., Schoep, T. D., Delaney, D. S., Feindel, K., Li, J., Kennedy, K. M., Allen, W. M., Kennedy, B. F., Larma, I., Sampson, D. D., Mahakian, L. M., Fite, B. Z., Zhang, H., Friman, T., Mann, A. P., Aziz, F. A., Kumarasinghe, M. P., Johansson, M., Ee, H. C., Yeoh, G., Mou, L., Ferrara, K. W., Billiran, H., Ganss, R., Ruoslahti, E., Hamzah, J. 2019: e10923

  Abstract

  High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune-modulating cytokine, tumour necrosis factor alpha (TNFalpha), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin-nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFalpha-CSG fusion protein to tumour ECM in tumour-bearing mice. Intravenously injected TNFalpha-CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM-rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour-bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFalpha.

  View details for DOI 10.15252/emmm.201910923

  View details for PubMedID 31709774

• Nonviral ultrasound-mediated gene delivery in small and large animal models NATURE PROTOCOLS Bez, M., Foiret, J., Shapiro, G., Pelled, G., Ferrara, K. W., Gazit, D. 2019; 14 (4): 1015–26

  View details for DOI 10.1038/s41596-019-0125-y

  View details for Web of Science ID 000462482100002

• CD8(+) T-Cell Density Imaging with Cu-64-Labeled Cys-Diabody Informs Immunotherapy Protocols CLINICAL CANCER RESEARCH Seo, J., Tavare, R., Mahakian, L. M., Silvestrini, M. T., Tam, S., Ingham, E. S., Salazar, F. B., Borowsky, A. D., Wu, A. M., Ferrara, K. W. 2018; 24 (20): 4976–87

  View details for DOI 10.1158/1078-0432.CCR-18-0261

  View details for Web of Science ID 000447598900009

• Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction MOLECULAR THERAPY Bez, M., Kremen, T. J., Tawackoli, W., Avalos, P., Sheyn, D., Shapiro, G., Giaconi, J. C., Ben David, S., Snedeker, J. G., Gazit, Z., Ferrara, K. W., Gazit, D., Pelled, G. 2018; 26 (7): 1746–55

  Abstract

  Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between the ligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8 weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting.

  View details for PubMedID 29784586

  View details for PubMedCentralID PMC6035740

• Distinct immune signatures in directly treated and distant tumors result from TLR adjuvants and focal ablation THERANOSTICS Chavez, M., Silvestrini, M. T., Ingham, E. S., Fite, B. Z., Mahakian, L. M., Tam, S. M., Ilovitsh, A., Monjazeb, A. M., Murphy, W. J., Hubbard, N. E., Davis, R. R., Tepper, C. G., Borowsky, A. D., Ferrara, K. W. 2018; 8 (13): 3611–28

  Abstract

  Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

  View details for PubMedID 30026870

• In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs SCIENCE TRANSLATIONAL MEDICINE Bez, M., Sheyn, D., Tawackoli, W., Avalos, P., Shapiro, G., Giaconi, J. C., Da, X., Ben David, S., Gavrity, J., Awad, H. A., Bae, H. W., Ley, E. J., Kremen, T. J., Gazit, Z., Ferrara, K. W., Pelled, G., Gazit, D. 2017; 9 (390)

  Abstract

  More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

  View details for PubMedID 28515335

  View details for PubMedCentralID PMC5524999

• Priming is key to effective incorporation of image-guided thermal ablation into immunotherapy protocols JCI INSIGHT Silvestrini, M. T., Ingham, E. S., Mahakian, L. M., Kheirolomoom, A., Liu, Y., Fite, B. Z., Tam, S. M., Tucci, S. T., Watson, K. D., Wong, A. W., Monjazeb, A. M., Hubbard, N. E., Murphy, W. J., Borowsky, A. D., Ferrara, K. W. 2017; 2 (6): e90521

  Abstract

  Focal therapies play an important role in the treatment of cancers where palliation is desired, local control is needed, or surgical resection is not feasible. Pairing immunotherapy with such focal treatments is particularly attractive; however, there is emerging evidence that focal therapy can have a positive or negative impact on the efficacy of immunotherapy. Thermal ablation is an appealing modality to pair with such protocols, as tumors can be rapidly debulked (cell death occurring within minutes to hours), tumor antigens can be released locally, and treatment can be conducted and repeated without the concerns of radiation-based therapies. In a syngeneic model of epithelial cancer, we found that 7 days of immunotherapy (TLR9 agonist and checkpoint blockade), prior to thermal ablation, reduced macrophages and myeloid-derived suppressor cells and enhanced IFN-γ-producing CD8+ T cells, the M1 macrophage fraction, and PD-L1 expression on CD45+ cells. Continued treatment with immunotherapy alone or with immunotherapy combined with ablation (primed ablation) then resulted in a complete response in 80% of treated mice at day 90, and primed ablation expanded CD8+ T cells as compared with all control groups. When the tumor burden was increased by implantation of 3 orthotopic tumors, successive primed ablation of 2 discrete lesions resulted in survival of 60% of treated mice as compared with 25% of mice treated with immunotherapy alone. Alternatively, when immunotherapy was begun immediately after thermal ablation, the abscopal effect was diminished and none of the mice within the cohort exhibited a complete response. In summary, we found that immunotherapy begun before ablation can be curative and can enhance efficacy in the presence of a high tumor burden. Two mechanisms have potential to impact the efficacy of immunotherapy when begun immediately after thermal ablation: mechanical changes in the tumor microenvironment and inflammatory-mediated changes in immune phenotype.

  View details for PubMedID 28352658

• Ultrasound ablation enhances drug accumulation and survival in mammary carcinoma models JOURNAL OF CLINICAL INVESTIGATION Wong, A. W., Fite, B. Z., Liu, Y., Kheirolomoom, A., Seo, J. W., Watson, K. D., Mahakian, L. M., Tam, S. M., Zhang, H., Foiret, J., Borowsky, A. D., Ferrara, K. W. 2016; 126 (1): 99–111

  Abstract

  Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates noninvasive image-guided conformal thermal therapy of cancer. Yet in many scenarios, the sensitive tissues surrounding the tumor constrain the margins of ablation; therefore, augmentation of MRgFUS with chemotherapy may be required to destroy remaining tumor. Here, we used 64Cu-PET-CT, MRI, autoradiography, and fluorescence imaging to track the kinetics of long-circulating liposomes in immunocompetent mammary carcinoma-bearing FVB/n and BALB/c mice. We observed a 5-fold and 50-fold enhancement of liposome and drug concentration, respectively, within MRgFUS thermal ablation-treated tumors along with dense accumulation within the surrounding tissue rim. Ultrasound-enhanced drug accumulation was rapid and durable and greatly increased total tumor drug exposure over time. In addition, we found that the small molecule gadoteridol accumulates around and within ablated tissue. We further demonstrated that dilated vasculature, loss of vascular integrity resulting in extravasation of blood cells, stromal inflammation, and loss of cell-cell adhesion and tissue architecture all contribute to the enhanced accumulation of the liposomes and small molecule probe. The locally enhanced liposome accumulation was preserved even after a multiweek protocol of doxorubicin-loaded liposomes and partial ablation. Finally, by supplementing ablation with concurrent liposomal drug therapy, a complete and durable response was obtained using protocols for which a sub-mm rim of tumor remained after ablation.

  View details for DOI 10.1172/JCI83312

  View details for Web of Science ID 000367765600013

  View details for PubMedID 26595815

  View details for PubMedCentralID PMC4701551

• Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE(-/-) Mice ACS NANO Kheirolomoom, A., Kim, C., Seo, J., Kumar, S., Son, D., Gagnon, M. J., Ingham, E. S., Ferrara, K. W., Jo, H. 2015; 9 (9): 8885–97

  Abstract

  The current study presents an effective and selective multifunctional nanoparticle used to deliver antiatherogenic therapeutics to inflamed pro-atherogenic regions without off-target changes in gene expression or particle-induced toxicities. MicroRNAs (miRNAs) regulate gene expression, playing a critical role in biology and disease including atherosclerosis. While anti-miRNA are emerging as therapeutics, numerous challenges remain due to their potential off-target effects, and therefore the development of carriers for selective delivery to diseased sites is important. Yet, co-optimization of multifunctional nanoparticles with high loading efficiency, a hidden cationic domain to facilitate lysosomal escape and a dense, stable incorporation of targeting moieties is challenging. Here, we create coated, cationic lipoparticles (CCLs), containing anti-miR-712 (∼1400 molecules, >95% loading efficiency) within the core and with a neutral coating, decorated with 5 mol % of peptide (VHPK) to target vascular cell adhesion molecule 1 (VCAM1). Optical imaging validated disease-specific accumulation as anti-miR-712 was efficiently delivered to inflamed mouse aortic endothelial cells in vitro and in vivo. As with the naked anti-miR-712, the delivery of VHPK-CCL-anti-miR-712 effectively downregulated the d-flow induced expression of miR-712 and also rescued the expression of its target genes tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) in the endothelium, resulting in inhibition of metalloproteinase activity. Moreover, an 80% lower dose of VHPK-CCL-anti-miR-712 (1 mg/kg dose given twice a week), as compared with naked anti-miR-712, prevented atheroma formation in a mouse model of atherosclerosis. While delivery of naked anti-miR-712 alters expression in multiple organs, miR-712 expression in nontargeted organs was unchanged following VHPK-CCL-anti-miR-712 delivery.

  View details for PubMedID 26308181

  View details for PubMedCentralID PMC4581466

• Dual inhibition of cyclooxygenase-2 and soluble epoxide hydrolase synergistically suppresses primary tumor growth and metastasis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, G., Panigrahy, D., Hwang, S., Yang, J., Mahakian, L. M., Wettersten, H. I., Liu, J., Wang, Y., Ingham, E. S., Tam, S., Kieran, M. W., Weiss, R. H., Ferrara, K. W., Hammock, B. D. 2014; 111 (30): 11127–32

  Abstract

  Prostaglandins derived from the cyclooxygenase (COX) pathway and epoxyeicosatrienoic acids (EETs) from the cytochrome P450/soluble epoxide hydrolase (sEH) pathway are important eicosanoids that regulate angiogenesis and tumorigenesis. COX-2 inhibitors, which block the formation of prostaglandins, suppress tumor growth, whereas sEH inhibitors, which increase endogenous EETs, stimulate primary tumor growth and metastasis. However, the functional interactions of these two pathways in cancer are unknown. Using pharmacological inhibitors as probes, we show here that dual inhibition of COX-2 and sEH synergistically inhibits primary tumor growth and metastasis by suppressing tumor angiogenesis. COX-2/sEH dual pharmacological inhibitors also potently suppress primary tumor growth and metastasis by inhibiting tumor angiogenesis via selective inhibition of endothelial cell proliferation. These results demonstrate a critical interaction of these two lipid metabolism pathways on tumorigenesis and suggest dual inhibition of COX-2 and sEH as a potential therapeutic strategy for cancer therapy.

  View details for PubMedID 25024195

• The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis NATURE COMMUNICATIONS Son, D., Kumar, S., Takabe, W., Kim, C., Ni, C., Alberts-Grill, N., Jang, I., Kim, S., Kim, W., Kang, S., Baker, A. H., Seo, J., Ferrara, K. W., Jo, H. 2013; 4: 3000

  Abstract

  MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge syndrome critical region 8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase 3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same 'seed sequence' as murine-specific miR-712 and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive 'athero-miRs' may provide a new treatment paradigm in atherosclerosis.

  View details for PubMedID 24346612

• Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angiogenesis, tumor growth, and metastasis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, G., Panigrahy, D., Mahakian, L. M., Yang, J., Liu, J., Lee, K., Wettersten, H. I., Ulu, A., Hu, X., Tam, S., Hwang, S., Ingham, E. S., Kieran, M. W., Weiss, R. H., Ferrara, K. W., Hammock, B. D. 2013; 110 (16): 6530–35

  Abstract

  Epidemiological and preclinical evidence supports that omega-3 dietary fatty acids (fish oil) reduce the risks of macular degeneration and cancers, but the mechanisms by which these omega-3 lipids inhibit angiogenesis and tumorigenesis are poorly understood. Here we show that epoxydocosapentaenoic acids (EDPs), which are lipid mediators produced by cytochrome P450 epoxygenases from omega-3 fatty acid docosahexaenoic acid, inhibit VEGF- and fibroblast growth factor 2-induced angiogenesis in vivo, and suppress endothelial cell migration and protease production in vitro via a VEGF receptor 2-dependent mechanism. When EDPs (0.05 mg · kg(-1) · d(-1)) are coadministered with a low-dose soluble epoxide hydrolase inhibitor, EDPs are stabilized in circulation, causing ~70% inhibition of primary tumor growth and metastasis. Contrary to the effects of EDPs, the corresponding metabolites derived from omega-6 arachidonic acid, epoxyeicosatrienoic acids, increase angiogenesis and tumor progression. These results designate epoxyeicosatrienoic acids and EDPs as unique endogenous mediators of an angiogenic switch to regulate tumorigenesis and implicate a unique mechanistic linkage between omega-3 and omega-6 fatty acids and cancers.

  View details for PubMedID 23553837

• Ultrasound Increases Nanoparticle Delivery by Reducing Intratumoral Pressure and Increasing Transport in Epithelial and Epithelial-Mesenchymal Transition Tumors CANCER RESEARCH Watson, K. D., Lai, C., Qin, S., Kruse, D. E., Lin, Y., Seo, J., Cardiff, R. D., Mahakian, L. M., Beegle, J., Inghanm, E. S., Curry, F., Reed, R. K., Ferrara, K. W. 2012; 72 (6): 1485–93

  Abstract

  Acquisition of the epithelial-mesenchymal transition (EMT) tumor phenotype is associated with impaired chemotherapeutic delivery and a poor prognosis. In this study, we investigated the application of therapeutic ultrasound methods available in the clinic to increase nanotherapeutic particle accumulation in epithelial and EMT tumors by labeling particles with a positron emission tomography tracer. Epithelial tumors were highly vascularized with tight cell-cell junctions, compared with EMT tumors where cells displayed an irregular, elongated shape with loosened cell-cell adhesions and a reduction in E-cadherin and cytokeratins 8/18 and 19. Without ultrasound, the accumulation of liposomal nanoparticles administered to tumors in vivo was approximately 1.5 times greater in epithelial tumors than EMT tumors. When ultrasound was applied, both nanoaccumulation and apparent tumor permeability were increased in both settings. Notably, ultrasound effects differed with thermal and mechanical indices, such that increasing the thermal ultrasound dose increased nanoaccumulation in EMT tumors. Taken together, our results illustrate how ultrasound can be used to enhance nanoparticle accumulation in tumors by reducing their intratumoral pressure and increasing their vascular permeability.

  View details for PubMedID 22282664

• Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hamzah, J., Kotamraju, V. R., Seo, J. W., Agemy, L., Fogal, V., Mahakian, L. M., Peters, D., Roth, L., Gagnon, M. J., Ferrara, K. W., Ruoslahti, E. 2011; 108 (17): 7154–59

  Abstract

  The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1(+) cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1-coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[(18)F]fluorobenzoic acid ([(18)F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [(18)F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [(18)F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.

  View details for PubMedID 21482787

• Longitudinal Investigation of Permeability and Distribution of Macromolecules in Mouse Malignant Transformation Using PET CLINICAL CANCER RESEARCH Rygh, C. B., Qin, S., Seo, J. W., Mahakian, L. M., Zhang, H., Adamson, R., Chen, J. Q., Borowsky, A. D., Cardiff, R. D., Reed, R. K., Curry, F. E., Ferrara, K. W. 2011; 17 (3): 550–59

  Abstract

  We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease.Albumin and liposomes were labeled with (64)Cu (half-life 12.7 hours), and longitudinal PET and CT imaging studies were conducted in a mouse model of ductal carcinoma in situ. A pharmacokinetic model was applied to estimate the tumor vascular volume and permeability.From early time points characterized by disseminated hyperproliferation, the enhanced vascular permeability facilitated lesion detection. During disease progression, the vascular volume fraction increased 1.6-fold and the apparent vascular permeability to albumin and liposomes increased ∼2.5-fold to 6.6 × 10(-8) and 1.3 × 10(-8) cm/s, respectively, with the accumulation of albumin increasing earlier in the disease process. In the malignant tumor, both tracers reached similar mean intratumoral concentrations of ∼6% ID/cc but the distribution of liposomes was more heterogeneous, ranging from 1% to 18% ID/cc compared with 1% to 9% ID/cc for albumin. The tumor-to-muscle ratio was 17.9 ± 8.1 and 7.1 ± 0.5 for liposomes and albumin, respectively, indicating a more specific delivery of liposomes than with albumin.PET imaging of radiolabeled particles, validated by confocal imaging and histology, detected the transition from premalignant to malignant lesions and effectively quantified the associated changes in vascular permeability.

  View details for PubMedID 21106723

• Lipid-Shelled Vehicles: Engineering for Ultrasound Molecular Imaging and Drug Delivery ACCOUNTS OF CHEMICAL RESEARCH Ferrara, K. W., Borden, M. A., Zhang, H. 2009; 42 (7): 881–92

  Abstract

  Ultrasound pressure waves can map the location of lipid-stabilized gas micro-bubbles after their intravenous administration in the body, facilitating an estimate of vascular density and microvascular flow rate. Microbubbles are currently approved by the Food and Drug Administration as ultrasound contrast agents for visualizing opacification of the left ventricle in echocardiography. However, the interaction of ultrasound waves with intravenously-injected lipid-shelled particles, including both liposomes and microbubbles, is a far richer field. Particles can be designed for molecular imaging and loaded with drugs or genes; the mechanical and thermal properties of ultrasound can then effect localized drug release. In this Account, we provide an overview of the engineering of lipid-shelled microbubbles (typical diameter 1000-10 000 nm) and liposomes (typical diameter 65-120 nm) for ultrasound-based applications in molecular imaging and drug delivery. The chemistries of the shell and core can be optimized to enhance stability, circulation persistence, drug loading and release, targeting to and fusion with the cell membrane, and therapeutic biological effects. To assess the biodistribution and pharmacokinetics of these particles, we incorporated positron emission tomography (PET) radioisotopes on the shell. The radionuclide (18)F (half-life approximately 2 h) was covalently coupled to a dipalmitoyl lipid, followed by integration of the labeled lipid into the shell, facilitating short-term analysis of particle pharmacokinetics and metabolism of the lipid molecule. Alternately, labeling a formed particle with (64)Cu (half-life 12.7 h), after prior covalent incorporation of a copper-chelating moiety onto the lipid shell, permits pharmacokinetic study of particles over several days. Stability and persistence in circulation of both liposomes and microbubbles are enhanced by long acyl chains and a poly(ethylene glycol) coating. Vascular targeting has been demonstrated with both nano- and microdiameter particles. Targeting affinity of the microbubble can be modulated by burying the ligand within a polymer brush layer; the application of ultrasound then reveals the ligand, enabling specific targeting of only the insonified region. Microbubbles and liposomes require different strategies for both drug loading and release. Microbubble loading is inhibited by the gas core and enhanced by layer-by-layer construction or conjugation of drug-entrapped particles to the surface. Liposome loading is typically internal and is enhanced by drug-specific loading techniques. Drug release from a microbubble results from the oscillation of the gas core diameter produced by the sound wave, whereas that from a liposome is enhanced by heat produced from the local absorption of acoustic energy within the tissue microenvironment. Biological effects induced by ultrasound, such as changes in cell membrane and vascular permeability, can enhance drug delivery. In particular, as microbubbles oscillate near a vessel wall, shock waves or liquid jets enhance drug transport. Mild heating induced by ultrasound, either before or after injection of the drug, facilitates the transport of liposomes from blood vessels to the tissue interstitium, thus increasing drug accumulation in the target region. Lipid-shelled vehicles offer many opportunities for chemists and engineers; ultrasound-based applications beyond the few currently in common use will undoubtedly soon multiply as molecular construction techniques are further refined.

  View details for PubMedID 19552457

• Microbubble-Enhanced Focused Ultrasound-Assisted Delivery of Adeno-Associated Viruses in Brain Tumors with PET Imaging Monitoring Guo, Y., Foiret, J., Seo, J., Zhang, N., Wu, B., Raie, M., Tumbale, S., Ferrara, K. CELL PRESS. 2024: 606-607

  View details for Web of Science ID 001332783402204

• PET Imaging of <i>In Situ</i>-Engineered CAR-T Cells in B Cell Lymphoma with <SUP>68</SUP>Ga-PSMA-11 Zhang, N., Seo, J., Robinson, E., Rivera-Rodriguez, A., Wang, J., Guo, Y., Wu, B., Tumbale, S. K., Raie, M. N., Inayathullah, M., Czerwinski, D. K., Levy, R., Ferrara, K. W. CELL PRESS. 2024: 69-70

  View details for Web of Science ID 001332783400130

• Pyroptosis Induction with Nanosonosensitizer-Augmented Sonodynamic Therapy Combined with PD-L1 Blockade Boosts Efficacy Against Liver Cancer. Advanced healthcare materials Zhang, N., Zeng, W., Xu, Y., Li, R., Wang, M., Liu, Y., Qu, S., Ferrara, K. W., Dai, Z. 2023: e2302606

  Abstract

  Induction of pyroptosis could promote anti-PD-L1 therapeutic efficacy due to the release of pro-inflammatory cytokines, but current approaches can cause off target toxicity. Herein, we design a phthalocyanine-conjugated mesoporous silicate nanoparticle (PMSN) for amplifying sonodynamic therapy (SDT) to augment oxidative stress and induce robust pyroptosis in tumors. The sub-10 nm diameter structure and c(RGDyC)-PEGylated modification enhance tumor targeting and renal clearance. The unique porous architecture of PMSN doubles ROS yield and enhances pyroptotic cell populations in tumors (25.0%) via a cavitation effect. PMSN-mediated SDT treatment efficiently reduces tumor mass and suppressed residual tumors in treated and distant sites by synergizing with PD-L1 blockade (85.93% and 77.09%, respectively). Furthermore, loading the chemotherapeutic, doxorubicin, into PMSN intensifies SDT-pyroptotic effects and increased efficacy. To our knowledge, this is the first report of the use of SDT regimens to induce pyroptosis in liver cancer. This noninvasive and effective strategy has potential for clinical translation. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/adhm.202302606

  View details for PubMedID 37987462

• Protocol for in vitro sonoporation validation using non-targeted microbubbles for human studies of ultrasound-mediated gene delivery. STAR protocols Zhang, N., Guo, Y., Foiret, J., Tumbale, S. K., Paulmurugan, R., Ferrara, K. W. 2023; 4 (4): 102723

  Abstract

  Microbubbles are currently approved for diagnostic ultrasound imaging and are under evaluation in therapeutic protocols. Here, we present a protocol for in vitro sonoporation validation using non-targeted microbubbles for gene delivery. We describe steps for computational simulation, experimental calibration, reagent preparation, ultrasound treatment, validation, and gene expression analysis. This protocol uses approved diagnostic microbubbles and parameters that are applicable for human use. For complete details on the use and execution of this protocol, please refer to Bez et al. (2017).1.

  View details for DOI 10.1016/j.xpro.2023.102723

  View details for PubMedID 37976155

• PAM and FUS-assisted local AAV gene delivery into the brain of Alzheimer's disease murine models assessed by quantitative PET reporter gene Ajenjo, J., Seo, J., Foiret, J., Wu, B., Raie, M., Wang, J., Guo, Y., Zhang, N., Tumbale, S., Ferrara, K. ELSEVIER SCIENCE INC. 2023: S141-S142

  View details for Web of Science ID 001128725600183

• Assessment of [<SUP>64</SUP>Cu]Cu-FAP-2286 in immunocompetent mice bearing pancreatic ductal adenocarcinoma (PDAC) tumors Seo, J., Ajenjo, J., Wang, J., Wu, B., Raie, M., Tumbale, S., Ferrara, K. ELSEVIER SCIENCE INC. 2023: S196

  View details for Web of Science ID 001128725600255

• OMIP-095: 40-Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues. Cytometry. Part A : the journal of the International Society for Analytical Cytology Kare, A. J., Nichols, L., Zermeno, R., Raie, M. N., Tumbale, S. K., Ferrara, K. W. 2023

  Abstract

  High-dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40-color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co-stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre-clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40-color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.

  View details for DOI 10.1002/cyto.a.24788

  View details for PubMedID 37768325

• Optical Breast Imaging: A Review of Physical Principles, Technologies, and Clinical Applications. Journal of breast imaging Poplack, S. P., Park, E. Y., Ferrara, K. W. 2023; 5 (5): 520-537

  Abstract

  Optical imaging involves the propagation of light through tissue. Current optical breast imaging technologies, including diffuse optical spectroscopy, diffuse optical tomography, and photoacoustic imaging, capitalize on the selective absorption of light in the near-infrared spectrum by deoxygenated and oxygenated hemoglobin. They provide information on the morphological and functional characteristics of different tissues based on their varied interactions with light, including physiologic information on lesion vascular content and anatomic information on tissue vascularity. Fluorescent contrast agents, such as indocyanine green, are used to visualize specific tissues, molecules, or proteins depending on how and where the agent accumulates. In this review, we describe the physical principles, spectrum of technologies, and clinical applications of the most common optical systems currently being used or developed for breast imaging. Most notably, US co-registered photoacoustic imaging and US-guided diffuse optical tomography have demonstrated efficacy in differentiating benign from malignant breast masses, thereby improving the specificity of diagnostic imaging. Diffuse optical tomography and diffuse optical spectroscopy have shown promise in assessing treatment response to preoperative systemic therapy, and photoacoustic imaging and diffuse optical tomography may help predict tumor phenotype. Lastly, fluorescent imaging using indocyanine green dye performs comparably to radioisotope mapping of sentinel lymph nodes and appears to improve the outcomes of autologous tissue flap breast reconstruction.

  View details for DOI 10.1093/jbi/wbad057

  View details for PubMedID 37981994

  View details for PubMedCentralID PMC10655724

• Optical Breast Imaging: A Review of Physical Principles, Technologies, and Clinical Applications JOURNAL OF BREAST IMAGING Poplack, S. P., Park, E., Ferrara, K. W. 2023; 5 (5): 520-537

  View details for DOI 10.1093/jbi/wbad057

  View details for Web of Science ID 001075554400003

• Gold-Nanostar-Chitosan-Mediated Delivery of SARS-CoV-2 DNA Vaccine for Respiratory Mucosal Immunization: Development and Proof-of-Principle (vol 15, pg 17582, 2021) ACS NANO Kumar, U. S., Afjei, R., Ferrara, K., Massoud, T. F., Paulmurugan, R. 2023

  View details for DOI 10.1021/acsnano.3c07103

  View details for Web of Science ID 001049111100001

  View details for PubMedID 37582219

• Large Array Deep Abdominal Imaging in Fundamental and Harmonic Mode. IEEE transactions on ultrasonics, ferroelectrics, and frequency control Ahmed, R., Foiret, J., Ferrara, K., Trahey, G. 2023; PP

  Abstract

  Deep abdominal images suffer from poor diffraction-limited lateral resolution. Extending the aperture size can improve resolution. However, phase distortion and clutter can limit the benefits of larger arrays. Previous studies have explored these effects using numerical simulations, multiple transducers and mechanically swept arrays. In this work, we used an 8.8 cm linear array transducer to investigate the effects of aperture size when imaging through the abdominal wall. We acquired channel data in fundamental and harmonic mode using five aperture sizes. To avoid motion and increase the parameter sampling, we decoded the full-synthetic aperture data and retrospectively synthesized nine apertures (2.9 cm to 8.8 cm). We imaged a wire target and a phantom through ex vivo porcine abdominal samples and scanned the livers of 13 healthy subjects. We applied bulk sound speed correction to the wire target data. Although point resolution improved from 2.12 mm to 0.74 mm at 10.5 cm depth, contrast resolution often degraded with aperture size. In subjects, larger apertures resulted in an average maximum contrast degradation of 5.5 dB at 9-11 cm depth. However, larger apertures often led to visual detection of vascular targets unseen with conventional apertures. An average 3.7 dB contrast improvement over fundamental mode in subjects showed that the known benefits of tissue-harmonic imaging extend to larger arrays.

  View details for DOI 10.1109/TUFFC.2023.3255800

  View details for PubMedID 37028314

• Molecular imaging and multi-cancer detection for precision medicine. Ferrara, K. AMER ASSOC CANCER RESEARCH. 2023: 2-3

  View details for Web of Science ID 000920673200021

• Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression Zengel, J. R., Wang, Y. X., Wseo, J., Hamilton, J. N., Wu, B., Raie, M., Holbrook, C., Su, S., Pillay, S., Puschnik, A. S., Nagamine, C., Ferrara, K., Blau, H. M., Carette, J. E. MARY ANN LIEBERT, INC. 2022: A27

  View details for Web of Science ID 000899950600084

• Multiomic analysis for optimization of combined focal and immunotherapy protocols in murine pancreatic cancer. Theranostics Wang, J., Fite, B. Z., Kare, A. J., Wu, B., Raie, M., Tumbale, S. K., Zhang, N., Davis, R. R., Tepper, C. G., Aviran, S., Newman, A. M., King, D. A., Ferrara, K. W. 2022; 12 (18): 7884-7902

  Abstract

  Background: Although combination immunotherapies incorporating local and systemic components have shown promising results in treating solid tumors, varied tumor microenvironments (TMEs) can impact immunotherapeutic efficacy. Method: We designed and evaluated treatment strategies for breast and pancreatic cancer combining magnetic resonance-guided focused ultrasound (MRgFUS) ablation and antibody therapies. With a combination of single-cell sequencing, spectral flow cytometry, and histological analyses, we profiled an immune-suppressed KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) pancreatic adenocarcinoma (MT4) model and a dense epithelial neu deletion (NDL) HER2+ mammary adenocarcinoma model with a greater fraction of lymphocytes, natural killer cells and activated dendritic cells. We then performed gene ontology analysis, spectral and digital cytometry to assess the immune response to combination immunotherapies and correlation with survival studies. Result: Based on gene ontology analysis, adding ablation to immunotherapy enriched immune cell migration pathways in the pancreatic cancer model and extensively enriched wound healing pathways in the breast cancer model. With CIBERSORTx digital cytometry, aCD40 + aPD-1 immunotherapy combinations enhanced dendritic cell activation in both models. In the MT4 TME, adding the combination of aCD40 antibody and checkpoint inhibitors (aPD-1 and aCTLA-4) with ablation was synergistic, increasing activated natural killer cells and T cells in distant tumors. Furthermore, ablation with immunotherapy upregulated critical Ly6c myeloid remodeling phenotypes that enhance T-cell effector function and increased granzyme and protease encoding genes by as much as 100-fold. Ablation combined with immunotherapy then extended survival in the MT4 model to a greater extent than immunotherapy alone. Conclusion: In summary, TME profiling informed a successful multicomponent treatment protocol incorporating ablation and facilitated differentiation of TMEs in which ablation is most effective.

  View details for DOI 10.7150/thno.73218

  View details for PubMedID 36451859

  View details for PubMedCentralID PMC9706583

• Improving plane wave ultrasound imaging through real-time beamformation across multiple arrays. Scientific reports Foiret, J., Cai, X., Bendjador, H., Park, E., Kamaya, A., Ferrara, K. W. 2022; 12 (1): 13386

  Abstract

  Ultrasound imaging is a widely used diagnostic tool but has limitations in the imaging of deep lesions or obese patients where the large depth to aperture size ratio (f-number) reduces image quality. Reducing the f-number can improve image quality, and in this work, we combined three commercial arrays to create a large imaging aperture of 100mm and 384 elements. To maintain the frame rate given the large number of elements, plane wave imaging was implemented with all three arrays transmitting a coherent wavefront. On wire targets at a depth of 100mm, the lateral resolution is significantly improved; the lateral resolution was 1.27mm with one array (1/3 of the aperture) and 0.37mm with the full aperture. After creating virtual receiving elements to fill the inter-array gaps, an autoregressive filter reduced the grating lobes originating from the inter-array gaps by -5.2dB. On a calibrated commercial phantom, the extended field-of-view and improved spatial resolution were verified. The large aperture facilitates aberration correction using a singular value decomposition-based beamformer. Finally, after approval of the Stanford Institutional Review Board, the three-array configuration was applied in imaging the liver of a volunteer, validating the potential for enhanced resolution.

  View details for DOI 10.1038/s41598-022-16961-2

  View details for PubMedID 35927389

• CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells. Robinson, E. R., Kare, A. J., Kheirolomoom, A., Inayathullah, M., Tumbale, S. K., Wu, B., Raie, M. N., Seo, J. W., Salazar, F. B., Paulmurugan, R., Wu, A. M., Ferrara, K. W. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509502651

• TME subtype impacts response to combined thermal ablation and immunotherapy Wang, J., Fite, B. Z., Kare, A., Wu, B., Raie, M. N., Tumbale, S., Davis, R. R., Tepper, C. G., Ferrara, K. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509503226

• TME subtype impacts response to combined thermal ablation and immunotherapy. Wang, J., Fite, B. Z., Kare, A., Wu, B., Raie, M. N., Tumbale, S., Davis, R. R., Tepper, C. G., Ferrara, K. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509501163

• CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells Robinson, E. R., Kare, A. J., Kheirolomoom, A., Inayathullah, M., Tumbale, S. K., Wu, B., Raie, M. N., Seo, J. W., Salazar, F. B., Paulmurugan, R., Wu, A. M., Ferrara, K. W. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509500599

• Alternative medicine: therapeutic effects on gastric original signet ring carcinoma via ascorbate and combination with sodium alpha lipoate. BMC complementary medicine and therapies Chen, W., Xu, L., Chang, E., Gowrishankar, G., Ferrara, K. W., Gambhir, S. S. 2022; 22 (1): 58

  Abstract

  BACKGROUND: Gastric signet ring cell carcinoma (SRCC) is an aggressive gastric adenocarcinoma with a poor prognosis when diagnosed at an advanced stage. As alternative medicine, two natural supplements (ascorbate (AA) and sodium alpha lipoate (LA)) have been shown to inhibit various cancers with mild side effects.METHODS: These two natural supplements and a series of combinations (AA&LA, AA+LA and LA+AA) were incubated with non-SRCC cells (GPM-1), patient-derived gastric origin SRCC (GPM-2), gastric-origin SRCCs (HSC-39 and KATO-3), human pancreatic (MIA PaCa-2) and ovarian (SKOV-3) cells for evaluating their therapeutic effects. Moreover, these treatments were applied in 3D-cultured organoids to reveal the feasibility of these approaches for in vivo study.RESULTS: Analyzing their antioxidant capabilities and dose-response curves, we observed that all four gastric cell lines, including three patient-derived cell lines were sensitive to ascorbate (~10mM). The influence of ascorbate incubation time was studied, with a 16-h incubation found to be optimal for in vitro studies. Moreover, a simultaneous combination of AA and LA (AA&LA) did not significantly inhibit cell proliferation, while prior LA treatment increased the growth inhibition of AA therapy (LA+AA). Anti-cancer efficacy of AA was further confirmed in 3D-cultured SRCC (KATO-3) organoids.CONCLUSIONS: This study highlights the potential of AA and LA+AA in treating gastric origin SRCC, and demonstrates the influence of order in which the drugs are administered.

  View details for DOI 10.1186/s12906-022-03541-0

  View details for PubMedID 35255889

• A Review of Imaging Methods to Assess Ultrasound-Mediated Ablation BMEF: A Science Partner Journal Fite, B. Z., Wang, J., Ghanouni, P., Ferrara, K. W. 2022

  View details for DOI 10.34133/2022/9758652

• Row-Multiplexed 1,024 Element Large Aperture Array for Electronic Scanning in Elevation Wodnicki, R., Bendjador, H., Kang, H., Foiret, J., Notard, C., Zhou, Q., Ferrara, K. W., IEEE IEEE. 2022

  View details for DOI 10.1109/IUS54386.2022.9958124

  View details for Web of Science ID 000896080400317

• Highly Integrated Multiplexing and Buffering Electronics for Large Aperture Ultrasonic Arrays BMEF: A Science Partner Journal Wodnicki, R., Kang, H., Li, D., Stephens, D. N., Jung, H., Sun, Y., Chen, R., Jiang, L., Cabrera-Munoz, N., Foiret, J., Zhou, Q., Ferrara, K. W. 2022

  View details for DOI 10.34133/2022/9870386

• Optimization of microbubble-based DNA vaccination with low-frequency ultrasound for enhanced cancer immunotherapy. Advanced therapeutics Zhang, N., Foiret, J., Kheirolomoom, A., Liu, P., Feng, Y., Tumbale, S., Raie, M., Wu, B., Wang, J., Fite, B. Z., Dai, Z., Ferrara, K. W. 2021; 4 (9)

  Abstract

  Immunotherapy is an important cancer treatment strategy; nevertheless, the lack of robust immune cell infiltration in the tumor microenvironment remains a factor in limiting patient response rates. In vivo gene delivery protocols can amplify immune responses and sensitize tumors to immunotherapies, yet non-viral transfection methods often sacrifice transduction efficiency for improved safety tolerance. To improve transduction efficiency, we optimized a strategy employing low ultrasound transmission frequency-induced bubble oscillation to introduce plasmids into tumor cells. Differential centrifugation isolated size-specific microbubbles. The diameter of the small microbubble population was 1.27 ± 0.89 μm and that of larger population was 4.23 ± 2.27 μm. Upon in vitro insonation with the larger microbubble population, 29.7% of cancer cells were transfected with DNA plasmids, higher than that with smaller microbubbles (18.9%, P <0.05) or positive control treatments with a commercial transfection reagent (12%, P < 0.01). After 48 h, gene expression increased more than two-fold in tumors treated with large, as compared with small, microbubbles. Furthermore, the immune response, including tumor infiltration of CD8+ T cells and F4/80+ macrophages, was enhanced. We believe that this safe and efficacious method can improve preclinical procedures and outcomes for DNA vaccines in cancer immunotherapy in the future.

  View details for DOI 10.1002/adtp.202100033

  View details for PubMedID 34632048

  View details for PubMedCentralID PMC8494128

• Synergies between therapeutic ultrasound, gene therapy and immunotherapy in cancer treatment. Advanced drug delivery reviews Zhang, N., Wang, J., Foiret, J., Dai, Z., Ferrara, K. W. 2021: 113906

  Abstract

  Due to the ease of use and excellent safety profile, ultrasound is a promising technique for both diagnosis and site-specific therapy. Ultrasound-based techniques have been developed to enhance the pharmacokinetics and efficacy of therapeutic agents in cancer treatment. In particular, transfection with exogenous nucleic acids has the potential to stimulate an immune response in the tumor microenvironment. Ultrasound-mediated gene transfection is a growing field, and recent work has incorporated this technique into cancer immunotherapy. Compared with other gene transfection methods, ultrasound-mediated gene transfection has a unique opportunity to augment the intracellular uptake of nucleic acids while safely and stably modulating the expression of immunostimulatory cytokines. The development and commercialization of therapeutic ultrasound systems further enhance the potential translation. In this Review, we introduce the underlying mechanisms and ongoing preclinical studies of ultrasound-based techniques in gene transfection for cancer immunotherapy. Furthermore, we expand on aspects of therapeutic ultrasound that impact gene therapy and immunotherapy, including tumor debulking, enhancing cytokines and chemokines and altering nanoparticle pharmacokinetics as these effects of ultrasound cannot be fully dissected from targeted gene therapy. We finally explore the outlook for this rapidly developing field.

  View details for DOI 10.1016/j.addr.2021.113906

  View details for PubMedID 34333075

• Optimization of Microbubble-Based DNA Vaccination with Low-Frequency Ultrasound for Enhanced Cancer Immunotherapy ADVANCED THERAPEUTICS Zhang, N., Foiret, J., Kheirolomoom, A., Liu, P., Feng, Y., Tumbale, S., Raie, M., Wu, B., Wang, J., Fite, B. Z., Dai, Z., Ferrara, K. W. 2021

  View details for DOI 10.1002/adtp.202100033

  View details for Web of Science ID 000657667500001

• Pre-clinical evaluation of immunoPET imaging using agonist CD40 monoclonal antibody in pancreatic tumor-bearing mice. Nuclear medicine and biology Aghevlian, S., Wu, B., Raie, M. N., Tumbale, S. K., Kare, A. J., Seo, J. W., Ferrara, K. W. 2021; 98-99: 8–17

  Abstract

  BACKGROUND: A novel [64Cu]Cu-NOTA-aCD40 immunoPET tracer was developed to image a CD40+ pancreatic tumor model in C57BL/6 mice and to study the biodistribution profile of the agonist CD40 (aCD40) monoclonal antibody (mAb) alone or combined with other mAbs.PROCEDURES: Copper-64 ([64Cu]Cu) labeled NOTA-aCD40 and NOTA-IgG (10 mug; 7 MBq) were injected intravenously into C57BL/6 mice with subcutaneous mT4 tumors to assess specificity 48 h post injection (p.i.) through positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies (n = 5). [64Cu]Cu-NOTA-aCD40 was injected alone or simultaneously in combination with a therapeutic mass of cold aCD40 (100 mug), aPD-1 (200 mug) and aCTLA-4 (200 mug) mAbs. A group of mice with or without tumor received the second round of injections 1 or 3 weeks apart, respectively. PET/CT imaging and biodistribution studies were performed at 48 h p.i. The organ dose for [64Cu]Cu was estimated based on biodistribution studies with 2 mug [64Cu]Cu-NOTA-aCD40 (corresponds to 5 mg patient dose) in non-tumor bearing mice.RESULTS: [64Cu]Cu-NOTA-aCD40 accumulation was 2.3- and 7.8-fold higher than [64Cu]Cu-NOTA-IgG in tumors and spleen, respectively, indicating the specificity of aCD40 mAb in a mouse pancreatic tumor model. Tumor accumulation of [64Cu]Cu-NOTA-aCD40 was 21.2 ± 7.3%ID/g at 48 h after injection. Co-injection of [64Cu]Cu-NOTA-aCD40 with cold aCD40 mAb alone or with PD-1 and CTLA-4 mAbs reduced both spleen and tumor uptake, whereas liver uptake was increased. With the second round of injections, the liver was the only organ with substantial uptake. With a 2 mug administered dose of [64Cu]Cu-NOTA-aCD40 in a dosimetry study, the liver to spleen ratio was greater compared to the 10 mug dose (2.8 vs 0.37; respectively). The human equivalent for the highest dose organ (liver) was 198 ± 28.7 muSv/MBq.CONCLUSIONS: A CD40-immunoreactive [64Cu]Cu-NOTA-aCD40 probe was developed. The ratio of spleen to liver accumulation exceeded that of the IgG isotype and was greatest with a single small, injected mass. The safety of human patient imaging with [64Cu]Cu was established based on extrapolation of the organ specificity to human imaging.

  View details for DOI 10.1016/j.nucmedbio.2021.04.001

  View details for PubMedID 33962357

• Estimation of Tissue Attenuation from Ultrasonic B-Mode Images-Spectral-Log-Difference and Method-of-Moments Algorithms Compared SENSORS Brandner, D., Cai, X., Foiret, J., Ferrara, K. W., Zagar, B. G. 2021; 21 (7)

  Abstract

  We report on results from the comparison of two algorithms designed to estimate the attenuation coefficient from ultrasonic B-mode scans obtained from a numerical phantom simulating an ultrasound breast scan. It is well documented that this parameter significantly diverges between normal tissue and malignant lesions. To improve the diagnostic accuracy it is of great importance to devise and test algorithms that facilitate the accurate, low variance and spatially resolved estimation of the tissue's attenuation properties. A numerical phantom is realized using k-Wave, which is an open source Matlab toolbox for the time-domain simulation of acoustic wave fields that facilitates both linear and nonlinear wave propagation in homogeneous and heterogeneous tissue, as compared to strictly linear ultrasound simulation tools like Field II. k-Wave allows to simulate arbitrary distributions, resolved down to single voxel sizes, of parameters including the speed of sound, mass density, scattering strength and to include power law acoustic absorption necessary for simulation tasks in medical diagnostic ultrasound. We analyze the properties and the attainable accuracy of both the spectral-log-difference technique, and a statistical moments based approach and compare the results to known reference values from the sound field simulation.

  View details for DOI 10.3390/s21072548

  View details for Web of Science ID 000638847600001

  View details for PubMedID 33916496

• Immune modulation resulting from MR-guided high intensity focused ultrasound in a model of murine breast cancer. Scientific reports Fite, B. Z., Wang, J., Kare, A. J., Ilovitsh, A., Chavez, M., Ilovitsh, T., Zhang, N., Chen, W., Robinson, E., Zhang, H., Kheirolomoom, A., Silvestrini, M. T., Ingham, E. S., Mahakian, L. M., Tam, S. M., Davis, R. R., Tepper, C. G., Borowsky, A. D., Ferrara, K. W. 2021; 11 (1): 927

  Abstract

  High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65°C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1beta and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.

  View details for DOI 10.1038/s41598-020-80135-1

  View details for PubMedID 33441763

• Systemic Immunotherapy with Micellar Resiquimod-Polymer Conjugates Triggers a Robust Antitumor Response in a Breast Cancer Model. Advanced healthcare materials Kakwere, H. n., Zhang, H. n., Ingham, E. S., Nura-Raie, M. n., Tumbale, S. K., Allen, R. n., Tam, S. M., Wu, B. n., Liu, C. n., Kheirolomoom, A. n., Fite, B. Z., Ilovitsh, A. n., Lewis, J. S., Ferrara, K. W. 2021: e2100008

  Abstract

  Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due to toxicity. Integrating resiquimod into nanoparticles presents an avenue to circumvent the toxicity problem. Herein, the preparation of degradable nanoparticles with covalently bound resiquimod and their systemic application in cancer immunotherapy is reported. Dispersion in water of amphiphilic constructs integrating resiquimod covalently bound via degradable amide or ester linkages yields immune-activating nanoparticles. The degradable agonist-nanoparticle bonds allow the release of resiquimod from the carrier nanoparticles. In vitro assays with antigen presenting cells demonstrate that the nanoparticles retain the immunostimulatory activity of resiquimod. Systemic administration of the nanoparticles and checkpoint blockade (aPD-1) to a breast cancer mouse model with multiple established tumors triggers antitumor activity evidenced by suppressed tumor growth and enhanced CD8+ T-cell infiltration. Nanoparticles with ester links, which hydrolyze more readily, yield a stronger immune response with 75% of tumors eliminated when combined with aPD-1. The reduced tumor growth and the presence of activated CD8+ T-cells across multiple tumors suggest the potential for treating metastatic cancer.

  View details for DOI 10.1002/adhm.202100008

  View details for PubMedID 33646600

• Modular Large Array for Liver Cancer Imaging in Handheld Form Factor Wodnicki, R., Kang, H., Zhang, J., Foiret, J., Notard, C., Ratsimandresy, L., Auclair, P., Zhou, Q., Ferrara, K. W., IEEE IEEE. 2021

  View details for DOI 10.1109/IUS52206.2021.9593539

  View details for Web of Science ID 000832095000212

• Minicircles for a two-step blood biomarker and PET imaging early cancer detection strategy. Journal of controlled release : official journal of the Controlled Release Society Robinson, E. R., Gowrishankar, G., D'Souza, A., Kheirolomoom, A., Haywood, T., Hori, S. S., Chuang, H. Y., Zeng, Y., Tumbale, S., Aalipour, A., Beinat, C., Alam, I. S., Sathirachinda, A., Kanada, M., Paulmurugan, R., Ferrara, K. W., Gambhir, S. S. 2021

  Abstract

  Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection agent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection agent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.

  View details for DOI 10.1016/j.jconrel.2021.05.026

  View details for PubMedID 34029631

• Gemcitabine-retinoid prodrug loaded nanoparticles display in vitro antitumor efficacy towards drug-resilient human PANC-1 pancreatic cancer cells. Materials science & engineering. C, Materials for biological applications Kakwere, H., Ingham, E. S., Tumbale, S. K., Ferrara, K. W. 2020; 117: 111251

  Abstract

  The treatment of pancreatic cancer with gemcitabine is hampered by its rapid metabolism in vivo, the dense stroma around the tumor site which prevents the drug from reaching the cancerous cells and drug resistance. To address these challenges, this study describes the preparation of a retinoid prodrug of gemcitabine, GemRA (gemcitabine conjugated to retinoic acid), and its formulation into a nanoparticulate system applicable for pancreatic cancer treatment. Retinoic acid targets stellate cells which are part of the stroma and can thus augment the delivery of gemcitabine. GemRA dissolved in dimethylsulfoxide presented efficacy towards PANC-1 (human) and mT4 (mouse) pancreatic cancer cell lines but its poor solubility in aqueous solution affects its applicability. Thus, the preparation of the nanoparticles was initially attempted through self-assembly of GemRA, which resulted in the formation of unstable aggregates that precipitated during preparation. As a result, encapsulation of the drug into micelles of polyethylene glycol-retinoic acid (PGRA) amphiphilic conjugates was accomplished and resulted in successful incorporation of GemRA into nanoparticles of ca. 33nm by dynamic light scattering and 25nm by transmission electron microscopy. The nanoparticles had good stability in aqueous media and protected gemcitabine from the enzymatic action of cytidine deaminase, which converts gemcitabine to its inactive metabolite upon circulation. Cellular uptake of the nanoparticles by PANC-1 cells was confirmed by fluorescence spectroscopy and flow cytometry. Treatment of PANC-1 cells in vitro with the prodrug-loaded nanoparticles resulted in a significant reduction in cell viability (IC50 ca. 5muM) compared to treatment with gemcitabine (IC50>1000muM). The ability of the GemRA-loaded nanoparticles to induce cellular apoptosis of treated PANC-1 cells was ascertained via a TUNEL assay suggesting these nanoparticles are effective in pancreatic cancer treatment.

  View details for DOI 10.1016/j.msec.2020.111251

  View details for PubMedID 32919625

• Development of thermosensitive resiquimod-loaded liposomes for enhanced cancer immunotherapy. Journal of controlled release : official journal of the Controlled Release Society Zhang, H., Tang, W., Kheirolomoom, A., Fite, B. Z., Wu, B., Lau, K., Baikoghli, M., Raie, M. N., Tumbale, S. K., Foiret, J., Ingham, E. S., Mahakian, L. M., Tam, S. M., Cheng, R. H., Borowsky, A. D., Ferrara, K. W. 2020

  Abstract

  Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100 mM FeSO4 as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSL released 80% of R848 within 5 min at 42 °C. These TSLs were then combined with alphaPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2+, ER/PR negative). Combined with alphaPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100 days. Immunohistochemistry confirmed enhanced CD8+ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and alphaPD-1, inhibited tumor growth and extended median survival from 28 days (no-treatment control) to 94 days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10 mug for intra-tumoral injection or 6 mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with alphaPD-1 in mouse models of breast cancer.

  View details for DOI 10.1016/j.jconrel.2020.11.013

  View details for PubMedID 33189786

• The effective coupling coefficient for a completed PIN-PMN-PT array. Ultrasonics Stephens, D. N., Wodnicki, R., Chen, R., Liang, L., Zhou, Q., Morrison, K., Ferrara, K. W. 2020; 109: 106258

  Abstract

  The computation of the electromechanical coupling coefficient (EMCC) of a fully assembled medical ultrasound transducer array is directly computed with closed form expressions. The Levenberg-Marquardt non-linear regression algorithm (LMA) is employed to help confirm the EMCC calculated prediction (kEFF) and provide statistical insights. The complex electrical impedance spectra of a 1-3 composite array with two matching layers operating at a 3.75MHz center frequency using PIN-PMN-PT single crystal material is measured in air both before and after oven heating at 160°C for 15min. The oven heating produces changes in the EMCC of -4.9%, clamped dielectric constant of -11%, and effective transducer longitudinal velocity of -2.5%. Utilizing the pre- and post-heating array impedance data, the calculated EMCC values from the new closed form expressions agree well with the complete KLM model based LMA, and also exhibit approximately one tenth the error as compared to the formulas for a flat, unloaded transducer.

  View details for DOI 10.1016/j.ultras.2020.106258

  View details for PubMedID 33011614

• Targeted Mir-146a; an Innovative Treatment Modality for Shear Stress-induced Vascular Inflammation Mohamed, I., Rooney, K., Ferrara, K., Searles, C. D. LIPPINCOTT WILLIAMS & WILKINS. 2020

  View details for Web of Science ID 000607042600011

• Large Area 1.75D Array for Liver Cancer by Tiling of Multi-Generation ASIC Array Modules Wodnicki, R., Sun, Y., Li, R., Stephens, D. N., Kang, H., Chen, R., Foiret, J., Zhou, Q., Ferrara, K. W., IEEE IEEE. 2020

  View details for Web of Science ID 000635688900263

• Engineering personalized peptide-based cancer nanovaccines Kakwere, H., Zhang, H., Kheirolomoom, A., Ferrara, K. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000525061504439

• Tumor-specific delivery of gemcitabine with activatable liposomes. Journal of controlled release : official journal of the Controlled Release Society Tucci, S. T., Kheirolomoom, A., Ingham, E. S., Mahakian, L. M., Tam, S. M., Foiret, J., Hubbard, N. E., Borowsky, A. D., Baikoghli, M., Cheng, R. H., Ferrara, K. W. 2019

  Abstract

  Gemcitabine delivery to pancreatic ductal adenocarcinoma is limited by poor pharmacokinetics, dense fibrosis and hypo-vascularization. Activatable liposomes, with drug release resulting from local heating, enhance serum stability and circulation, and the released drug retains the ability to diffuse within the tumor. A limitation of liposomal gemcitabine has been the low loading efficiency. To address this limitation, we used the superior solubilizing potential of copper (II) gluconate to form a complex with gemcitabine at copper:gemcitabine (1:4). Thermosensitive liposomes composed of DPPC:DSPC:DSPE-PEG2k (80:15:5, mole%) then reached 12 wt% loading, 4-fold greater than previously reported values. Cryo transmission electron microscopy confirmed the presence of a liquid crystalline gemcitabine‑copper mixture. The optimized gemcitabine liposomes released 60% and 80% of the gemcitabine within 1 and 5 min, respectively, at 42 °C. Liposomal encapsulation resulted in a circulation half-life of ~2 h in vivo (compared to reported circulation of 16 min for free gemcitabine in mice), and free drug was not detected within the plasma. The resulting gemcitabine liposomes were efficacious against both murine breast cancer and pancreatic cancer in vitro. Three repeated treatments of activatable gemcitabine liposomes plus ultrasound hyperthermia regressed or eliminated tumors in the neu deletion model of murine breast cancer with limited toxicity, enhancing survival when compared to treatment with gemcitabine alone. With 5% of the free gemcitabine dose (5 rather than 100 mg/kg), tumor growth was suppressed to the same degree as gemcitabine. Additionally, in a more aggressive tumor model of murine pancreatic cancer, liposomal gemcitabine combined with local hyperthermia induced cell death and regions of apoptosis and necrosis.

  View details for DOI 10.1016/j.jconrel.2019.07.014

  View details for PubMedID 31301340

• Combining activatable nanodelivery with immunotherapy in a murine breast cancer model JOURNAL OF CONTROLLED RELEASE Kheirolomoom, A., Silvestrini, M. T., Ingham, E. S., Mahakian, L. M., Tam, S. M., Tumbale, S. K., Foiret, J., Hubbard, N. E., Borowsky, A. D., Ferrara, K. W. 2019; 303: 42–54

  View details for DOI 10.1016/j.jconrel.2019.04.008

  View details for Web of Science ID 000471239600005

• Polymeric perfluorocarbon nanoemulsions are ultrasound-activated wireless drug infusion catheters BIOMATERIALS Zhong, Q., Yoon, B. C., Aryal, M., Wang, J. B., Ilovitsh, T., Baikoghli, M. A., Hosseini-Nassab, N., Karthik, A., Cheng, R. H., Ferrara, K. W., Airan, R. D. 2019; 206: 73–86

  View details for DOI 10.1016/j.biomaterials.2019.03.021

  View details for Web of Science ID 000467669700007

• Acoustic radiation force imaging using a single-shot spiral readout PHYSICS IN MEDICINE AND BIOLOGY Ilovitsh, A., Fite, B. Z., Ilovitsh, T., Ferrara, K. W. 2019; 64 (12)

  View details for DOI 10.1088/1361-6560/ab1e21

  View details for Web of Science ID 000471166400004

• Carson-Zagzebski Distinguished Lectureship On Medical Ultrasound Diederich, C., Liu, T., Ferrara, K. WILEY. 2019: E349

  View details for Web of Science ID 000471277702224

• Positron emission tomography imaging of adeno-associated virus serotype 9-tetracystein (AAV9-TC) labeled with a multichelator Seo, J., Mahakian, L., Ingham, E., Tumbale, S., Shams, S., Silva, E., Ferrara, K. WILEY. 2019: S40–S41

  View details for Web of Science ID 000468965200023

• Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis ACTA NEUROPATHOLOGICA COMMUNICATIONS Sundstrom, T., Prestegarden, L., Azuaje, F., Aasen, S., Rosland, G., Varughese, J. K., Bahador, M., Bernatz, S., Braun, Y., Harter, P. N., Skaftnesmo, K., Ingham, E. S., Mahakian, L. M., Tam, S., Tepper, C. G., Petersen, K., Ferrara, K. W., Tronstad, K., Lund-Johansen, M., Beschorner, R., Bjerkvig, R., Thorsen, F. 2019; 7

  View details for DOI 10.1186/s40478-019-0712-8

  View details for Web of Science ID 000464707800001

• Simultaneous Axial Multifocal Imaging Using a Single Acoustical Transmission: A Practical Implementation IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Ilovitsh, A., Ilovitsh, T., Foiret, J., Stephens, D. N., Ferrara, K. W. 2019; 66 (2): 273–84

  Abstract

  Standard ultrasound imaging techniques rely on sweeping a focused beam across a field of view; however, outside the transmission focal depth, image resolution and contrast are degraded. High-quality deep tissue in vivo imaging requires focusing the emitted field at multiple depths, yielding high-resolution and high-contrast ultrasound images but at the expense of a loss in frame rate. Recent developments in ultrasound technologies have led to user-programmable systems, which enable real-time dynamic control over the phase and apodization of each individual element in the imaging array. In this paper, we present a practical implementation of a method to achieve simultaneous axial multifoci using a single acoustical transmission. Our practical approach relies on the superposition of axial multifoci waveforms in a single transmission. The delay in transmission between different elements is set such that pulses constructively interfere at multiple focal depths. The proposed method achieves lateral resolution similar to successive focusing, but with an enhanced frame rate. The proposed method uses standard dynamic receive beamforming, identical to two-way focusing, and does not require additional postprocessing. Thus, the method can be implemented in real time on programmable ultrasound systems that allow different excitation signals for each element. The proposed method is described analytically and validated by laboratory experiments in phantoms and ex vivo biological samples.

  View details for DOI 10.1109/TUFFC.2018.2885080

  View details for Web of Science ID 000458775800003

  View details for PubMedID 30530361

  View details for PubMedCentralID PMC6375789

• Enhanced delivery of AAV-like nanoparticles after blood-brain barrier disruption in a mouse model Foiret, J., Seo, J., Zhang, H., Kakwere, H., Mahakian, L. M., Tam, S., Ferrara, K. W., IEEE IEEE. 2019: 884–87

  View details for Web of Science ID 000510220100226

• Co-integrated PIN-PMN-PT 2D Array and Transceiver Electronics by Direct-Assembly Using a 3D Printed Interposer Grid Frame. IEEE transactions on ultrasonics, ferroelectrics, and frequency control Wodnicki, R. n., Kang, H. n., Chen, R. n., Cabrera-Munoz, N. E., Jong, H. n., Jiang, L. n., Foiret, J. n., Liu, Y. n., Chiu, V. n., Stephens, D. N., Zhou, Q. n., Ferrara, K. W. 2019

  Abstract

  Tiled modular 2D ultrasound arrays have the potential for realizing large apertures for novel diagnostic applications. This work presents an architecture for fabrication of tileable 2D array modules implemented using 1-3 composites of high bandwidth PIN-PMN-PT single crystal piezoelectric material closely coupled with high voltage CMOS Application Specific Integrated Circuit (ASIC) electronics for buffering and multiplexing functions. The module, which is designed to be operated as a λ-pitch 1.75D array, benefits from an improved electromechanical coupling coefficient and increased Curie temperature and is assembled directly on top of the ASIC silicon substrate using an interposer backing. The interposer consists of a novel 3D printed acrylic frame that is filled with conducting and acoustically absorbing silver epoxy material. The ASIC comprises a high voltage switching matrix with locally integrated buffering and is interfaced to a Verasonics Vantage 128, using a local FPGA controller. Multiple prototype 5 × 6 element array modules have been fabricated by this process. The combined acoustic array and ASIC module was configured electronically by programming the switches to operate as a 1D array with elements grouped in elevation for imaging and pulse-echo testing. The resulting array configuration had an average center frequency of 4.55 MHz, azimuthal element pitch of 340 #gm, and exhibited average -20dB pulse-width of 592 ns, and average -6dB fractional bandwidth of 77%.

  View details for DOI 10.1109/TUFFC.2019.2944668

  View details for PubMedID 31567082

• Multiplexed ultrasound beam summation for side lobe reduction. Scientific reports Ilovitsh, A. n., Ilovitsh, T. n., Ferrara, K. W. 2019; 9 (1): 13961

  Abstract

  Two-way focusing, which relies on sweeping a focused beam across a field of view, is the conventional method for performing high-quality ultrasound imaging. Side lobes resulting from diffraction reduce the image contrast, thus degrade the image quality. In this paper, we present a new method for beam shaping the transmitted ultrasound waveform in order to reduce side lobes and improve image quality. The beam shaping is achieved by transmitting two different waveforms that are interlaced between the odd and even elements. One waveform generates a standard diffraction-limited single focus, and the second waveform generates two foci at the same focal depth as the single focus. The distance between the two foci is selected such that they will destructively interfere with the first order side lobes of the single focus, effectively eliminating these side lobes. A 7.5 dB side lobe reduction was measured experimentally at a depth of 60 mm, using a phased array transducer with a center frequency of 3 MHz. This real-time method utilizes standard receive beamforming, identical to traditional two-way focusing, and does not require post-processing. The method can be implemented with conventional ultrasound systems, without the need for additional components. The proposed method is described analytically, optimized via numerical simulation, and validated by experiments using wire targets, tissue-mimicking phantoms, and in vivo imaging of the rat bladder.

  View details for DOI 10.1038/s41598-019-50317-7

  View details for PubMedID 31562381

• 3D monitoring and control of microbubble cavitation for gene delivery Cai, X., Foiret, J., Roth, J., Gazit, Z., Pelled, G., Gazit, D., Ferrara, K. W., IEEE IEEE. 2019: 888–90

  View details for Web of Science ID 000510220100227

• Tiled Large Element 1.75D Aperture with Dual Array Modules by Adjacent Integration of PIN-PMN-PT Transducers and Custom High Voltage Switching ASICs Wodnicki, R., Kang, H., Li, D., Sun, Y., Jiang, L., Chen, R., Chen, Z., Jong, H., Foiret, J., Liu, Y., Chiu, V., Stephens, D. N., Zhou, Q., Ferrara, K. W., IEEE IEEE. 2019: 1955–58

  View details for Web of Science ID 000510220100501

• Enhanced microbubble contrast agent oscillation following 250kHz insonation. Scientific reports Ilovitsh, T., Ilovitsh, A., Foiret, J., Caskey, C. F., Kusunose, J., Fite, B. Z., Zhang, H., Mahakian, L. M., Tam, S., Butts-Pauly, K., Qin, S., Ferrara, K. W. 2018; 8 (1): 16347

  Abstract

  Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuationcompared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250kHz transmission as compared with the 1MHz center frequency. Following 250kHz insonation, microbubble expansion increased nonlinearly with increasing ultrasonic pressure, and was accurately predicted by either the modified Rayleigh-Plesset equation for a clean bubble or the Marmottant model of a lipid-shelledmicrobubble. The expansion ratio reached 30-fold with 250kHz at a peak negative pressure of 400kPa, as compared to a measured expansion ratio of 1.6 fold for 1MHz transmission at a similar peak negative pressure. Further, the range of peak negative pressure yielding stable cavitation in vitro was narrow (~100kPa) for the 250kHz transmission frequency. Blood brain barrier opening using in vivo transcranial ultrasound in mice followed the same trend as the in vitro experiments, and the pressure range for safe and effective treatment was 75-150kPa. For pressures above 150kPa, inertial cavitation and hemorrhage occurred. Therefore, we conclude that (1) at this low frequency, and for the large oscillations, lipid-shelled microbubbles can be approximately modeled as clean gas microbubbles and (2) the development of safe and successful protocols for therapeutic delivery to the brain utilizing 250kHz or a similar center frequency requires consideration of the narrow pressure window between stable and inertial cavitation.

  View details for PubMedID 30397280

• Unimicellar hyperstars as multi-antigen cancer nanovaccines displaying clustered epitopes of immunostimulating peptides BIOMATERIALS SCIENCE Kakwere, H., Ingham, E. S., Allen, R., Mahakian, L. M., Tam, S. M., Zhang, H., Silvestrini, M. T., Lewis, J. S., Ferrara, K. W. 2018; 6 (11): 2850–58

  View details for DOI 10.1039/c8bm00891d

  View details for Web of Science ID 000448822800005

• Delivery of an Artificial Transcription Factor for Angelman Syndrome Using AAV-PHP.B, AAV-PHP.eB and AAV-DJ/8J with Focused Ultrasound Beitnere, U., Pyles, B., O'Geen, H., Foiret, J., Zhang, H., Fite, B. Z., Ilovitch, T., Mahakian, L., Tam, S., Ferrara, K. W., Segal, D. J. CELL PRESS. 2018: 140

  View details for Web of Science ID 000435342201168

• Imaging beyond ultrasonically-impenetrable objects SCIENTIFIC REPORTS Ilovitsh, T., Ilovitsh, A., Foiret, J., Ferrara, K. W. 2018; 8: 5759

  Abstract

  Ultrasound images are severely degraded by the presence of obstacles such as bones and air gaps along the beam path. This paper describes a method for imaging structures that are distal to obstacles that are otherwise impenetrable to ultrasound. The method uses an optically-inspired holographic algorithm to beam-shape the emitted ultrasound field in order to bypass the obstacle and place the beam focus beyond the obstruction. The resulting performance depends on the transducer aperture, the size and position of the obstacle, and the position of the target. Improvement compared to standard ultrasound imaging is significant for obstacles for which the width is larger than one fourth of the transducer aperture and the depth is within a few centimeters of the transducer. For such cases, the improvement in focal intensity at the location of the target reaches 30-fold, and the improvement in peak-to-side-lobe ratio reaches 3-fold. The method can be implemented in conventional ultrasound systems, and the entire process can be performed in real time. This method has applications in the fields of cancer detection, abdominal imaging, imaging of vertebral structure and ultrasound tomography. Here, its effectiveness is demonstrated using wire targets, tissue mimicking phantoms and an ex vivo biological sample.

  View details for PubMedID 29636513

• Acoustical structured illumination for super-resolution ultrasound imaging. Communications biology Ilovitsh, T., Ilovitsh, A., Foiret, J., Fite, B. Z., Ferrara, K. W. 2018; 1

  Abstract

  Structured illumination microscopy is an optical method to increase the spatial resolution of wide-field fluorescence imaging beyond the diffraction limit by applying a spatially structured illumination light. Here, we extend this concept to facilitate super-resolution ultrasound imaging by manipulating the transmitted sound field to encode the high spatial frequencies into the observed image through aliasing. Post processing is applied to precisely shift the spectral components to their proper positions in k-space and effectively double the spatial resolution of the reconstructed image compared to one-way focusing. The method has broad application, including the detection of small lesions for early cancer diagnosis, improving the detection of the borders of organs and tumors, and enhancing visualization of vascular features. The method can be implemented with conventional ultrasound systems, without the need for additional components. The resulting image enhancement is demonstrated with both test objects and ex vivo rat metacarpals and phalanges.

  View details for PubMedID 29888748

• Assessment of Electromechanical Coupling Coefficient for a Completed PIN-PMN-PT Array Stephens, D. N., Wodnicki, R., Chen, R., Liang, L., Zhou, Q., Morrison, K., Ferrara, K. W., IEEE IEEE. 2018

  View details for Web of Science ID 000458693001055

• Modular fabrication and assembly of large 2D arrays with interface ASICs, PIN-PMN-PT composite, and 3D printed backing Wodnicki, R., Kang, H., Adhikari, J., Jung, H., Chen, R., Chen, Z., Jiang, L., Cabrera-Munoz, N., Zhang, R., Liu, Y., Chiu, V., Foiret, J., Stephens, D. N., Zhou, Q., Ferrera, K., IEEE IEEE. 2018

  View details for Web of Science ID 000458693000009

• Acoustical structured illumination for super-resolution ultrasound imaging COMMUNICATIONS BIOLOGY Ilovitsh, T., Ilovitsh, A., Foiret, J., Fite, B. Z., Ferrara, K. W. 2018; 1

  View details for DOI 10.1038/s42003-017-0003-5

  View details for Web of Science ID 000461126500003

• A Scalable Method for Squalenoylation and Assembly of Multifunctional 64Cu-Labeled Squalenoylated Gemcitabine Nanoparticles. Nanotheranostics Tucci, S. T., Seo, J. W., Kakwere, H., Kheirolomoom, A., Ingham, E. S., Mahakian, L. M., Tam, S., Tumbale, S., Baikoghli, M., Cheng, R. H., Ferrara, K. W. 2018; 2 (4): 387–402

  Abstract

  Squalenoylation of gemcitabine, a front-line therapy for pancreatic cancer, allows for improved cellular-level and system-wide drug delivery. The established methods to conjugate squalene to gemcitabine and to form nanoparticles (NPs) with the squalenoylated gemcitabine (SqGem) conjugate are cumbersome, time-consuming and can be difficult to reliably replicate. Further, the creation of multi-functional SqGem-based NP theranostics would facilitate characterization of in vivo pharmacokinetics and efficacy. Methods: Squalenoylation conjugation chemistry was enhanced to improve reliability and scalability using tert-butyldimethylsilyl (TBDMS) protecting groups. We then optimized a scalable microfluidic mixing platform to produce SqGem-based NPs and evaluated the stability and morphology of select NP formulations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity was evaluated in both PANC-1 and KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre) pancreatic cancer cell lines. A 64Cu chelator (2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) was squalenoylated and used with positron emission tomography (PET) imaging to monitor the in vivo fate of SqGem-based NPs. Results: Squalenoylation yields of gemcitabine increased from 15% to 63%. Cholesterol-PEG-2k inclusion was required to form SqGem-based NPs using our technique, and additional cholesterol inclusion increased particle stability at room temperature; after 1 week the PDI of SqGem NPs with cholesterol was ~ 0.2 while the PDI of SqGem NPs lacking cholesterol was ~ 0.5. Similar or superior cytotoxicity was achieved for SqGem-based NPs compared to gemcitabine or Abraxane when evaluated at a concentration of 10 M. Squalenoylation of NOTA enabled in vivo monitoring of SqGem-based NP pharmacokinetics and biodistribution. Conclusion: We present a scalable technique for fabricating efficacious squalenoylated-gemcitabine nanoparticles and confirm their pharmacokinetic profile using a novel multifunctional 64Cu-SqNOTA-SqGem NP.

  View details for PubMedID 30324084

• Toward Personalized Peptide-Based Cancer Nanovaccines: A Facile and Versatile Synthetic Approach BIOCONJUGATE CHEMISTRY Kakwere, H., Ingham, E. S., Allen, R., Mahakian, L. M., Tam, S. M., Zhang, H., Silvestrini, M. T., Lewis, J. S., Ferrara, K. W. 2017; 28 (11): 2756–71

  Abstract

  Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and improved survival (up to 24 days, TRP2) compared to the untreated group (14 days).

  View details for PubMedID 28956907

• Ultrasound localization microscopy to image and assess microvasculature in a rat kidney SCIENTIFIC REPORTS Foiret, J., Zhang, H., Ilovitsh, T., Mahakian, L., Tam, S., Ferrara, K. W. 2017; 7: 13662

  Abstract

  The recent development of ultrasound localization microscopy, where individual microbubbles (contrast agents) are detected and tracked within the vasculature, provides new opportunities for imaging the vasculature of entire organs with a spatial resolution below the diffraction limit. In stationary tissue, recent studies have demonstrated a theoretical resolution on the order of microns. In this work, single microbubbles were localized in vivo in a rat kidney using a dedicated high frame rate imaging sequence. Organ motion was tracked by assuming rigid motion (translation and rotation) and appropriate correction was applied. In contrast to previous work, coherence-based non-linear phase inversion processing was used to reject tissue echoes while maintaining echoes from very slowly moving microbubbles. Blood velocity in the small vessels was estimated by tracking microbubbles, demonstrating the potential of this technique to improve vascular characterization. Previous optical studies of microbubbles in vessels of approximately 20 microns have shown that expansion is constrained, suggesting that microbubble echoes would be difficult to detect in such regions. We therefore utilized the echoes from individual MBs as microscopic sensors of slow flow associated with such vessels and demonstrate that highly correlated, wideband echoes are detected from individual microbubbles in vessels with flow rates below 2 mm/s.

  View details for PubMedID 29057881

• Gemcitabine nanoparticles show in vitro efficacy in murine pancreatic ductal adenocarcinoma Tucci, S. T., Kakwere, H., Kheirolomoom, A., Seo, J. W., Ingham, E., Hwang, C., Ferrara, K. AMER ASSOC CANCER RESEARCH. 2017

  View details for DOI 10.1158/1538-7445.AM2017-3108

  View details for Web of Science ID 000442513300389

• Neoadjuvant immunotherapy improves efficacy of image-guided thermal ablation to generate curative responses in a murine breast cancer model Silvestrini, M. T., Ingham, E. S., Mahakian, L. M., Kheirolomoom, A., Liu, Y., Fite, B. Z., Tam, S. M., Tucci, S., Watson, K. D., Wong, A. W., Monjazeb, A. M., Hubbard, N. E., Murphy, W. J., Borowsky, A. D., Ferrara, K. W. AMER ASSOC CANCER RESEARCH. 2017

  View details for DOI 10.1158/1538-7445.AM2017-576

  View details for Web of Science ID 000442496702029

• Protocol optimization combining activatable nanodelivery with immunotherapy in a murine breast cancer model Kheirolomoom, A., Silvestrini, M., Mahakian, L., Ingham, E., Foiret, J., Tumbale, S., Tam, S., Hubbard, N., Borowsky, A., Ferrara, K. AMER ASSOC CANCER RESEARCH. 2017

  View details for DOI 10.1158/1538-7445.AM2017-4603

  View details for Web of Science ID 000442513303283

• Dynamic contrast enhanced MRI detects changes in vascular transport rate constants following treatment with thermally-sensitive liposomal doxorubicin JOURNAL OF CONTROLLED RELEASE Fite, B. Z., Kheirolomoom, A., Foiret, J. L., Seo, J. W., Mahakian, L. M., Ingham, E. S., Tam, S. M., Borowsky, A. D., Curry, F. E., Ferrara, K. W. 2017; 256: 203–13

  Abstract

  Temperature-sensitive liposomal formulations of chemotherapeutics, such as doxorubicin, can achieve locally high drug concentrations within a tumor and tumor vasculature while maintaining low systemic toxicity. Further, doxorubicin delivery by temperature-sensitive liposomes can reliably cure local cancer in mouse models. Histological sections of treated tumors have detected red blood cell extravasation within tumors treated with temperature-sensitive doxorubicin and ultrasound hyperthermia. We hypothesize that the local release of drug into the tumor vasculature and resulting high drug concentration can alter vascular transport rate constants along with having direct tumoricidal effects. Dynamic contrast enhanced MRI (DCE-MRI) coupled with a pharmacokinetic model can detect and quantify changes in such vascular transport rate constants. Here, we set out to determine whether changes in rate constants resulting from intravascular drug release were detectable by MRI. We found that the accumulation of gadoteridol was enhanced in tumors treated with temperature-sensitive liposomal doxorubicin and ultrasound hyperthermia. While the initial uptake rate of the small molecule tracer was slower (k1=0.0478±0.011s-1 versus 0.116±0.047s-1) in treated compared to untreated tumors, the tracer was retained after treatment due to a larger reduction in the rate of clearance (k2=0.291±0.030s-1 versus 0.747±0.24s-1). While DCE-MRI assesses a combination of blood flow and permeability, ultrasound imaging of microvascular flow rate is sensitive only to changes in vascular flow rate; based on this technique, blood flow was not significantly altered 30min after treatment. In summary, DCE-MRI provides a means to detect changes that are associated with treatment by thermally-activated particles and such changes can be exploited to enhance local delivery.

  View details for PubMedID 28395970

  View details for PubMedCentralID PMC5545100

• Supersonic transient magnetic resonance elastography for quantitative assessment of tissue elasticity PHYSICS IN MEDICINE AND BIOLOGY Liu, Y., Liu, J., Fite, B. Z., Foiret, J., Ilovitsh, A., Leach, J., Dumont, E., Caskey, C. F., Ferrara, K. W. 2017; 62 (10): 4083–4106

  Abstract

  Non-invasive, quantitative methods to assess the properties of biological tissues are needed for many therapeutic and tissue engineering applications. Magnetic resonance elastography (MRE) has historically relied on external vibration to generate periodic shear waves. In order to focally assess a biomaterial or to monitor the response to ablative therapy, the interrogation of a specific region of interest by a focused beam is desirable and transient MRE (t-MRE) techniques have previously been developed to accomplish this goal. Also, strategies employing a series of discrete ultrasound pulses directed to increasing depths along a single line-of-sight have been designed to generate a quasi-planar shear wave. Such 'supersonic' excitations have been applied for ultrasound elasticity measurements. The resulting shear wave is higher in amplitude than that generated from a single excitation and the properties of the media are simply visualized and quantified due to the quasi-planar wave geometry and the opportunity to generate the wave at the site of interest. Here for the first time, we extend the application of supersonic methods by developing a protocol for supersonic transient magnetic resonance elastography (sst-MRE) using an MR-guided focused ultrasound system capable of therapeutic ablation. We apply the new protocol to quantify tissue elasticity in vitro using biologically-relevant inclusions and tissue-mimicking phantoms, compare the results with elasticity maps acquired with ultrasound shear wave elasticity imaging (US-SWEI), and validate both methods with mechanical testing. We found that a modified time-of-flight (TOF) method efficiently quantified shear modulus from sst-MRE data, and both the TOF and local inversion methods result in similar maps based on US-SWEI. With a three-pulse excitation, the proposed sst-MRE protocol was capable of visualizing quasi-planar shear waves propagating away from the excitation location and detecting differences in shear modulus of 1 kPa. The techniques demonstrated here have potential application in real-time in vivo lesion detection and monitoring, with particular significance for image-guided interventions.

  View details for PubMedID 28426437

  View details for PubMedCentralID PMC5545104

• Anatomical image-guided fluorescence molecular tomography reconstruction using kernel method JOURNAL OF BIOMEDICAL OPTICS Baikejiang, R., Zhao, Y., Fite, B. Z., Ferrara, K. W., Li, C. 2017; 22 (5): 55001

  Abstract

  Fluorescence molecular tomography (FMT) is an important in vivo imaging modality to visualize physiological and pathological processes in small animals. However, FMT reconstruction is ill-posed and ill-conditioned due to strong optical scattering in deep tissues, which results in poor spatial resolution. It is well known that FMT image quality can be improved substantially by applying the structural guidance in the FMT reconstruction. An approach to introducing anatomical information into the FMT reconstruction is presented using the kernel method. In contrast to conventional methods that incorporate anatomical information with a Laplacian-type regularization matrix, the proposed method introduces the anatomical guidance into the projection model of FMT. The primary advantage of the proposed method is that it does not require segmentation of targets in the anatomical images. Numerical simulations and phantom experiments have been performed to demonstrate the proposed approach’s feasibility. Numerical simulation results indicate that the proposed kernel method can separate two FMT targets with an edge-to-edge distance of 1 mm and is robust to false-positive guidance and inhomogeneity in the anatomical image. For the phantom experiments with two FMT targets, the kernel method has reconstructed both targets successfully, which further validates the proposed kernel method.

  View details for PubMedID 28464120

  View details for PubMedCentralID PMC5629124

• In vitro characterization and in vivo ultrasound molecular imaging of nucleolin-targeted microbubbles BIOMATERIALS Zhang, H., Ingham, E. S., Gagnon, M. J., Mahakian, L. M., Liu, J., Foiret, J. L., Willmann, J. K., Ferrara, K. W. 2017; 118: 63–73

  Abstract

  Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p < 0.001. We then set out to create a method to extract both the tumor wash-in and wash-out kinetics and tumor accumulation following a single injection of targeted MBs. In order to accomplish this, a series of ultrasound frames (a clip) was recorded at the time of injection and subsequent time points. Each pixel within this clip was analyzed for the minimum intensity projection (MinIP) and average intensity projection (AvgIP). We found that the MinIP robustly demonstrates enhanced accumulation of F3-conjugated MBs over the range of tumor diameters evaluated here (2-8 mm), and the difference between the AvgIP and the MinIP quantifies inflow and kinetics. The inflow and clearance were similar for unbound F3-conjugated MBs, control (non-targeted) and scrambled control agents. Targeted agent accumulation was confirmed by a high amplitude pulse and by a two-dimensional Fourier Transform technique. In summary, F3-conjugated MBs provide a new imaging agent for ultrasound molecular imaging of cancer vasculature, and we have validated metrics to assess performance using low mechanical index strategies that have potential for use in human molecular imaging studies.

  View details for PubMedID 27940383

  View details for PubMedCentralID PMC5279957

• Feasibility of quantitative contrast ultrasound imaging of bladder tumors in dogs CANADIAN VETERINARY JOURNAL-REVUE VETERINAIRE CANADIENNE Pollard, R. E., Watson, K. D., Hu, X., Ingham, E., Ferrara, K. W. 2017; 58 (1): 70–72

  Abstract

  The purpose of this pilot study was to assess the feasibility of Cadence contrast pulse sequencing ultrasound to predict clinical and angiogenic tumor response in dogs undergoing chemotherapy. Contrast ultrasound facilitated visualization of bladder tumors but failed to identify a straightforward relationship between ultrasound measures and clinical outcome.

  View details for PubMedID 28042158

• PIN-PMN-PT single crystal composite and 3D printed interposer backing for ASIC integration of large aperture 2D array Wodnicki, R., Kang, H., Zhang, R., Chen, R., Cabrera-Munoz, N., Adhikari, J., Neher, C., Chiu, C., Liu, Y., Stephens, D. N., Zhou, Q., Ferrera, K., IEEE IEEE. 2017

  View details for Web of Science ID 000416948401175

• Blood-brain barrier disruption for the delivery of noninfectious viral vectors and proteins, preliminary study Foiret, J., Zhang, H., Fite, B. Z., Ilovitsh, T., Mahakian, L. M., Tam, S., Beitnere, U., Pyles, B., Segal, D. J., Ferrara, K. W., IEEE IEEE. 2017

  View details for Web of Science ID 000416948401095

• Intracellular trafficking of a pH-responsive drug metal complex JOURNAL OF CONTROLLED RELEASE Kheirolomoom, A., Ingham, E. S., Commisso, J., Abushaban, N., Ferrara, K. W. 2016; 243: 232–42

  Abstract

  We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1.4-fold in both cancer and endothelial cells. Endothelial cytotoxicity was similarly augmented with mild hyperthermia applied prior to treatment with released CuDox. In summary, we find that the drug-metal complex formed in temperature-sensitive particles can be internalized by cancer and endothelial cells resulting in therapeutic efficacy that is similar to free doxorubicin, and this efficacy can be enhanced by elevated temperature.

  View details for PubMedID 27746275

  View details for PubMedCentralID PMC5191948

• Novel theranostic nanoporphyrins for photodynamic diagnosis and trimodal therapy for bladder cancer BIOMATERIALS Lin, T., Li, Y., Liu, Q., Chen, J., Zhang, H., Lac, D., Zhang, H., Ferrara, K. W., Wachsmann-Hogiu, S., Li, T., Airhart, S., White, R., Lam, K. S., Pan, C. 2016; 104: 339–51

  Abstract

  The overall prognosis of bladder cancer has not been improved over the last 30 years and therefore, there is a great medical need to develop novel diagnosis and therapy approaches for bladder cancer. We developed a multifunctional nanoporphyrin platform that was coated with a bladder cancer-specific ligand named PLZ4. PLZ4-nanoporphyrin (PNP) integrates photodynamic diagnosis, image-guided photodynamic therapy, photothermal therapy and targeted chemotherapy in a single procedure. PNPs are spherical, relatively small (around 23 nm), and have the ability to preferably emit fluorescence/heat/reactive oxygen species upon illumination with near infrared light. Doxorubicin (DOX) loaded PNPs possess slower drug release and dramatically longer systemic circulation time compared to free DOX. The fluorescence signal of PNPs efficiently and selectively increased in bladder cancer cells but not normal urothelial cells in vitro and in an orthotopic patient derived bladder cancer xenograft (PDX) models, indicating their great potential for photodynamic diagnosis. Photodynamic therapy with PNPs was significantly more potent than 5-aminolevulinic acid, and eliminated orthotopic PDX bladder cancers after intravesical treatment. Image-guided photodynamic and photothermal therapies synergized with targeted chemotherapy of DOX and significantly prolonged overall survival of mice carrying PDXs. In conclusion, this uniquely engineered targeting PNP selectively targeted tumor cells for photodynamic diagnosis, and served as effective triple-modality (photodynamic/photothermal/chemo) therapeutic agents against bladder cancers. This platform can be easily adapted to individualized medicine in a clinical setting and has tremendous potential to improve the management of bladder cancer in the clinic.

  View details for PubMedID 27479049

  View details for PubMedCentralID PMC5412594

• Development of a spherically focused phased array transducer for ultrasonic image-guided hyperthermia PHYSICS IN MEDICINE AND BIOLOGY Liu, J., Foiret, J., Stephens, D. N., Le Baron, O., Ferrara, K. W. 2016; 61 (14): 5275–96

  Abstract

  A 1.5 MHz prolate spheroidal therapeutic array with 128 circular elements was designed to accommodate standard imaging arrays for ultrasonic image-guided hyperthermia. The implementation of this dual-array system integrates real-time therapeutic and imaging functions with a single ultrasound system (Vantage 256, Verasonics). To facilitate applications involving small animal imaging and therapy the array was designed to have a beam depth of field smaller than 3.5 mm and to electronically steer over distances greater than 1 cm in both the axial and lateral directions. In order to achieve the required f number of 0.69, 1-3 piezocomposite modules were mated within the transducer housing. The performance of the prototype array was experimentally evaluated with excellent agreement with numerical simulation. A focal volume (2.70 mm (axial)  ×  0.65 mm (transverse)  ×  0.35 mm (transverse)) defined by the  -6 dB focal intensity was obtained to address the dimensions needed for small animal therapy. An electronic beam steering range defined by the  -3 dB focal peak intensity (17 mm (axial)  ×  14 mm (transverse)  ×  12 mm (transverse)) and  -8 dB lateral grating lobes (24 mm (axial)  ×  18 mm (transverse)  ×  16 mm (transverse)) was achieved. The combined testing of imaging and therapeutic functions confirmed well-controlled local heating generation and imaging in a tissue mimicking phantom. This dual-array implementation offers a practical means to achieve hyperthermia and ablation in small animal models and can be incorporated within protocols for ultrasound-mediated drug delivery.

  View details for PubMedID 27353347

  View details for PubMedCentralID PMC5028201

• Activatable nanodelivery combined with CpG-ODN and anti-PD-1 achieves a complete response in directly-treated and contralateral tumors in a murine breast cancer model Silvestrini, M. T., Kheirolomoom, A., Ingham, E. S., Mahakian, L. M., Tam, S. M., Foiret, J., Tucci, S., Hubbard, N. E., Borowsky, A. D., Ferrara, K. W. AMER ASSOC CANCER RESEARCH. 2016

  View details for DOI 10.1158/1538-7445.AM2016-LB-052

  View details for Web of Science ID 000389940600134

• Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation JOURNAL OF CONTROLLED RELEASE Shapiro, G., Wong, A. W., Bez, M., Yang, F., Tam, S., Even, L., Sheyn, D., Ben-David, S., Tawackoli, W., Pelled, G., Ferrara, K. W., Gazit, D. 2016; 223: 157–64

  Abstract

  More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of plasmid DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50μg luciferase plasmid DNA and 5×10(5) microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540 Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation-induced gene therapies.

  View details for PubMedID 26682505

  View details for PubMedCentralID PMC4724495

• Quantitative MR-guided transient shear wave imaging for tissue elasticity assessment Liu, Y., Liu, J., Fite, B. Z., Foiret, J., Leach, J., Ferrara, K. W., IEEE IEEE. 2016

  View details for Web of Science ID 000387497400484

• HIFU Power Network Optimization for Catheter Based Cardiac Interventions Stephens, D. N., Foiret, J., Lucero, S., Ferrara, K. W., Shivkumar, K., Khuri-Yakub, P., IEEE IEEE. 2016

  View details for Web of Science ID 000387497400461

• Super-localization of contrast agents in moving organs, first experiments in a rat kidney Foiret, J., Zhang, H., Mahakian, L., Tam, S., Ferrara, K. W., IEEE IEEE. 2016

  View details for Web of Science ID 000387497400257

• CpG expedites regression of local and systemic tumors when combined with activatable nanodelivery JOURNAL OF CONTROLLED RELEASE Kheirolomoom, A., Ingham, E. S., Mahakian, L. M., Tam, S. M., Silvestrini, M. T., Tumbale, S. K., Foiret, J., Hubbard, N. E., Borowsky, A. D., Murphy, W. J., Ferrara, K. W. 2015; 220: 253–64

  Abstract

  Ultrasonic activation of nanoparticles provides the opportunity to deliver a large fraction of the injected dose to insonified tumors and produce a complete local response. Here, we evaluate whether the local and systemic response to chemotherapy can be enhanced by combining such a therapy with locally-administered CpG as an immune adjuvant. In order to create stable, activatable particles, a complex between copper and doxorubicin (CuDox) was created within temperature-sensitive liposomes. Whereas insonation of the CuDox liposomes alone has been shown to produce a complete response in murine breast cancer after 8 treatments of 6 mg/kg delivered over 4 weeks, combining this treatment with CpG resolved local cancers within 3 treatments delivered over 7 days. Further, contralateral tumors regressed as a result of the combined treatment, and survival was extended in systemic disease. In both the treated and contralateral tumor site, the combined treatment increased leukocytes and CD4+ and CD8+ T-effector cells and reduced myeloid-derived suppressor cells (MDSCs). Taken together, the results suggest that this combinatorial treatment significantly enhances the systemic efficacy of locally-activated nanotherapy.

  View details for PubMedID 26471394

  View details for PubMedCentralID PMC4688109

• Self-assembled 20-nm Cu-64-micelles enhance accumulation in rat glioblastoma JOURNAL OF CONTROLLED RELEASE Seo, J., Ang, J., Mahakian, L. M., Tam, S., Fite, B., Ingham, E. S., Beyer, J., Forsayeth, J., Bankiewicz, K. S., Xu, T., Ferrara, K. W. 2015; 220: 51–60

  Abstract

  There is an urgent need to develop nanocarriers for the treatment of glioblastoma multiforme (GBM). Using co-registered positron emission tomography (PET) and magnetic resonance (MR) images, here we performed systematic studies to investigate how a nanocarrier's size affects the pharmacokinetics and biodistribution in rodents with a GBM xenograft. In particular, highly stable, long-circulating three-helix micelles (3HM), based on a coiled-coil protein tertiary structure, were evaluated as an alternative to larger nanocarriers. While the circulation half-life of the 3HM was similar to 110-nm PEGylated liposomes (t1/2=15.5 and 16.5h, respectively), the 20-nm micelles greatly enhanced accumulation within a U87MG xenograft in nu/nu rats after intravenous injection. After accounting for tumor blood volume, the extravasated nanoparticles were quantified from the PET images, yielding ~0.77%ID/cm(3) for the micelles and 0.45%ID/cm(3) for the liposomes. For GBM lesions with a volume greater than 100mm(3), 3HM accumulation was enhanced both within the detectable tumor and in the surrounding brain parenchyma. Further, the nanoparticle accumulation was shown to extend to the margins of the GBM xenograft. In summary, 3HM provides an attractive nanovehicle for carrying treatment to GBM.

  View details for PubMedID 26437259

  View details for PubMedCentralID PMC4688122

• Concurrent Visualization of Acoustic Radiation Force Displacement and Shear Wave Propagation with 7T MRI PLOS ONE Liu, Y., Fite, B. Z., Mahakian, L. M., Johnson, S. M., Larrat, B., Dumont, E., Ferrara, K. W. 2015; 10 (10): e0139667

  Abstract

  Manual palpation is a common and very informative diagnostic tool based on estimation of changes in the stiffness of tissues that result from pathology. In the case of a small lesion or a lesion that is located deep within the body, it is difficult for changes in mechanical properties of tissue to be detected or evaluated via palpation. Furthermore, palpation is non-quantitative and cannot be used to localize the lesion. Magnetic Resonance-guided Focused Ultrasound (MRgFUS) can also be used to evaluate the properties of biological tissues non-invasively. In this study, an MRgFUS system combines high field (7T) MR and 3 MHz focused ultrasound to provide high resolution MR imaging and a small ultrasonic interrogation region (~0.5 x 0.5 x 2 mm), as compared with current clinical systems. MR-Acoustic Radiation Force Imaging (MR-ARFI) provides a reliable and efficient method for beam localization by detecting micron-scale displacements induced by ultrasound mechanical forces. The first aim of this study is to develop a sequence that can concurrently quantify acoustic radiation force displacements and image the resulting transient shear wave. Our motivation in combining these two measurements is to develop a technique that can rapidly provide both ARFI and shear wave velocity estimation data, making it suitable for use in interventional radiology. Secondly, we validate this sequence in vivo by estimating the displacement before and after high intensity focused ultrasound (HIFU) ablation, and we validate the shear wave velocity in vitro using tissue-mimicking gelatin and tofu phantoms. Such rapid acquisitions are especially useful in interventional radiology applications where minimizing scan time is highly desirable.

  View details for PubMedID 26439259

• Spatial and Temporal Control of Hyperthermia Using Real Time Ultrasonic Thermal Strain Imaging with Motion Compensation, Phantom Study PLOS ONE Foiret, J., Ferrara, K. W. 2015; 10 (8): e0134938

  Abstract

  Mild hyperthermia has been successfully employed to induce reversible physiological changes that can directly treat cancer and enhance local drug delivery. In this approach, temperature monitoring is essential to avoid undesirable biological effects that result from thermal damage. For thermal therapies, Magnetic Resonance Imaging (MRI) has been employed to control real-time Focused Ultrasound (FUS) therapies. However, combined ultrasound imaging and therapy systems offer the benefits of simple, low-cost devices that can be broadly applied. To facilitate such technology, ultrasound thermometry has potential to reliably monitor temperature. Control of mild hyperthermia was previously achieved using a proportional-integral-derivative (PID) controller based on thermocouple measurements. Despite accurate temporal control of heating, this method is limited by the single position at which the temperature is measured. Ultrasound thermometry techniques based on exploiting the thermal dependence of acoustic parameters (such as longitudinal velocity) can be extended to create thermal maps and allow an accurate monitoring of temperature with good spatial resolution. However, in vivo applications of this technique have not been fully developed due to the high sensitivity to tissue motion. Here, we propose a motion compensation method based on the acquisition of multiple reference frames prior to treatment. The technique was tested in the presence of 2-D and 3-D physiological-scale motion and was found to provide effective real-time temperature monitoring. PID control of mild hyperthermia in presence of motion was then tested with ultrasound thermometry as feedback and temperature was maintained within 0.3°C of the requested value.

  View details for PubMedID 26244783

• Ultrasound molecular imaging of tumor angiogenesis with a neuropilin-1-targeted microbubble BIOMATERIALS Zhang, H., Tam, S., Ingham, E. S., Mahakian, L. M., Lai, C., Tumbale, S. K., Teesalu, T., Hubbard, N. E., Borowsky, A. D., Ferrara, K. W. 2015; 56: 104–13

  Abstract

  Ultrasound molecular imaging has great potential to impact early disease diagnosis, evaluation of disease progression and the development of target-specific therapy. In this paper, two neuropilin-1 (NRP) targeted peptides, CRPPR and ATWLPPR, were conjugated onto the surface of lipid microbubbles (MBs) to evaluate molecular imaging of tumor angiogenesis in a breast cancer model. Development of a molecular imaging agent using CRPPR has particular importance due to the previously demonstrated internalizing capability of this and similar ligands. In vitro, CRPPR MBs bound to an NRP-expressing cell line 2.6 and 15.6 times more than ATWLPPR MBs and non-targeted (NT) MBs, respectively, and the binding was inhibited by pretreating the cells with an NRP antibody. In vivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the ∼6 min circulation of the CRPPR-targeted contrast agents providing high contrast images of angiogenic tumors. Approximately 67% of the initial signal from CRPPR MBs remained bound after the majority of circulating MBs had cleared (8 min), 8 and 4.5 times greater than ATWLPPR and NT MBs, respectively. Finally, at 7-21 days after the first injection, we found that CRPPR MBs cleared faster from circulation and tumor accumulation was reduced likely due to a complement-mediated recognition of the targeted microbubble and a decrease in angiogenic vasculature, respectively. In summary, we find that CRPPR MBs specifically bind to NRP-expressing cells and provide an effective new agent for molecular imaging of angiogenesis.

  View details for PubMedID 25934284

  View details for PubMedCentralID PMC4417484

• Therapeutic Strategies and Image Guidance Ferrara, K., Chopra, R., Konofagou, E., Farahani, K. AMER ASSOC PHYSICISTS MEDICINE AMER INST PHYSICS. 2015: 3624–25

  View details for DOI 10.1118/1.4925710

  View details for Web of Science ID 000356998303133

• Magnetic Resonance Imaging Assessment of Effective Ablated Volume following High Intensity Focused Ultrasound PLOS ONE Fite, B. Z., Wong, A., Liu, Y., Mahakian, L. M., Tam, S. M., Aina, O., Hubbard, N. E., Borowsky, A., Cardiff, R. D., Dumont, E., Ferrara, K. W. 2015; 10 (3): e0120037

  Abstract

  Under magnetic resonance (MR) guidance, high intensity focused ultrasound (HIFU) is capable of precise and accurate delivery of thermal dose to tissues. Given the excellent soft tissue imaging capabilities of MRI, but the lack of data on the correlation of MRI findings to histology following HIFU, we sought to examine tumor response to HIFU ablation to determine whether there was a correlation between histological findings and common MR imaging protocols in the assessment of the extent of thermal damage. Female FVB mice (n = 34), bearing bilateral neu deletion tumors, were unilaterally insonated under MR guidance, with the contralateral tumor as a control. Between one and five spots (focal size 0.5 × 0.5 × 2.5 mm3) were insonated per tumor with each spot receiving approximately 74.2 J of acoustic energy over a period of 7 seconds. Animals were then imaged on a 7T MR scanner with several protocols. T1 weighted images (with and without gadolinium contrast) were collected in addition to a series of T2 weighted and diffusion weighted images (for later reconstruction into T2 and apparent diffusion coefficient maps), immediately following ablation and at 6, 24, and 48 hours post treatment. Animals were sacrificed at each time point and both insonated/treated and contralateral tumors removed and stained for NADH-diaphorase, caspase 3, or with hematoxylin and eosin (H&E). We found the area of non-enhancement on contrast enhanced T1 weighted imaging immediately post ablation correlated with the region of tissue receiving a thermal dose CEM43 ≥ 240 min. Moreover, while both tumor T2 and apparent diffusion coefficient values changed from pre-ablation values, contrast enhanced T1 weighted images appeared to be more senstive to changes in tissue viability following HIFU ablation.

  View details for PubMedID 25785992

• The pharmacokinetics of Zr-89 labeled liposomes over extended periods in a murine tumor model NUCLEAR MEDICINE AND BIOLOGY Seo, J., Mahakian, L. M., Tam, S., Qin, S., Ingham, E. S., Meares, C. F., Ferrara, K. W. 2015; 42 (2): 155–63

  Abstract

  (89)Zr (t1/2=78.4h), a positron-emitting metal, has been exploited for PET studies of antibodies because of its relatively long decay time and facile labeling procedures. Here, we used (89)Zr to evaluate the pharmacokinetics of long-circulating liposomes over 168h (1week). We first developed a liposomal-labeling method using p-isothiocyanatobenzyl-desferrioxamine (df-Bz-NCS) and df-PEG1k-DSPE. Df-Bz-NCS was conjugated to 1mol% amino- and amino-PEG2k-DSPE, where the 1mol% df-PEG1k-DSPE was incorporated when the liposomes were formulated. Incubation of (89)Zr with df, df-PEG1k, and df-PEG2k liposomes for one hour resulted in greater than 68% decay-corrected yield. The loss of the (89)Zr label from liposomes after incubation in 50% human serum for 48h ranged from ~1 to 3% across the three formulations. Tail vein administration of the three liposomal formulations in NDL tumor-bearing mice showed that the (89)Zr label at the end of the PEG2k brush was retained in the tumor, liver, spleen and whole body for a longer time interval than (89)Zr labels located under the PEG2k brush. The blood clearance rate of all three liposomal formulations was similar. Overall, the results indicate that the location of the (89)Zr label altered the clearance rate of intracellularly-trapped radioactivity and that df-PEG1k-DSPE provides a stable chelation site for liposomal or lipid-based particle studies over extended periods of time.

  View details for PubMedID 25451215

  View details for PubMedCentralID PMC4281498

• Microfluidic co-cultures with hydrogel-based ligand trap to study paracrine signals giving rise to cancer drug resistance LAB ON A CHIP Patel, D., Gao, Y., Son, K., Siltanen, C., Neve, R. M., Ferrara, K., Revzin, A. 2015; 15 (24): 4614–24

  Abstract

  Targeted cancer therapies are designed to deactivate signaling pathways used by cancer cells for survival. However, cancer cells are often able to adapt by activating alternative survival pathways, thereby acquiring drug resistance. An emerging theory is that autocrine or paracrine growth factor signaling in the cancer microenvironment represent an important mechanism of drug resistance. In the present study we wanted to examine whether paracrine interactions between groups of melanoma cells result in resistance to vemurafenib - an FDA approved drug targeting the BRAF mutation in metastatic melanoma. We used a vemurafenib-resistant melanoma model which secretes fibroblast growth factor (FGF)-2 to test our hypothesis that this is a key paracrine mediator of resistance to vemurafenib. Sensitive cells treated with media conditioned by resistant cells did not protect from the effects of vemurafenib. To query paracrine interactions further we fabricated a microfluidic co-culture device with two parallel compartments, separated by a 100 μm wide hydrogel barrier. The gel barrier prevented resorting/contact of cells while permitting paracrine cross-talk. In this microfluidic system, sensitive cells did become refractive to the effects of vemurafenib when cultured adjacent to resistant cells. Importantly, incorporation of FGF-2 capture probes into the gel barrier separating the two cell types prevented onset of resistance to vemurafenib. Microfluidic tools described here allow for more sensitive analysis of paracrine signals, may help better understand signaling in the cancer microenvironment and may enable development of more effective cancer therapies.

  View details for PubMedID 26542093

• Advances in thermal strain imaging: 3D motion and tumor validation studies Foiret, J., Ferrara, K., IEEE IEEE. 2015

  View details for DOI 10.1109/ULTSYM.2015.0041

  View details for Web of Science ID 000366045700546

• 10 MHz Catheter-based Annular Array for Thermal Strain Guided Intramural Cardiac Ablations Stephens, D. N., Foiret, J., Lucero, S., Ferrara, K. W., Shivkumar, K., Khuri-Yakub, P., IEEE IEEE. 2015

  View details for DOI 10.1109/ULTSYM.2015.0038

  View details for Web of Science ID 000366045700418

• NEW USES OF OLD DRUGS FOR THE CLINICAL TREATMENT OF BRAIN METASTASES Sundstrom, T., Varughese, J., Azuaje, F., Prestegarden, L., Petersen, K., Tepper, C., Ingham, E., Even, L., Johnson, S., Skaftnesmo, K., Lund-Johansen, M., Bjerkvig, R., Ferrara, K., Thorsen, F. OXFORD UNIV PRESS INC. 2014

  View details for DOI 10.1093/neuonc/nou240.33

  View details for Web of Science ID 000350452200151

• omega-3 Polyunsaturated fatty acids-derived lipid metabolites on angiogenesis, inflammation and cancer PROSTAGLANDINS & OTHER LIPID MEDIATORS Wang, W., Zhu, J., Lyu, F., Panigrahy, D., Ferrara, K. W., Hammock, B., Zhang, G. 2014; 113: 13–20

  Abstract

  Epidemiological and pre-clinical studies support the anti-tumor effects of ω-3 PUFAs; however, the results from human trials are mixed, making it difficult to provide dietary guidelines or recommendations of ω-3 PUFAs for disease prevention or treatment. Understanding the molecular mechanisms by which ω-3 PUFAs inhibit cancer could lead to better nutritional paradigms and human trials to clarify their health effects. The ω-3 PUFAs exert their biological activities mainly through the formation of bioactive lipid metabolites. Here we discuss the biology of cyclooxygenase, lipoxygenase and cytochrome P450 enzymes-derived ω-3-series lipid metabolites on angiogenesis, inflammation and cancer.

  View details for PubMedID 25019221

  View details for PubMedCentralID PMC4306447

• Drug repurposing to treat melanoma brain metastases Sundstrom, T., Varughese, J. K., Azuaje, F., Petersen, K., Tepper, C. G., Ingham, E., Even, L., Tam, S., Skaftnesmo, K., Lund-Johansen, M., Bjerkvig, R., Ferrara, K. W., Thorsen, F. A. AMER ASSOC CANCER RESEARCH. 2014

  View details for DOI 10.1158/1538-7445.AM2014-4036

  View details for Web of Science ID 000349910202019

• Effect of Alkyl Length of Peptide-Polymer Amphiphile on Cargo Encapsulation Stability and Pharmacokinetics of 3-Helix Micelles BIOMACROMOLECULES Dube, N., Seo, J. W., Dong, H., Shu, J. Y., Lund, R., Mahakian, L. M., Ferrara, K. W., Xu, T. 2014; 15 (8): 2963–70

  Abstract

  3-Helix micelles have demonstrated excellent in vitro and in vivo stability. Previous studies showed that the unique design of the peptide-polymer conjugate based on protein tertiary structure as the headgroup is the main design factor to achieve high kinetic stability. In this contribution, using amphiphiles with different alkyl tails, namely, C16 and C18, we quantified the effect of alkyl length on the stability of 3-helix micelles to delineate the contribution of the micellar core and shell on the micelle stability. Both amphiphiles form well-defined micelles, <20 nm in size, and show good stability, which can be attributed to the headgroup design. C18-micelles exhibit slightly higher kinetic stability in the presence of serum proteins at 37 °C, where the rate constant of subunit exchange is 0.20 h(-1) for C18-micelles vs 0.22 h(-1) for C16-micelles. The diffusion constant for drug release from C18-micelles is approximately half of that for C16-micelles. The differences between the two micelles are significantly more pronounced in terms of in vivo stability and extent of tumor accumulation. C18-micelles exhibit significantly longer blood circulation time of 29.5 h, whereas C16-micelles have a circulation time of 16.1 h. The extent of tumor accumulation at 48 h after injection is ∼43% higher for C18-micelles. The present studies underscore the importance of core composition on the biological behavior of 3-helix micelles. The quantification of the effect of this key design parameter on the stability of 3-helix micelles provides important guidelines for carrier selection and use in complex environment.

  View details for PubMedID 24988250

  View details for PubMedCentralID PMC4130244

• Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect (vol 306, pg H1011, 2014) AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Adamson, R. H., Clark, J. F., Radeva, M., Jiang, Y., Kheirolomoom, A., Ferrara, K. W., Curry, F. E. 2014; 307 (1): H120

  View details for DOI 10.1152/ajpheart.H-zh4-1176-corr.2014

  View details for Web of Science ID 000338917200014

• Comparison of PET Imaging with Cu-64-Liposomes and F-18-FDG in the 7,12-Dimethylbenz[a]anthracene (DMBA)-Induced Hamster Buccal Pouch Model of Oral Dysplasia and Squamous Cell Carcinoma MOLECULAR IMAGING AND BIOLOGY Mahakian, L. M., Farwell, D., Zhang, H., Seo, J., Poirier, B., Tinling, S. P., Afify, A. M., Haynam, E. M., Shaye, D., Ferrara, K. W. 2014; 16 (2): 284–92

  Abstract

  Currently, 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG) is the gold standard radiotracer for staging of head and neck cancer; however, the low sensitivity of this tracer can impede detection of early lesions. (64)Cu-liposomes accumulate in various cancers and provide both a sensitive tracer and an indication of the biodistribution of nanotherapeutics. Here, the accumulation of (64)Cu-liposomes in early and established cancers is assessed and compared with (18)F-FDG in a head and neck cancer model.Lesions ranging from mild dysplasia to squamous cell carcinoma were induced in a hamster model of head and neck cancer by topical application of 7,12-dimethylbenz[a]anthracene to the buccal pouch. The hamsters were imaged with micro-positron emission tomography using (18)F-FDG and (64)Cu-liposomes.At 24 h postinjection, (64)Cu-liposome accumulation exceeded the accumulation of (18)F-FDG in every pathologic grade. The lesion-to-cheek pouch (background) ratio and lesion-to-brain ratio were also higher for (64)Cu-liposomes than for (18)F-FDG.Imaging of a nanotracer such as (64)Cu-liposomes can improve the visualization of head and neck tumors. Accumulation of liposomal particles in head and neck tumors over various pathologic grades averaged 3.5%ID/cc demonstrating the potential for liposomal therapy with targeted chemotherapeutic agents.

  View details for PubMedID 24019092

  View details for PubMedCentralID PMC3984137

• Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Adamson, R. H., Clark, J. F., Radeva, M., Kheirolomoom, A., Ferrara, K. W., Curry, F. E. 2014; 306 (7): H1011–H1017

  Abstract

  Removal of plasma proteins from perfusates increases vascular permeability. The common interpretation of the action of albumin is that it forms part of the permeability barrier by electrostatic binding to the endothelial glycocalyx. We tested the alternate hypothesis that removal of perfusate albumin in rat venular microvessels decreased the availability of sphingosine-1-phosphate (S1P), which is normally carried in plasma bound to albumin and lipoproteins and is required to maintain stable baseline endothelial barriers (Am J Physiol Heart Circ Physiol 303: H825-H834, 2012). Red blood cells (RBCs) are a primary source of S1P in the normal circulation. We compared apparent albumin permeability coefficients [solute permeability (Ps)] measured using perfusates containing albumin (10 mg/ml, control) and conditioned by 20-min exposure to rat RBCs with Ps when test perfusates were in RBC-conditioned protein-free Ringer solution. The control perfusate S1P concentration (439 ± 46 nM) was near the normal plasma value at 37 °C and established a stable baseline Ps (0.9 ± 0.4 × 10(-6) cm/s). Ringer solution perfusate contained 52 ± 8 nM S1P and increased Ps more than 10-fold (16.1 ± 3.9 × 10(-6) cm/s). Consistent with albumin-dependent transport of S1P from RBCs, S1P concentrations in RBC-conditioned solutions decreased as albumin concentration, hematocrit, and temperature decreased. Protein-free Ringer solution perfusates that used liposomes instead of RBCs as flow markers failed to maintain normal permeability, reproducing the "albumin effect" in these mammalian microvessels. We conclude that the albumin effect depends on the action of albumin to facilitate the release and transport of S1P from RBCs that normally provide a significant amount of S1P to the endothelium.

  View details for PubMedID 24531813

  View details for PubMedCentralID PMC3962635

• Accumulation, internalization and therapeutic efficacy of neuropilin-1-targeted liposomes JOURNAL OF CONTROLLED RELEASE Paoli, E. E., Ingham, E. S., Zhang, H., Mahakian, L. M., Fite, B. Z., Gagnon, M., Tam, S., Kheirolomoom, A., Cardiff, R. D., Ferrara, K. W. 2014; 178: 108–17

  Abstract

  Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.

  View details for PubMedID 24434424

  View details for PubMedCentralID PMC4079909

• Creation and Characterization of an Ultrasound and CT Phantom for Noninvasive Ultrasound Thermometry Calibration IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Lai, C., Kruse, D. E., Ferrara, K. W., Caskey, C. F. 2014; 61 (2): 502–12

  View details for DOI 10.1109/TBME.2013.2282775

  View details for Web of Science ID 000333268000030

• A Physiological Perspective on the Use of Imaging to Assess the In Vivo Delivery of Therapeutics ANNALS OF BIOMEDICAL ENGINEERING Qin, S., Fite, B. Z., Gagnon, M. J., Seo, J. W., Curry, F., Thorsen, F., Ferrara, K. W. 2014; 42 (2): 280–98

  Abstract

  Our goal is to provide a physiological perspective on the use of imaging to optimize and monitor the accumulation of nanotherapeutics within target tissues, with an emphasis on evaluating the pharmacokinetics of organic particles. Positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound technologies, as well as methods to label nanotherapeutic constructs, have created tremendous opportunities for preclinical optimization of therapeutics and for personalized treatments in challenging disease states. Within the methodology summarized here, the accumulation of the construct is estimated directly from the image intensity. Particle extravasation is then estimated based on classical physiological measures. Specifically, the transport of nanotherapeutics is described using the concept of apparent permeability, which is defined as the net flux of solute across a blood vessel wall per unit surface area of the blood vessel and per unit solute concentration difference across the blood vessel wall. The apparent permeability to small molecule MRI constructs is accurately shown to be far larger than that estimated for proteins such as albumin or nanoconstructs such as liposomes. Further, the quantitative measurements of vascular permeability are shown to facilitate detection of the transition from a pre-malignant to a malignant cancer and to quantify the delivery enhancement resulting from interventions such as ultrasound. While PET-based estimates facilitate quantitative comparisons of many constructs, high field MRI proves useful in the visualization of model drugs within small lesions and in the evaluation of the release and intracellular trafficking of nanoparticles and cargo.

  View details for PubMedID 24018607

  View details for PubMedCentralID PMC3943857

• Cu-64-Labeled LyP-1-Dendrimer for PET-CT Imaging of Atherosclerotic Plaque BIOCONJUGATE CHEMISTRY Seo, J., Baek, H., Mahakian, L. M., Kusunose, J., Hamzah, J., Ruoslahti, E., Ferrara, K. W. 2014; 25 (2): 231–39

  Abstract

  The ability to detect and quantify macrophage accumulation can provide important diagnostic and prognostic information for atherosclerotic plaque. We have previously shown that LyP-1, a cyclic 9-amino acid peptide, binds to p32 proteins on activated macrophages, facilitating the visualization of atherosclerotic plaque with PET. Yet, the in vivo plaque accumulation of monomeric [(18)F]FBA-LyP-1 was low (0.31 ± 0.05%ID/g). To increase the avidity of LyP-1 constructs to p32, we synthesized a dendritic form of LyP-1 on solid phase using lysine as the core structural element. Imaging probes (FAM or 6-BAT) were conjugated to a lysine or cysteine on the dendrimer for optical and PET studies. The N-terminus of the dendrimer was further modified with an aminooxy group in order to conjugate LyP-1 and ARAL peptides bearing a ketone. Oxime ligation of peptides to both dendrimers resulted in (LyP-1)4- and (ARAL)4-dendrimers with optical (FAM) and PET probes (6-BAT). For PET-CT studies, (LyP-1)4- and (ARAL)4-dendrimer-6-BAT were labeled with (64)Cu (t1/2 = 12.7 h) and intravenously injected into the atherosclerotic (ApoE(-/-)) mice. After two hours of circulation, PET-CT coregistered images demonstrated greater uptake of the (LyP-1)4-dendrimer-(64)Cu than the (ARAL)4-dendrimer-(64)Cu in the aortic root and descending aorta. Ex vivo images and the biodistribution acquired at three hours after injection also demonstrated a significantly higher uptake of the (LyP-1)4-dendrimer-(64)Cu (1.1 ± 0.26%ID/g) than the (ARAL)4-dendrimer-(64)Cu (0.22 ± 0.05%ID/g) in the aorta. Similarly, subcutaneous injection of the LyP-1-dendrimeric carriers resulted in preferential accumulation in plaque-containing regions over 24 h. In the same model system, ex vivo fluorescence images within aortic plaque depict an increased accumulation and penetration of the (LyP-1)4-dendrimer-FAM as compared to the (ARAL)4-dendrimer-FAM. Taken together, the results suggest that the (LyP-1)4-dendrimer can be applied for in vivo PET imaging of plaque and that LyP-1 could be further exploited for the delivery of therapeutics with multivalent carriers or nanoparticles.

  View details for PubMedID 24433095

• Assessing the barriers to image-guided drug delivery WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY Lanza, G. M., Moonen, C., Baker, J. R., Chang, E., Cheng, Z., Grodzinski, P., Ferrara, K., Hynynen, K., Kelloff, G., Lee, Y. K., Patri, A. K., Sept, D., Schnitzer, J. E., Wood, B. J., Zhang, M., Zheng, G., Farahani, K. 2014; 6 (1): 1-14

  Abstract

  Imaging has become a cornerstone for medical diagnosis and the guidance of patient management. A new field called image-guided drug delivery (IGDD) now combines the vast potential of the radiological sciences with the delivery of treatment and promises to fulfill the vision of personalized medicine. Whether imaging is used to deliver focused energy to drug-laden particles for enhanced, local drug release around tumors, or it is invoked in the context of nanoparticle-based agents to quantify distinctive biomarkers that could risk stratify patients for improved targeted drug delivery efficiency, the overarching goal of IGDD is to use imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities. Over the last several years, innumerable reports and reviews covering the gamut of IGDD technologies have been published, but inadequate attention has been directed toward identifying and addressing the barriers limiting clinical translation. In this consensus opinion, the opportunities and challenges impacting the clinical realization of IGDD-based personalized medicine were discussed as a panel and recommendations were proffered to accelerate the field forward.

  View details for DOI 10.1002/wnan.1247

  View details for Web of Science ID 000328354300001

  View details for PubMedID 24339356

  View details for PubMedCentralID PMC3967549

• Spatial and temporal control of hyperthermia using real time thermal strain imaging with motion compensation Foiret, J., Ferrara, K. W., IEEE IEEE. 2014: 2133–36

  View details for DOI 10.1109/ULTSYM.2014.0531

  View details for Web of Science ID 000352792500529

• Quantitation of nanoparticle accumulation in flow using optimized microfluidic chambers JOURNAL OF DRUG TARGETING Kusunose, J., Gagnon, M. J., Seo, J. W., Ferrara, K. W. 2014; 22 (1): 48–56

  Abstract

  The vascular cell adhesion molecule-1 (VCAM-1) targeting peptide sequence, VHPKQHR, is a promising moiety for targeting atherosclerosis through incorporation into nanoparticles such as dendrimers and liposomes.We aim to develop VCAM-1-targeted nanoparticles that effectively accumulate on the endothelium under shear conditions and to develop robust microfluidic chambers able to house sufficient cells for flow cytometric measurements.Carboxyfluorescein-labeled monomeric VHP-peptide, tetrameric VHP-dendrimers (bisbidentate or radial architecture, with or without N-terminal acetylation) and VHP-peptide liposomes were prepared. Human umbilical vein endothelial cells were treated with nanoparticles under 0 or 2.9 dyne/cm(2) shear, and particle binding was quantified. Flow chambers cured at various temperatures, with or without glass backings were fabricated, characterized for deformation and applied in experiments.Although liposomes accumulated with highest efficiency, dendrimers also demonstrated specific binding. N-terminal acetylation significantly reduced dendrimer binding, and despite shorter movement range, bisbidentate dendrimers outperformed radial dendrimers, suggesting multiple epitope presence within its estimated arm-span of 57 Å. Under shear, while liposome binding increased 300%, dendrimer binding to cells decreased 65%. Through higher temperature curing and glass backing insertion, polydimethylsiloxane flow chambers maintaining rectangular cross-section with aspect-ratio as low as 1:111 were achieved.Optimized dendrimers and liposomal nanocarriers specifically accumulated onto cells within microfluidic chambers.

  View details for PubMedID 24079404

  View details for PubMedCentralID PMC4004627

• Multimodal imaging enables early detection and characterization of changes in tumor permeability of brain metastases JOURNAL OF CONTROLLED RELEASE Thorsen, F., Fite, B., Mahakian, L. M., Seo, J. W., Qin, S., Harrison, V., Johnson, S., Ingham, E., Caskey, C., Sundstrom, T., Meade, T. J., Harter, P. N., Skaftnesmo, K., Ferrara, K. W. 2013; 172 (3): 812–22

  Abstract

  Our goal was to develop strategies to quantify the accumulation of model therapeutics in small brain metastases using multimodal imaging, in order to enhance the potential for successful treatment. Human melanoma cells were injected into the left cardiac ventricle of immunodeficient mice. Bioluminescent, MR and PET imaging were applied to evaluate the limits of detection and potential for contrast agent extravasation in small brain metastases. A pharmacokinetic model was applied to estimate vascular permeability. Bioluminescent imaging after injecting d-luciferin (molecular weight (MW) 320 D) suggested that tumor cell extravasation had already occurred at week 1, which was confirmed by histology. 7T T1w MRI at week 4 was able to detect non-leaky 100 μm sized lesions and leaky tumors with diameters down to 200 μm after contrast injection at week 5. PET imaging showed that (18)F-FLT (MW 244 Da) accumulated in the brain at week 4. Gadolinium-based MRI tracers (MW 559 Da and 2.066 kDa) extravasated after 5 weeks (tumor diameter 600 μm), and the lower MW agent cleared more rapidly from the tumor (mean apparent permeabilities 2.27 × 10(-5)cm/s versus 1.12 × 10(-5)cm/s). PET imaging further demonstrated tumor permeability to (64)Cu-BSA (MW 65.55 kDa) at week 6 (tumor diameter 700 μm). In conclusion, high field T1w MRI without contrast may improve the detection limit of small brain metastases, allowing for earlier diagnosis of patients, although the smallest lesions detected with T1w MRI were permeable only to d-luciferin and the amphipathic small molecule (18)F-FLT. Different-sized MR and PET contrast agents demonstrated the gradual increase in leakiness of the blood tumor barrier during metastatic progression, which could guide clinicians in choosing tailored treatment strategies.

  View details for DOI 10.1016/j.jconrel.2013.10.019

  View details for Web of Science ID 000328492800024

  View details for PubMedID 24161382

  View details for PubMedCentralID PMC3922207

• Complete regression of local cancer using temperature-sensitive liposomes combined with ultrasound-mediated hyperthermia JOURNAL OF CONTROLLED RELEASE Kheirolomoom, A., Lai, C., Tam, S. M., Mahakian, L. M., Ingham, E. S., Watson, K. D., Ferrara, K. W. 2013; 172 (1): 266–73

  Abstract

  The development of treatment protocols that result in a complete response to chemotherapy has been hampered by free drug toxicity and the low bioavailability of nano-formulated drugs. Here, we explore the application of temperature-sensitive liposomes that have been formulated to enhance stability in circulation. We formed a pH-sensitive complex between doxorubicin (Dox) and copper (CuDox) in the core of lysolipid-containing temperature-sensitive liposomes (LTSLs). The complex remains associated at neutral pH but dissociates to free Dox in lower pH environments. The resulting CuDox-LTSLs were injected intravenously into a syngeneic murine breast cancer model (6 mg Dox/kg body weight) and intravascular release of the drug was triggered by ultrasound. The entire tumor was insonified for 5 min prior to drug administration and 20 min post drug injection. A single-dose administration of CuDox-LTSLs combined with insonation suppressed tumor growth. Moreover, after twice per week treatment over a period of 28 days, a complete response was achieved in which the NDL tumor cells and the tumor interstitium could no longer be detected. All mice treated with ultrasound combined with CuDox-LTSLs survived, and tumor was undetectable 8 months post treatment. Iron and copper-laden macrophages were observed at early time points following treatment with this temperature sensitive formulation. Systemic toxicity indicators, such as cardiac hypertrophy, leukopenia, and weight and hair loss were not detected with CuDox-LTSLs after the 28-day therapy.

  View details for PubMedID 23994755

  View details for PubMedCentralID PMC3938386

• Inhibition of Mechanosensitive MicroRNA, mir-712 Decreases Endothelial Dysfunction and Atherosclerosis Kumar, S., Son, D. J., Takabe, W., Kim, C., Ni, C. W., Alberts-Grill, N., Kim, S. O., Kim, W., Seo, J., Ferrara, K., Jo, H. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000332162905148

• EARLY TUMOR DETECTION AND CHARACTERIZATION OF CHANGES IN TUMOR PERMEABILITY OF BRAINMETASTASES USING MULTIMODAL IMAGING Thorsen, F., Fite, B., Mahakian, L. M., Seo, J. W., Qin, S., Harrison, V., Sundstrom, T., Harter, P. N., Johnson, S., Ingham, E., Caskey, C., Meade, T., Skaftnesmo, K., Ferrara, K. W. OXFORD UNIV PRESS INC. 2013: 241

  View details for Web of Science ID 000327456200941

• BRAIN-SPECIFIC GENE SIGNATURE OF MELANOMA METASTASIS Sundstrom, T., Varughese, J. K., Harter, P., Prestegarden, L., Petersen, K., Azuaje, F., Tepper, C., Ingham, E., Even, L., Johnson, S., Skaftnesmo, K., Lund-Johansen, M., Bjerkvig, R., Ferrara, K., Thorsen, F. OXFORD UNIV PRESS INC. 2013: 27

  View details for Web of Science ID 000327456200113

• Evaluation of Doxorubicin-Loaded 3-Helix Micelles as Nanocarriers BIOMACROMOLECULES Dube, N., Shu, J. Y., Dong, H., Seo, J. W., Ingham, E., Kheirolomoom, A., Chen, P., Forsayeth, J., Bankiewicz, K., Ferrara, K. W., Xu, T. 2013; 14 (10): 3697–3705

  Abstract

  Designing stable drug nanocarriers, 10-30 nm in size, would have significant impact on their transport in circulation, tumor penetration, and therapeutic efficacy. In the present study, biological properties of 3-helix micelles loaded with 8 wt % doxorubicin (DOX), ~15 nm in size, were characterized to validate their potential as a nanocarrier platform. DOX-loaded micelles exhibited high stability in terms of size and drug retention in concentrated protein environments similar to conditions after intravenous injections. DOX-loaded micelles were cytotoxic to PPC-1 and 4T1 cancer cells at levels comparable to free DOX. 3-Helix micelles can be disassembled by proteolytic degradation of peptide shell to enable drug release and clearance to minimize long-term accumulation. Local administration to normal rat striatum by convection enhanced delivery (CED) showed greater extent of drug distribution and reduced toxicity relative to free drug. Intravenous administration of DOX-loaded 3-helix micelles demonstrated improved tumor half-life and reduced toxicity to healthy tissues in comparison to free DOX. In vivo delivery of DOX-loaded 3-helix micelles through two different routes clearly indicates the potential of 3-helix micelles as safe and effective nanocarriers for cancer therapeutics.

  View details for PubMedID 24050265

  View details for PubMedCentralID PMC3923314

• A phantom for visualization of three-dimensional drug release by ultrasound-induced mild hyperthermia MEDICAL PHYSICS Lai, C., Kruse, D., Seo, J., Kheirolomoom, A., Ferrara, K. W. 2013; 40 (8): 083301

  Abstract

  Ultrasound-induced mild hyperthermia has advantages for noninvasive, localized and controlled drug delivery. In this study, a tissue-mimicking agarose-based phantom with a thermally sensitive indicator was developed for studying the spatial drug delivery profile using ultrasound-induced mild hyperthermia.Agarose powder, regular evaporated milk, Dulbecco's phosphate-buffered saline (DPBS), n-propanol, and silicon carbide powder were homogeneously mixed with low temperature sensitive liposomes (LTSLs) loaded with a self-quenched near-infrared (NIR) fluorescent dye. A dual-mode linear array ultrasound transducer was used for insonation at 1.54 MHz with a total acoustic power and acoustic pressure of 2.0 W and 1.5 MPa, respectively. After insonation, the dye release pattern in the phantom was quantified based on optical images, and the three-dimensional release profile was reconstructed and analyzed. A finite-difference time-domain-based algorithm was developed to simulate both the temperature distribution and spatial dye diffusion as a function of time. Finally, the simulated dye diffusion patterns were compared to experimental measurements.Self-quenching of the fluorescent dye in DPBS was substantial at a concentration of 6.25×10(-2) mM or greater. The transition temperature of LTSLs in the phantom was 35 °C, and the release reached 90% at 37 °C. The simulated temperature for hyperthermia correlated with the thermocouple measurements with a mean error between 0.03±0.01 and 0.06±0.02 °C. The R2 value between the experimental and simulated spatial extent of the dye diffusion, defined by the half-peak level in the elevation, lateral and depth directions, was 0.99 (slope=1.08), 0.95 (slope=0.99), and 0.80 (slope=1.04), respectively, indicating the experimental and simulated dye release profiles were similar.The combination of LTSLs encapsulating a fluorescent dye and an optically transparent phantom is useful for visualizing and modeling drug release in vitro following ultrasound-induced mild hyperthermia. The coupled temperature simulation and dye-diffusion simulation tools were validated with the experimental system and can be used to optimize the thermal dose and spatial and temporal dye release pattern.

  View details for PubMedID 23927360

• Advanced Novel Technologies & Therapeutic Strategies Ferrara, K., Griffin, R., Guha, C., Holland, C., Lafon, C. AMER ASSOC PHYSICISTS MEDICINE AMER INST PHYSICS. 2013

  View details for DOI 10.1118/1.4815443

  View details for Web of Science ID 000336849900281

• Cardiac myocyte exosomes: stability, HSP60, and proteomics AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Malik, Z. A., Kott, K. S., Poe, A. J., Kuo, T., Chen, L., Ferrara, K. W., Knowlton, A. A. 2013; 304 (7): H954–H965

  Abstract

  Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at "physiological" concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated.

  View details for PubMedID 23376832

  View details for PubMedCentralID PMC3625894

• Multimodal imaging of blood-brain barrier disruption during brain metastatic progression in a relevant experimental mouse model Thorsen, F., Fite, B., Mahakian, L., Harrison, V., Johnson, S., Ingham, E., Qin, S., Soo, J. W., Meade, T., Sundstrom, T., Ferrara, K. W. AMER ASSOC CANCER RESEARCH. 2013

  View details for DOI 10.1158/1538-7445.TIM2013-A39

  View details for Web of Science ID 000209496400034

• A comparison of image contrast with Cu-64-labeled long circulating liposomes and F-18-FDG in a murine model of mammary carcinoma AMERICAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Ormsby, A., Zhang, H., Seo, J., Mahakian, L. M., Caskey, C. F., Ferrara, K. W. 2013; 3 (1): 32–43

  Abstract

  Conjugation of the (64)Cu PET radioisotope (t(1/2) = 12.7 hours) to long circulating liposomes enables long term liposome tracking. To evaluate the potential clinical utility of this radiotracer in diagnosis and therapeutic guidance, we compare image contrast, tumor volume, and biodistribution of (64)Cu-liposomes to metrics obtained with the dominant clinical tracer, (18)F-FDG. Twenty four female FVB mice with MET1 mammary carcinoma tumor grafts were examined. First, serial PET images were obtained with the (18)F-FDG radiotracer at 0.5 hours after injection and with the (64)Cu-liposome radiotracer at 6, 18, 24, and 48 hours after injection (n = 8). Next, paired imaging and histology were obtained at four time points: 0.5 hours after (18)F-FDG injection and 6, 24, and 48 hours after (64)Cu-liposome injection (n = 16). Tissue biodistribution was assessed with gamma counting following necropsy and tumors were paraffin embedded, sectioned, and stained with hematoxylin and eosin. The contrast ratio of images obtained using (18)F-FDG was 0.88 ± 0.01 (0.5 hours after injection), whereas with the (64)Cu-liposome radiotracer the contrast ratio was 0.78 ± 0.01, 0.89 ± 0.01, 0.88 ± 0.01, and 0.94 ± 0.01 at 6, 18, 24, and 48 hours, respectively. Estimates of tumor diameter were comparable between (64)Cu-liposomes and (18)F-FDG, (64)Cu-liposomes and necropsy, and (64)Cu-liposomes and ultrasound with Pearson's r-squared values of 0.79, 0.79, and 0.80, respectively. Heterogeneity of tumor tracer uptake was observed with both tracers, correlating with regions of necrosis on histology. The average tumor volume of 0.41 ± 0.05 cc measured with (64)Cu-liposomes was larger than that estimated with (18)F-FDG (0.28 ± 0.04 cc), with this difference apparently resulting primarily from accumulation of the radiolabeled particles in the pro-angiogenic tumor rim. The imaging of radiolabeled nanoparticles can facilitate tumor detection, identification of tumor margins, therapeutic evaluation and interventional guidance.

  View details for PubMedID 23342299

• Ultrasound MEDICAL IMAGING: PRINCIPLES AND PRACTICES Goldberg, R. L., Smith, S. W., Mottley, J. G., Ferrara, K., Analoui, M., Bronzino, J. D., Peterson, D. R. 2013

  View details for Web of Science ID 000362981200009

• Blood Flow Measurement Using Ultrasound MEDICAL IMAGING: PRINCIPLES AND PRACTICES Ferrara, K., Analoui, M., Bronzino, J. D., Peterson, D. R. 2013

  View details for Web of Science ID 000362981200011

• Microfluidic System for Facilitated Quantification of Nanoparticle Accumulation to Cells Under Laminar Flow ANNALS OF BIOMEDICAL ENGINEERING Kusunose, J., Zhang, H., Gagnon, M. J., Pan, T., Simon, S. I., Ferrara, K. W. 2013; 41 (1): 89–99

  Abstract

  The identification of novel, synthetic targeting ligands to endothelial receptors has led to the rapid development of targeted nanoparticles for drug, gene and imaging probe delivery. Central to development and optimization are effective models for assessing particle binding in vitro. Here, we developed a simple and cost effective method to quantitatively assess nanoparticle accumulation under physiologically-relevant laminar flow. We designed reversibly vacuum-sealed PDMS microfluidic chambers compatible with 35 mm petri dishes, which deliver uniform or gradient shear stress. These chambers have sufficient surface area for facile cell collection for particle accumulation quantitation through FACS. We tested this model by synthesizing and flowing liposomes coated with APN (K (D) ~ 300 μM) and VCAM-1-targeting (K (D) ~ 30 μM) peptides over HUVEC. Particle binding significantly increased with ligand concentration (up to 6 mol%) and decreased with excess PEG. While the accumulation of particles with the lower affinity ligand decreased with shear, accumulation of those with the higher affinity ligand was highest in a low shear environment (2.4 dyne/cm(2)), as compared with greater shear or the absence of shear. We describe here a robust flow chamber model that is applied to optimize the properties of 100 nm liposomes targeted to inflamed endothelium.

  View details for PubMedID 22855121

• Ultrasonic enhancement of drug penetration in solid tumors FRONTIERS IN ONCOLOGY Lai, C., Fite, B. Z., Ferrara, K. W. 2013; 3: 204

  Abstract

  Increasing the penetration of drugs within solid tumors can be accomplished through multiple ultrasound-mediated mechanisms. The application of ultrasound can directly change the structure or physiology of tissues or can induce changes in a drug or vehicle in order to enhance delivery and efficacy. With each ultrasonic pulse, a fraction of the energy in the propagating wave is absorbed by tissue and results in local heating. When ultrasound is applied to achieve mild hyperthermia, the thermal effects are associated with an increase in perfusion or the release of a drug from a temperature-sensitive vehicle. Higher ultrasound intensities locally ablate tissue and result in increased drug accumulation surrounding the ablated region of interest. Further, the mechanical displacement induced by the ultrasound pulse can result in the nucleation, growth and collapse of gas bubbles. As a result of such cavitation, the permeability of a vessel wall or cell membrane can be increased. Finally, the radiation pressure of the propagating pulse can translate particles or tissues. In this perspective, we will review recent progress in ultrasound-mediated tumor delivery and the opportunities for clinical translation.

  View details for PubMedID 23967400

• In vivo validation and 3D visualization of broadband ultrasound molecular imaging AMERICAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Hu, X., Caskey, C. F., Mahakian, L. M., Kruse, D. E., Beegle, J. R., Decleves, A., Rychak, J. J., Sutcliffe, P. L., Sharma, K., Ferrara, K. W. 2013; 3 (4): 336–49

  Abstract

  Ultrasound can selectively and specifically visualize upregulated vascular receptors through the detection of bound microbubbles. However, most current ultrasound molecular imaging methods incur delays that result in longer acquisition times and reduced frame rates. These delays occur for two main reasons: 1) multi-pulse imaging techniques are used to differentiate microbubbles from tissue and 2) acquisition occurs after free bubble clearance (>6 minutes) in order to differentiate bound from freely circulating microbubbles. In this paper, we validate tumor imaging with a broadband single pulse molecular imaging method that is faster than the multi-pulse methods typically implemented on commercial scanners. We also combine the single pulse method with interframe filtering to selectively image targeted microbubbles without waiting for unbound bubble clearance, thereby reducing acquisition time from 10 to 2 minutes. The single pulse imaging method leverages non-linear bubble behavior by transmitting at low and receiving at high frequencies (TLRH). We implemented TLRH imaging and visualized the accumulation of intravenously administrated integrin-targeted microbubbles in a phantom and a Met-1 mouse tumor model. We found that the TLRH contrast imaging has a ~2-fold resolution improvement over standard contrast pulse sequencing (CPS) imaging. By using interframe filtering, the tumor contrast was 24.8±1.6 dB higher after the injection of integrin-targeted microbubbles than non-targeted control MBs, while echoes from regions lacking the target integrin were suppressed by 26.2±2.1 dB as compared with tumor echoes. Since real-time three-dimensional (3D) molecular imaging provides a more comprehensive view of receptor distribution, we generated 3D images of tumors to estimate their volume, and these measurements correlated well with expected tumor sizes. We conclude that TLRH combined with interframe filtering is a feasible method for 3D targeted ultrasound imaging that is faster than current multi-pulse strategies.

  View details for PubMedID 23901359

• The cargo of CRPPR-conjugated liposomes crosses the intact murine cardiac endothelium Zhang, H., Li, N., Sirish, P., Mahakian, L., Ingham, E., Curry, F., Yamada, S., Chiamvimonvat, N., Ferrara, K. W. ELSEVIER SCIENCE BV. 2012: 10–17

  Abstract

  Ligand-conjugated liposomes and other nano-sized constructs are attractive drug carriers due to their extended plasma circulation; however, limited data are available as to whether their cargo can traverse the endothelium of solid organs. To determine whether the cargo of endothelially targeted liposomes is internalized by endothelial cells and transported into tissue, and to evaluate whether such liposomes can accumulate in models of cardiovascular disease, we tracked the fate of the cargo (a hydrophilic fluorescent dye) and shell (conjugated with a radioisotope) of a heart-homing liposome (CRPPR-conjugated). The ex vivo heart was imaged with confocal microscopy and the in vivo heart with positron emission tomography in sham-treated mice and models of ischemia/reperfusion (I/R) and myocardial infarction (MI). Within 30 min of injection of 20mg/kg CRPPR liposomes, fluorescence increased by 47 fold in the tissue surrounding the vascular lumen, as compared with non-targeted liposomes. Both the accumulation on the endothelium and the interstitial fluorescence saturated at an injected dose of 20mg/kg. In both I/R and MI models, CRPPR liposomes accumulated in diseased sites, although less than in surrounding healthy tissue. The accumulation in the diseased sites increased with time post-injury: the ratio of accumulated radioactivity in the diseased and healthy cardiac tissue increased from 0.20±0.04, to 0.58±0.12 and 0.61±0.19 for 1, 7, and 99 days post-MI, indicating the potential for adequate delivery and therapeutic efficacy if the targeted particles are injected at 7 or more days post-MI. In summary, CRPPR- liposomes accumulated in normal and diseased hearts, and the cargo accumulated in the tissue within minutes and remained detectable after 24 h.

  View details for PubMedID 22776291

• Ultrasound Imaging of Oxidative Stress In Vivo with Chemically-Generated Gas Microbubbles ANNALS OF BIOMEDICAL ENGINEERING Perng, J., Lee, S., Kundu, K., Caskey, C. F., Knight, S. F., Satir, S., Ferrara, K. W., Taylor, W., Degertekin, F., Sorescu, D., Murthy, N. 2012; 40 (9): 2059–68

  Abstract

  Ultrasound contrast agents (UCAs) have tremendous potential for in vivo molecular imaging because of their high sensitivity. However, the diagnostic potential of UCAs has been difficult to exploit because current UCAs are based on pre-formed microbubbles, which can only detect cell surface receptors. Here, we demonstrate that chemical reactions that generate gas forming molecules can be used to perform molecular imaging by ultrasound in vivo. This new approach was demonstrated by imaging reactive oxygen species in vivo with allylhydrazine, a liquid compound that is converted into nitrogen and propylene gas after reacting with radical oxidants. We demonstrate that allylhydrazine encapsulated within liposomes can detect a 10 micromolar concentration of radical oxidants by ultrasound, and can image oxidative stress in mice, induced by lipopolysaccharide, using a clinical ultrasound system. We anticipate numerous applications of chemically-generated microbubbles for molecular imaging by ultrasound, given ultrasound's ability to detect small increments above the gas saturation limit, its spatial resolution and widespread clinical use.

  View details for DOI 10.1007/s10439-012-0573-9

  View details for Web of Science ID 000307400900019

  View details for PubMedID 22562306

  View details for PubMedCentralID PMC4029424

• Insonation of Targeted Microbubbles Produces Regions of Reduced Blood Flow Within Tumor Vasculature INVESTIGATIVE RADIOLOGY Hu, X., Kheirolomoom, A., Mahakian, L. M., Beegle, J. R., Kruse, D. E., Lam, K. S., Ferrara, K. W. 2012; 47 (7): 398–405

  Abstract

  In ultrasound molecular imaging, a sequence of high-pressure ultrasound pulses is frequently applied to destroy bound targeted microbubbles, to quantify accumulated microbubbles or to prepare for successive microbubble injections; however, the potential for biological effects from such a strategy has not been fully investigated. Here, we investigate the effect of high-pressure insonation of bound microbubbles and the potential for thrombogenic effects.A total of 114 mice carrying either Met-1 or neu deletion mutant (NDL) tumors was insonified (Siemens Sequoia system, 15L8 transducer, 5-MHz color-Doppler pulses, 4 or 2 MPa peak-negative pressure, 8.1-millisecond pulse repetition period, 6-cycle pulse length, and 900-millisecond insonation). Microbubbles conjugated with cyclic-arginine-glycine-aspartic acid (cRGD) or cyclic-aspartic-acid-glycine-tyrosine (3-NO)-glycine-hydroxyproline-asparagine (LXY-3) peptides or control (no peptide) microbubbles were injected, and contrast pulse sequencing was used to visualize the flowing and bound microbubbles. An anti-CD41 antibody was injected in a subset of animals to block potential thrombogenic effects.After the accumulation of targeted microbubbles and high-pressure (4 MPa) insonation, reduced blood flow, as demonstrated by a reduction in echoes from flowing microbubbles, was observed in 20 Met-1 mice (71%) and 4 NDL mice (40%). The area of low image intensity increased from 22 ± 13% to 63 ± 17% of the observed plane in the Met-1 model (P < 0.01) and from 16 ± 3% to 45 ± 24% in the NDL model (P < 0.05). Repeated microbubble destruction at 4 MPa increased the area of low image intensity to 76.7 ± 13.4% (P < 0.05). The fragmentation of bound microbubbles with a lower peak-negative pressure (2 MPa) reduced the occurrence of the blood flow alteration to 28% (5/18 Met-1 tumor mice). The persistence of the observed blood flow change was approximately 30 minutes after the microbubble destruction event. Dilated vessels and enhanced extravasation of 150 kDa fluorescein-isothiocyanate (FITC)-dextran were observed by histology and confocal microscopy. Preinjection of an anti-CD41 antibody blocked the reduction of tumor blood flow, where a reduction in blood flow was observed in only 1 of 26 animals.High-pressure fragmentation of microbubbles bound to tumor endothelial receptors reduced blood flow within 2 syngeneic mouse tumor models for ∼30 minutes. Platelet activation, likely resulting from the injury of small numbers of endothelial cells, was the apparent mechanism for the flow reduction.

  View details for PubMedID 22659591

• Long-Circulating 15 nm Micelles Based on Amphiphilic 3-Helix Peptide-PEG Conjugates ACS NANO Dong, H., Dube, N., Shu, J. Y., Seo, J. W., Mahakian, L. M., Ferrara, K. W., Xu, T. 2012; 6 (6): 5320–29

  Abstract

  Generating stable, multifunctional organic nanocarriers will have a significant impact on drug formulation. However, it remains a significant challenge to generate organic nanocarriers with a long circulation half-life, effective tumor penetration, and efficient clearance of metabolites. We have advanced this goal by designing a new family of amphiphiles based on coiled-coil 3-helix bundle forming peptide-poly(ethylene glycol) conjugates. The amphiphiles self-assemble into monodisperse micellar nanoparticles, 15 nm in diameter. Using the 3-helix micelles, a drug loading of ∼8 wt % was obtained using doxorubicin and the micelles showed minimal cargo leakage after 12 h of incubation with serum proteins at 37 °C. In vivo pharmacokinetics studies using positron emission tomography showed a circulation half-life of 29.5 h and minimal accumulation in the liver and spleen. The demonstrated strategy, by incorporating unique protein tertiary structure in the headgroup of an amphiphile, opens new avenues to generate organic nanoparticles with tunable stability, ligand clustering, and controlled disassembly to meet current demands in nanomedicine.

  View details for PubMedID 22545944

• Magnetic Resonance Thermometry at 7T for Real-Time Monitoring and Correction of Ultrasound Induced Mild Hyperthermia PLOS ONE Fite, B. Z., Liu, Y., Kruse, D. E., Caskey, C. F., Walton, J. H., Lai, C., Mahakian, L. M., Larrat, B., Dumont, E., Ferrara, K. W. 2012; 7 (4): e35509

  Abstract

  While Magnetic Resonance Thermometry (MRT) has been extensively utilized for non-invasive temperature measurement, there is limited data on the use of high field (≥7T) scanners for this purpose. MR-guided Focused Ultrasound (MRgFUS) is a promising non-invasive method for localized hyperthermia and drug delivery. MRT based on the temperature sensitivity of the proton resonance frequency (PRF) has been implemented in both a tissue phantom and in vivo in a mouse Met-1 tumor model, using partial parallel imaging (PPI) to speed acquisition. An MRgFUS system capable of delivering a controlled 3D acoustic dose during real time MRT with proportional, integral, and derivative (PID) feedback control was developed and validated. Real-time MRT was validated in a tofu phantom with fluoroptic temperature measurements, and acoustic heating simulations were in good agreement with MR temperature maps. In an in vivo Met-1 mouse tumor, the real-time PID feedback control is capable of maintaining the desired temperature with high accuracy. We found that real time MR control of hyperthermia is feasible at high field, and k-space based PPI techniques may be implemented for increasing temporal resolution while maintaining temperature accuracy on the order of 1°C.

  View details for PubMedID 22536396

• Leveraging the power of ultrasound for therapeutic design and optimization JOURNAL OF CONTROLLED RELEASE Caskey, C. F., Hu, X., Ferrara, K. W. 2011; 156 (3): 297–306

  Abstract

  Contrast agent-enhanced ultrasound can facilitate personalized therapeutic strategies by providing the technology to measure local blood flow rate, to selectively image receptors on the vascular endothelium, and to enhance localized drug delivery. Ultrasound contrast agents are micron-diameter encapsulated bubbles that circulate within the vascular compartment and can be selectively imaged with ultrasound. Microbubble transport-based estimates of local blood flow can quantify changes resulting from anti-angiogenic therapies and facilitate differentiation of angiogenic mechanisms. Microbubbles that are conjugated with targeting ligands attach to endothelial surface receptors that are upregulated in disease, providing high signal-to-noise ratio images of pathological vasculature. In addition to imaging applications, microbubbles can be used to enhance localized gene and drug delivery, either by changing membrane and vascular permeability or by carrying and locally releasing cargo. Our goal in this review is to provide an overview of the use of contrast-enhanced ultrasound methodologies in the design and evaluation of therapeutic strategies with emphases on quantitative blood flow mapping, molecular imaging, and enhanced drug delivery.

  View details for PubMedID 21835212

• An Open Environment CT-US Fusion for Tissue Segmentation during Interventional Guidance PLOS ONE Caskey, C. F., Hlawitschka, M., Qin, S., Mahakian, L. M., Cardiff, R. D., Boone, J. M., Ferrara, K. W. 2011; 6 (11): e27372

  Abstract

  Therapeutic ultrasound (US) can be noninvasively focused to activate drugs, ablate tumors and deliver drugs beyond the blood brain barrier. However, well-controlled guidance of US therapy requires fusion with a navigational modality, such as magnetic resonance imaging (MRI) or X-ray computed tomography (CT). Here, we developed and validated tissue characterization using a fusion between US and CT. The performance of the CT/US fusion was quantified by the calibration error, target registration error and fiducial registration error. Met-1 tumors in the fat pads of 12 female FVB mice provided a model of developing breast cancer with which to evaluate CT-based tissue segmentation. Hounsfield units (HU) within the tumor and surrounding fat pad were quantified, validated with histology and segmented for parametric analysis (fat: -300 to 0 HU, protein-rich: 1 to 300 HU, and bone: HU>300). Our open source CT/US fusion system differentiated soft tissue, bone and fat with a spatial accuracy of ∼1 mm. Region of interest (ROI) analysis of the tumor and surrounding fat pad using a 1 mm(2) ROI resulted in mean HU of 68±44 within the tumor and -97±52 within the fat pad adjacent to the tumor (p<0.005). The tumor area measured by CT and histology was correlated (r(2) = 0.92), while the area designated as fat decreased with increasing tumor size (r(2) = 0.51). Analysis of CT and histology images of the tumor and surrounding fat pad revealed an average percentage of fat of 65.3% vs. 75.2%, 36.5% vs. 48.4%, and 31.6% vs. 38.5% for tumors <75 mm(3), 75-150 mm(3) and >150 mm(3), respectively. Further, CT mapped bone-soft tissue interfaces near the acoustic beam during real-time imaging. Combined CT/US is a feasible method for guiding interventions by tracking the acoustic focus within a pre-acquired CT image volume and characterizing tissues proximal to and surrounding the acoustic focus.

  View details for PubMedID 22132098

• Fast Ultrasound Beam Prediction for Linear and Regular Two-Dimensional Arrays IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Hlawitschka, M., McGough, R. J., Ferrara, K. W., Kruse, D. E. 2011; 58 (9): 2001–12

  Abstract

  Real-time beam predictions are highly desirable for the patient-specific computations required in ultrasound therapy guidance and treatment planning. To address the longstanding issue of the computational burden associated with calculating the acoustic field in large volumes, we use graphics processing unit (GPU) computing to accelerate the computation of monochromatic pressure fields for therapeutic ultrasound arrays. In our strategy, we start with acceleration of field computations for single rectangular pistons, and then we explore fast calculations for arrays of rectangular pistons. For single-piston calculations, we employ the fast near-field method (FNM) to accurately and efficiently estimate the complex near-field wave patterns for rectangular pistons in homogeneous media. The FNM is compared with the Rayleigh-Sommerfeld method (RSM) for the number of abscissas required in the respective numerical integrations to achieve 1%, 0.1%, and 0.01% accuracy in the field calculations. Next, algorithms are described for accelerated computation of beam patterns for two different ultrasound transducer arrays: regular 1-D linear arrays and regular 2-D linear arrays. For the array types considered, the algorithm is split into two parts: 1) the computation of the field from one piston, and 2) the computation of a piston-array beam pattern based on a pre-computed field from one piston. It is shown that the process of calculating an array beam pattern is equivalent to the convolution of the single-piston field with the complex weights associated with an array of pistons. Our results show that the algorithms for computing monochromatic fields from linear and regularly spaced arrays can benefit greatly from GPU computing hardware, exceeding the performance of an expensive CPU by more than 100 times using an inexpensive GPU board. For a single rectangular piston, the FNM method facilitates volumetric computations with 0.01% accuracy at rates better than 30 ns per field point. Furthermore, we demonstrate array calculation speeds of up to 11.5 X 10(9) field-points per piston per second (0.087 ns per field point per piston) for a 512-piston linear array. Beam volumes containing 256(3) field points are calculated within 1 s for 1-D and 2-D arrays containing 512 and 20(2) pistons, respectively, thus facilitating future real-time thermal dose predictions.

  View details for DOI 10.1109/TUFFC.2011.2044

  View details for Web of Science ID 000295101600038

  View details for PubMedID 21937338

  View details for PubMedCentralID PMC3306819

• Design Aspects of Focal Beams From High-Intensity Arrays IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Stephens, D. N., Kruse, D. E., Qin, S., Ferrara, K. W. 2011; 58 (8): 1590–1602

  Abstract

  As the applications of ultrasonic thermal therapies expand, the design of the high-intensity array must address both the energy delivery of the main beam and the character and relevance of off-target beam energy. We simulate the acoustic field performance of a selected set of circular arrays organized by array format, including flat versus curved arrays, periodic versus random arrays, and center void diameter variations. Performance metrics are based on the -3-dB focal main lobe (FML) positioning range, axial grating lobe (AGL) temperatures, and side lobe levels. Using finite-element analysis, we evaluate the relative heating of the FML and the AGLs. All arrays have a maximum diameter of 100λ, with element count ranging from 64 to 1024 and continuous wave frequency of 1.5 MHz. First, we show that a 50% spherical annulus produces focus beam side lobes which decay as a function of lateral distance at nearly 87% of the exponential rate of a full aperture. Second, for the arrays studied, the efficiency of power delivery over the -3-dB focus positioning range for spherical arrays is at least 2-fold greater than for flat arrays; the 256-element case shows a 5-fold advantage for the spherical array. Third, AGL heating can be significant as the focal target is moved to its distal half-intensity depth from the natural focus. Increasing the element count of a randomized array to 256 elements decreases the AGL-to-FML heating ratio to 0.12 at the distal half-intensity depth. Further increases in element count yield modest improvements. A 49% improvement in the AGL-to-peak heating ratio is predicted by using the Sumanaweera spiral element pattern with randomization.

  View details for PubMedID 21859578

• A Radio-Frequency Coupling Network for Heating of Citrate-Coated Gold Nanoparticles for Cancer Therapy: Design and Analysis IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Kruse, D. E., Stephens, D. N., Lindfors, H. A., Ingham, E. S., Paoli, E. E., Ferrara, K. W. 2011; 58 (7): 2002–12

  Abstract

  Gold nanoparticles (GNPs) are nontoxic, can be functionalized with ligands, and preferentially accumulate in tumors. We have developed a 13.56-MHz RF-electromagnetic field (RF-EM) delivery system capable of generating high E-field strengths required for noninvasive, noncontact heating of GNPs. The bulk heating and specific heating rates were measured as a function of NP size and concentration. It was found that heating is both size and concentration dependent, with 5 nm particles producing a 50.6 ± 0.2 °C temperature rise in 30 s for 25 μg/mL gold (125 W input). The specific heating rate was also size and concentration dependent, with 5 nm particles producing a specific heating rate of 356 ± 78 kW/g gold at 16 μg/mL (125 W input). Furthermore, we demonstrate that cancer cells incubated with GNPs are killed when exposed to 13.56 MHz RF-EM fields. Compared to cells that were not incubated with GNPs, three out of four RF-treated groups showed a significant enhancement of cell death with GNPs (p<0.05). GNP-enhanced cell killing appears to require temperatures above 50 °C for the experimental parameters used in this study. Transmission electron micrographs show extensive vacuolization with the combination of GNPs and RF treatment.

  View details for PubMedID 21402506

• NOVEL ULTRASOUND AND DCE-MRI ANALYSES AFTER ANTIANGIOGENIC TREATMENT WITH A SELECTIVE VEGF RECEPTOR INHIBITOR ULTRASOUND IN MEDICINE AND BIOLOGY Watson, K. D., Hu, X., Lai, C., Lindfors, H. A., Hu-Lowe, D. D., Tuthill, T. A., Shalinsky, D. R., Ferrara, K. W. 2011; 37 (6): 909–21

  Abstract

  We report a comparison between tumor perfusion estimates acquired using contrast-enhanced MRI and motion-corrected contrast-enhanced ultrasound before and after treatment with AG-028262, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor. Antiangiogenic activity was determined by assessing weekly ultrasound and MRI images of rats with bilateral hind flank mammary adenocarcinomas before and after treatment with AG-028262. Images were acquired with a spoiled gradient, 1.5 T magnetic resonance sequence and a destruction-replenishment ultrasound protocol. For ultrasound, a time to 80% contrast replenishment was calculated for each tumor voxel; for MR imaging, a measure of local flow rate was estimated from a linear fit of minimum to maximum intensities. AG-028262 significantly decreased tumor growth and increased the time required to replenish tumor voxels with an ultrasound contrast agent from 2.66 to 4.54 s and to fill with an MR contrast agent from 29.5 to 50.8 s. Measures of flow rate derived from MRI and ultrasound demonstrated a positive linear correlation of r2 = 0.86.

  View details for PubMedID 21531499

  View details for PubMedCentralID PMC3198831

• PET-guided and ultrasound enhanced nanotherapy Ferrara, K. W., Kheirolomoom, A., Seo, J. W., Qin, S., Zhang, H., Even, L., Rygh, C. AMER ASSOC CANCER RESEARCH. 2011

  View details for DOI 10.1158/1538-7445.AM2011-SY20-01

  View details for Web of Science ID 000209701303231

• Positron emission tomography imaging of the stability of Cu-64 labeled dipalmitoyl and distearoyl lipids in liposomes JOURNAL OF CONTROLLED RELEASE Seo, J., Qin, S., Mahakian, L. M., Watson, K. D., Kheirolomoom, A., Ferrara, K. W. 2011; 151 (1): 28–34

  Abstract

  Changes in lipid acyl chain length can result in desorption of lipid from the liposomal anchorage and interaction with blood components. PET studies of the stability of such lipids have not been performed previously although such studies can map the pharmacokinetics of unstable lipids non-invasively in vivo. The purpose of this study was to characterize the in vivo clearance of (64)Cu-labeled distearoyl- and dipalmitoyl lipid included within long circulating liposomes. Distearoyl and dipalmitoyl maleimide lipids (1mol%) in liposomes were labeled with a (64)Cu-incorporated bifunctional chelator (TETA-PDP) after the activation of pyridine disulfide to thiol by TCEP. Long circulating liposomes containing HSPC:DSPE-PEG2k-OMe:cholesterol: x (55:5:39:1), where x was (64)Cu-DSPE or (64)Cu-DPPE, or HSPC:DSPE-PEG2k-OMe:cholesterol:(64)Cu-DSPE:DPPC (54:5:39:1:1) were evaluated in serum (in vitro) and via intravenous injection to FVB mice. The time-activity curves for the blood, liver, and kidney were measured from PET images and the biodistribution was performed at 48h. In vitro assays showed that (64)Cu-DPPE transferred from liposomes to serum with a 7.9h half-life but (64)Cu-DSPE remained associated with the liposomes. The half clearance of radioactivity from the blood pool was 18 and 5h for (64)Cu-DSPE- and (64)Cu-DPPE liposome-injected mice, respectively. The clearance of radioactivity from the liver and kidney was significantly greater following the injection of (64)Cu-DPPE-labeled liposomes than (64)Cu-DSPE-labeled liposomes at 6, 18 and 28h. Forty eight hours after injection, the whole body radioactivity was 57 and 17% ID/cc for (64)Cu-DSPE and (64)Cu-DPPE, respectively. These findings suggest that the acyl chain length of the radiolabel should be considered for liposomal PET studies and that PET is an effective tool for evaluating the stability of nanoformulations in vivo.

  View details for PubMedID 21241753

• Novel Method to Label Solid Lipid Nanoparticles with Cu-64 for Positron Emission Tomography Imaging BIOCONJUGATE CHEMISTRY Andreozzi, E., Seo, J., Ferrara, K., Louie, A. 2011; 22 (4): 808–18

  Abstract

  Solid lipid nanoparticles (SLNs) are submicrometer (1-1000 nm) colloidal carriers developed in the past decade as an alternative system to traditional carriers (emulsions, liposomes, and polymeric nanoparticles) for intravenous applications. Because of their potential as drug carriers, there is much interest in understanding the in vivo biodistribution of SLNs following intravenous (i.v.) injection. Positron emission tomography (PET) is an attractive method for investigating biodistribution but requires a radiolabeled compound. In this work, we describe a method to radiolabel SLN for in vivo PET studies. A copper specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT), conjugated with a synthetic lipid, was incorporated into the SLN. Following incubation with (64)CuCl(2) for 1 h at 25 °C in 0.1 M NH(4)OAc buffer (pH 5.5), the SLNs (∼150 nm) were successfully radiolabeled with (64)Cu (66.5% radiolabeling yield), exhibiting >95% radiolabeled particles following purification. The (64)Cu-SLNs were delivered intravenously to mice and imaged with PET at 0.5, 3, 20, and 48 h post injection. Gamma counting was utilized post imaging to confirm organ distributions. Tissue radioactivity (% injected dose/gram, %ID/g), obtained by quantitative analysis of the images, suggests that the (64)Cu-SLNs are circulating in the bloodstream after 3 h (blood half-life ∼1.4 h), but are almost entirely cleared by 48 h. PET and gamma counting demonstrate that approximately 5-7%ID/g (64)Cu-SLNs remain in the liver at 48 h post injection. Stability assays confirm that copper remains associated with the SLN over the 48 h time period and that the biodistribution patterns observed are not from free, dissociated copper. Our results indicate that SLNs can be radiolabeled with (64)Cu, and their biodistribution can be quantitatively evaluated by in vivo PET imaging and ex vivo gamma counting.

  View details for PubMedID 21388194

• Novel Targeted Contrast Agent for Ultrasound Molecular Imaging of alpha v beta 3 Integrin Anderson, C. R., Rychak, J. J., Hu, X., Ferrara, K. FEDERATION AMER SOC EXP BIOL. 2011

  View details for Web of Science ID 000310708405317

• Ultrasound Molecular Imaging of Tumor Angiogenesis With an Integrin Targeted Microbubble Contrast Agent INVESTIGATIVE RADIOLOGY Anderson, C. R., Hu, X., Zhang, H., Tlaxca, J., Decleves, A., Houghtaling, R., Sharma, K., Lawrence, M., Ferrara, K. W., Rychak, J. J. 2011; 46 (4): 215–24

  Abstract

  Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of antiangiogenic therapies. Our objectives are to develop a robust ultrasound contrast agent platform using microbubbles (MB) to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index (MI) imaging processing method and corresponding quantification method. The MB and the imaging methods were evaluated in a mouse model of breast cancer in vivo.We used a cyclic arginine-glycine-aspartic acid (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting αvβ3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine αvβ3 integrin protein and αvβ3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for αvβ3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted MB were visualized using a low MI contrast imaging pulse sequence, and quantified by intensity normalization and 2-dimensional Fourier transform analysis.The cRGD ligand concentration on the MB surface was ∼8.2 × 10(6) molecules per MB. At a wall shear stress of 1.0 dynes/cm, cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant αvβ3 integrin relative to nontargeted MB and cRAD-MB controls. Similarly, cRGD-MB showed significantly greater adhesion to bEnd.3 EC compared with nontargeted MB and cRAD-MB. In addition, cRGD-MB, but not nontargeted MB or cRAD-MB, showed significantly enhanced contrast signals with a high tumor-to-background ratio. The adhesion of cRGD-MB to bEnd.3 was reduced by 80% after using anti-αv monoclonal antibody to treat bEnd.3. The normalized image intensity amplitude was ∼0.8, 7 minutes after the administration of cRGD-MB relative to the intensity amplitude at the time of injection, while the spatial variance in image intensity improved the detection of bound agents. The accumulation of cRGD-MB was blocked by preadministration with an anti-αv blocking antibody.The results demonstrate the functionality of a novel MB contrast agent covalently coupled to an RGD peptide for ultrasound molecular imaging of αvβ3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low MI.

  View details for DOI 10.1097/RLI.0b013e3182034fed

  View details for Web of Science ID 000287984600002

  View details for PubMedID 21343825

  View details for PubMedCentralID PMC3075480

• Triggered complement activation using liposome flip-flop Day, J. J., Paoli, E. E., Ferrara, K. W., Meares, C. F. AMER CHEMICAL SOC. 2011

  View details for Web of Science ID 000291982802230

• Targeting Activin Receptor-Like Kinase 1 Inhibits Angiogenesis and Tumorigenesis through a Mechanism of Action Complementary to Anti-VEGF Therapies CANCER RESEARCH Hu-Lowe, D. D., Chen, E., Zhang, L., Watson, K. D., Mancuso, P., Lappin, P., Wickman, G., Chen, J. H., Wang, J., Jiang, X., Amundson, K., Simon, R., Erbersdobler, A., Bergqvist, S., Feng, Z., Swanson, T. A., Simmons, B. H., Lippincott, J., Casperson, G. F., Levin, W. J., Stampino, C., Shalinsky, D. R., Ferrara, K. W., Fiedler, W., Bertolini, F. 2011; 71 (4): 1362–73

  Abstract

  Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

  View details for PubMedID 21212415

  View details for PubMedCentralID PMC3269003

• MR-Guided Focused Ultrasound with Spatial and Temporal Temperature Control for Hyperthermia Liu, Y., Fite, B. Z., Kruse, D. E., Mahakian, L., Dumont, E., Caskey, C. F., Ferrara, K. W., IEEE IEEE. 2011: 1641–44

  View details for DOI 10.1109/ULTSYM.2011.0408

  View details for Web of Science ID 000309918400389

• Reduced blood flow in murine tumors after the destruction of bound, targeted microbubbles Hu, X., Kheirolomoom, A., Mahakian, L. M., Beegle, J. R., Ingham, E. S., Caskey, C. F., Kruse, D. E., Ferrara, K. W., IEEE IEEE. 2011: 955–58

  View details for DOI 10.1109/ULTSYM.2011.0234

  View details for Web of Science ID 000309918400217

• A Fast Ultrasound Molecular Imaging Method and its 3D Visualization in vivo Hu, X., Mahakian, L., Caskey, C., Beegle, J., Kruse, D., Rychak, J., Sutcliffe, P., Ferrara, K., IEEE IEEE. 2011: 1618–21

  View details for DOI 10.1109/ULTSYM.2011.0402

  View details for Web of Science ID 000309918400383

• Noninvasive Thermometry Assisted by a Dual-Function Ultrasound Transducer for Mild Hyperthermia IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Lai, C., Kruse, D. E., Caskey, C. F., Stephens, D. N., Sutcliffe, P. L., Ferrara, K. W. 2010; 57 (12): 2671–84

  Abstract

  Mild hyperthermia is increasingly important for the activation of temperature-sensitive drug delivery vehicles. Noninvasive ultrasound thermometry based on a 2-D speckle tracking algorithm was examined in this study. Here, a commercial ultrasound scanner, a customized co-linear array transducer, and a controlling PC system were used to generate mild hyperthermia. Because the co-linear array transducer is capable of both therapy and imaging at widely separated frequencies, RF image frames were acquired during therapeutic insonation and then exported for off-line analysis. For in vivo studies in a mouse model, before temperature estimation, motion correction was applied between a reference RF frame and subsequent RF frames. Both in vitro and in vivo experiments were examined; in the in vitro and in vivo studies, the average temperature error had a standard deviation of 0.7°C and 0.8°C, respectively. The application of motion correction improved the accuracy of temperature estimation, where the error range was 1.9 to 4.5°C without correction compared with 1.1 to 1.0°C following correction. This study demonstrates the feasibility of combining therapy and monitoring using a commercial system. In the future, real-time temperature estimation will be incorporated into this system.

  View details for PubMedID 21156363

• Copper-Doxorubicin as a Nanoparticle Cargo Retains Efficacy with Minimal Toxicity MOLECULAR PHARMACEUTICS Kheirolomoom, A., Mahakian, L. M., Lai, C., Lindfors, H. A., Seo, J., Paoli, E. E., Watson, K. D., Haynam, E. M., Ingham, E. S., Xing, L., Cheng, R., Borowsky, A. D., Cardiff, R. D., Ferrara, K. W. 2010; 7 (6): 1948–58

  Abstract

  Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multidose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 h (∼40% of the injected dose), indicating stable circulation of the complex. Highly quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 h after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ∼10% ID/g and was enhanced approximately 2-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a premalignant phenotype of ∼(1 mm)(3) or could not be detected.

  View details for PubMedID 20925429

• A model for the dynamics of ultrasound contrast agents in vivo JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Qin, S., Ferrara, K. W. 2010; 128 (3): 1511–21

  Abstract

  The Rayleigh-Plesset (RP) equation for a clean gas bubble in an incompressible and infinite liquid has previously been applied to approximately simulate the behavior of ultrasound contrast agents (UCA) in vivo, and extended RP equations have been proposed to account for the effects of the UCA shell or surrounding soft tissue. These models produce results that are consistent with experimental measurements for low acoustic pressure scenarios. For applications of UCAs in therapeutic medicine, the transmitted acoustic pulse can have a peak negative pressure (PNP) up to a few megapascals, resulting in discrepancies between measurements and predictions using these extended RP equations. Here, a model was developed to describe the dynamics of UCAs in vivo while taking account of the effects of liquid compressibility, the shell and the surrounding tissue. Liquid compressibility is approximated to first order and the shell is treated either as a Voigt viscoelastic solid or a Newtonian viscous liquid. Finite deformation of the shell and tissue is derived. Dynamics of UCAs with a shell of lipid, polymer, albumin and liquid are investigated for typical therapeutic ultrasound pulses. The effects of liquid compressibility and shell and tissue parameters are analyzed.

  View details for PubMedID 20815486

• Liposomal Cu-64 Labeling Method Using Bifunctional Chelators: Poly(ethylene glycol) Spacer and Chelator Effects BIOCONJUGATE CHEMISTRY Seo, J., Mahakian, L. M., Kheirolomoom, A., Zhang, H., Meares, C. F., Ferdani, R., Anderson, C. J., Ferrara, K. W. 2010; 21 (7): 1206–15

  Abstract

  Two bifunctional Cu-64 chelators (BFCs), (6-(6-(3-(2-pyridyldithio)propionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA-PDP) and 4-(2-(2-pyridyldithioethyl)ethanamido)-11-carboxymethyl-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A-PDEA), were synthesized and conjugated to long-circulating liposomes (LCLs) via attachment to a maleimide lipid. An in vitro stability assay of (64)Cu-TETA, (64)Cu-TETA-PEG2k, and (64)Cu-CB-TE2A-PEG2k liposomes showed that more than 86% of the radioactivity remains associated with the liposomal fraction after 48 h of incubation with mouse serum. The in vivo time activity curves (TAC) for the three liposomal formulations showed that approximately 50% of the radioactivity cleared from the blood pool in 16-18 h. As expected, the in vivo biodistribution and TAC data obtained at 48 h demonstrate that the clearance of radioactivity from the liver slows with the incorporation of a poly(ethylene glycol)-2k (PEG2k) brush. Our data suggest that (64)Cu-TETA and (64)Cu-CB-TE2A are similarly stable in the blood pool and accumulation of radioactivity in the liver and spleen is not related to the stability of Cu-64 chelator complex; however, clearance of Cu-64 from the liver and spleen are faster when injected as (64)Cu-TETA-chelated liposomes rather than (64)Cu-CB-TE2A-chelated liposomes.

  View details for PubMedID 20568726

• An optical and microPET assessment of thermally-sensitive liposome biodistribution in the Met-1 tumor model: Importance of formulation JOURNAL OF CONTROLLED RELEASE Paoli, E. E., Kruse, D. E., Seo, J. W., Zhang, H., Kheirolomoom, A., Watson, K. D., Chiu, P., Stahlberg, H., Ferrara, K. W. 2010; 143 (1): 13–22

  Abstract

  The design of delivery vehicles that are stable in circulation but can be activated by exogenous energy sources is challenging. Our goals are to validate new imaging methods for the assessment of particle stability, to engineer stable and activatable particles and to assess accumulation of a hydrophilic model drug in an orthotopic tumor. Here, liposomes were injected into the tail vein of FVB mice containing bilateral Met-1 tumors and imaged in vivo using microPET and optical imaging techniques. Cryo-electron microscopy was applied to assess particle shape prior to injection, ex vivo fluorescence images of dissected tissues were acquired, excised tissue was further processed with a cell-digest preparation and assayed for fluorescence. We find that for a stable particle, in vivo tumor images of a hydrophilic model drug were highly correlated with PET images of the particle shell and ex vivo fluorescence images of processed tissue, R(2)=0.95 and R(2)=0.99 respectively. We demonstrate that the accumulation of a hydrophilic model drug is increased by up to 177 fold by liposomal encapsulation, as compared to accumulation of the drug at 24 hours.

  View details for PubMedID 20006659

• Longitudinal investigation of permeability and distribution of macromolecules in mouse tumor development and malignant transformation using PET Rygh, C. B., Seo, J. W., Qin, S. P., Mahakian, L. M., Zhang, H., Kukis, D. L., Chan, J. Q., Cheng-Liao, J., Reed, R. K., Ferrara, K. W. FEDERATION AMER SOC EXP BIOL. 2010

  View details for Web of Science ID 000208675505600

• Radiolabeled lipids assess liposomal stability in vitro and in vivo Seo, J., Mahakian, L., Kheirolomoom, A., Ferrara, K. W. AMER CHEMICAL SOC. 2010

  View details for Web of Science ID 000208189300584

• A Sensitive TLRH Targeted Imaging Technique for Ultrasonic Molecular Imaging IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Hu, X., Zheng, H., Kruse, D. E., Sutcliffe, P., Stephens, D. N., Ferrara, K. W. 2010; 57 (2): 305–16

  Abstract

  The primary goals of ultrasound molecular imaging are the detection and imaging of ultrasound contrast agents (microbubbles), which are bound to specific vascular surface receptors. Imaging methods that can sensitively and selectively detect and distinguish bound microbubbles from freely circulating microbubbles (free microbubbles) and surrounding tissue are critically important for the practical application of ultrasound contrast molecular imaging. Microbubbles excited by low-frequency acoustic pulses emit wide-band echoes with a bandwidth extending beyond 20 MHz; we refer to this technique as transmission at a low frequency and reception at a high frequency (TLRH). Using this wideband, transient echo, we have developed and implemented a targeted imaging technique incorporating a multifrequency colinear array and the Siemens Antares imaging system. The multifrequency colinear array integrates a center 5.4-MHz array, used to receive echoes and produce radiation force, and 2 outer 1.5-MHz arrays used to transmit low-frequency incident pulses. The targeted imaging technique makes use of an acoustic radiation force subsequence to enhance accumulation and a TLRH imaging subsequence to detect bound microbubbles. The radiofrequency (RF) data obtained from the TLRH imaging subsequence are processed to separate echo signatures between tissue, free microbubbles, and bound microbubbles. By imaging biotin-coated microbubbles targeted to avidin-coated cellulose tubes, we demonstrate that the proposed method has a high contrast-to-tissue ratio (up to 34 dB) and a high sensitivity to bound microbubbles (with the ratio of echoes from bound microbubbles versus free microbubbles extending up to 23 dB). The effects of the imaging pulse acoustic pressure, the radiation force subsequence, and the use of various slow-time filters on the targeted imaging quality are studied. The TLRH targeted imaging method is demonstrated in this study to provide sensitive and selective detection of bound microbubbles for ultrasound molecularly targeted imaging.

  View details for PubMedID 20178897

  View details for PubMedCentralID PMC2892275

• Enhanced in vivo bioluminescence imaging using liposomal luciferin delivery system JOURNAL OF CONTROLLED RELEASE Kheirolomoom, A., Kruse, D. E., Qin, S., Watson, K. E., Lai, C., Young, L. T., Cardiff, R. D., Ferrara, K. W. 2010; 141 (2): 128–36

  Abstract

  To provide a continuous and prolonged delivery of the substrate D-luciferin for bioluminescence imaging in vivo, luciferin was encapsulated into liposomes using either the pH gradient or acetate gradient method. Under optimum loading conditions, 0.17 mg luciferin was loaded per mg of lipid with 90-95% encapsulation efficiency, where active loading was 6 to 18-fold higher than that obtained with passive loading. Liposomal luciferin in a long-circulating formulation had good shelf stability, with 10% release over 3-month storage at 4 degrees C. Pharmacokinetic profiles of free and liposomal luciferin were then evaluated in transgenic mice expressing luciferase. In contrast to rapid in vivo clearance of free luciferin (t(1/2)=3.54 min), luciferin encapsulated into long-circulating liposomes showed a prolonged release over 24h. The first-order release rate constant of luciferin from long-circulating liposomes, as estimated from the best fit of the analytical model to the experimental data, was 0.01 h(-1). Insonation of luciferin-loaded temperature-sensitive liposomes directly injected into one tumor of Met1-luc tumor-bearing mice resulted in immediate emission of light. Systemic injection of luciferin-loaded long-circulating liposomes into Met1-luc tumor-bearing mice, followed by unilateral ultrasound-induced hyperthermia, produced a gradual increase in radiance over time, reaching a peak at 4-7 h post-ultrasound.

  View details for PubMedID 19748536

• An Imaging-Driven Model for Liposomal Stability and Circulation MOLECULAR PHARMACEUTICS Qin, S., Seo, J., Zhang, H., Qi, J., Curry, F. E., Ferrara, K. W. 2010; 7 (1): 12–21

  Abstract

  Simultaneous labeling of the drug compartment and shell of delivery vehicles with optical and positron emission tomography (PET) probes is developed and employed to inform a hybrid physiologically based pharmacokinetic model. Based on time-dependent estimates of the concentration of these tracers within the blood pool, reticuloendothelial system (RES) and tumor interstitium, we compare the stability and circulation of long-circulating and temperature-sensitive liposomes. We find that rates of transport to the RES for long-circulating and temperature-sensitive particles are 0.046 and 0.19 h(-1), respectively. Without the application of exogenous heat, the rates of release from the long-circulating and temperature-sensitive particles circulating within the blood pool are 0.003 and 0.2 h(-1), respectively. Prolonged lifetime in circulation and slow drug release from liposomes result in a significantly greater drug area under the curve for the long-circulating particles. Future studies will couple these intrinsic parameters with exogenous heat-based release. Finally, we develop a transport constant for the transport of liposomes from the blood pool to the tumor interstitium, which is on the order of 0.01 h(-1) for the Met-1 tumor system.

  View details for PubMedID 19621944

• Spatial and Temporal-Controlled Tissue Heating on a Modified Clinical Ultrasound Scanner for Generating Mild Hyperthermia in Tumors IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Kruse, D. E., Lai, C., Stephens, D. N., Sutcliffe, P., Paoli, E. E., Barnes, S. H., Ferrara, K. W. 2010; 57 (1): 155–66

  Abstract

  A new system is presented for generating controlled tissue heating with a clinical ultrasound scanner, and initial in vitro and in vivo results are presented that demonstrate both transient and sustained heating in the mild-hyperthermia range of 37 ( degrees )C-42 ( degrees )C. The system consists of a Siemens Antares ultrasound scanner, a custom dual-frequency three-row transducer array and an external temperature feedback control system. The transducer has two outer rows that operate at 1.5 MHz for tissue heating and a center row that operates at 5 MHz for B-mode imaging to guide the therapy. We compare the field maps obtained using a hydrophone against calculations of the ultrasound beam based on monochromatic and linear assumptions. Using the finite-difference time-domain (FDTD) method, we compare predicted time-dependent thermal profiles to measured profiles for soy tofu as a tissue-mimicking phantom. In vitro results show differential heating of 6 ( degrees )C for chicken breast and tofu. In vivo tests of the system were performed on three mice bearing Met-1 tumors, which is a model of aggressive, metastatic, and highly vascular breast cancer. In superficially implanted tumors, we demonstrate controlled heating to 42 ( degrees )C. We show that the system is able to maintain the temperature to within 0.1 ( degrees )C of the desired temperature both in vitro and in vivo.

  View details for PubMedID 20064754

• Motion Corrected Cadence CPS Ultrasound for Quantifying Response to Vasoactive Drugs in a Rat Kidney Model UROLOGY Pollard, R. E., Dayton, P. A., Watson, K. D., Hu, X., Guracar, I. M., Ferrara, K. W. 2009; 74 (3): 675–81

  Abstract

  To establish the ability of contrast-enhanced motion corrected cadence pulse sequencing (CPS) to detect changes in renal blood flow induced by vasoactive substances in rats.Ultrasound contrast media was administered as a constant rate infusion into a phantom at a known rate and CPS data acquired. Rats were anesthetized and predrug CPS estimates of replenishment rate were made for the right kidney. Real-time motion correction was applied, and parametric images were generated from the CPS data. Group 1 rats (n = 7) were administered a vasodilator and group 2 rats (n = 3) were administered a vasoconstrictor. The CPS imaging of the kidney was repeated after ample time for drug effects to occur.Contrast CPS accurately estimated flow velocity in the phantom model. In addition, CPS defined statistically significant differences between pre- and postdrug blood flow in the renal medulla (vasodilator, P < .01; vasoconstrictor, P < .0001) and cortex (vasoconstrictor, P < .0001).We conclude that motion-corrected CPS ultrasound provides real-time quantification of renal blood flow alterations and may prove useful for the assessment of blood flow in transplanted kidneys.

  View details for PubMedID 19589583

• Motion Corrected Cadence CPS Ultrasound for Quantifying Response to Vasoactive Drugs in a Rat Kidney Model REPLY UROLOGY Pollard, R. E., Ferrara, K. W. 2009; 74 (3): 682

  View details for DOI 10.1016/j.urology.2009.03.020

  View details for Web of Science ID 000270207100071

• BIOL 63 - Temperature triggered release of payload covalently bound to liposomes by flip-flop mechanism Day, J. J., Paoli, E., Ferrara, K. W., Meares, C. F. AMER CHEMICAL SOC. 2009: 867

  View details for Web of Science ID 000207861900779

• Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering (vol 54, pg R27, 2009) PHYSICS IN MEDICINE AND BIOLOGY Qin, S., Caskey, C. F., Ferrara, K. W. 2009; 54 (14): 4621

  View details for DOI 10.1088/0031-9155/54/14/C01

  View details for Web of Science ID 000267770100020

• Systems and Probes for Ultrasound Molecular Imaging Ferrara, K., Hu, X., Zhang, H., Stephens, D., Kruse, D. WILEY. 2009: 2780-+

  View details for DOI 10.1118/1.3182548

  View details for Web of Science ID 000411593200011

• Microbubble tunneling in gel phantoms JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Caskey, C. F., Qin, S., Dayton, P. A., Ferrara, K. W. 2009; 125 (5): EL183–EL189

  Abstract

  Insonified microbubbles were observed in vessels within a gel with a Young's modulus similar to that of tissue, demonstrating shape instabilities, liquid jets, and the formation of small tunnels. In this study, tunnel formulation occurred in the direction of the propagating ultrasound wave, where radiation pressure directed the contact of the bubble and gel, facilitating the activity of the liquid jets. Combinations of ultrasonic parameters and microbubble concentrations that are relevant for diagnostic imaging and drug delivery and that lead to tunnel formation were applied and the resulting tunnel formation was quantified.

  View details for PubMedID 19425620

• Angiogenic Response to Bioactive Glass Promotes Bone Healing in an Irradiated Calvarial Defect TISSUE ENGINEERING PART A Leu, A., Stieger, S. M., Dayton, P., Ferrara, K. W., Leach, J. 2009; 15 (4): 877–85

  Abstract

  Localized radiation is an effective treatment modality for carcinomas, yet the associated reduction of the host vasculature significantly inhibits the tissue's regenerative capacity. Low concentrations of bioactive glass (BG) possess angiogenic potential, and we hypothesized that localized BG presentation would increase neovascularization and promote healing in an irradiated bone defect. An isolated calvarial region of Sprague-Dawley rats was irradiated 2 weeks before surgery. Bilateral critical-sized defects were created and immediately filled with a BG-loaded collagen sponge or an empty sponge as an internal control. Histological analysis of calvaria collected after 2 weeks demonstrated greater neovascularization within the defect in the presence of BG than with collagen alone. Noninvasive ultrasound imaging at 4 weeks detected less contrast agent in the brain below BG-treated defects than in the nearby untreated defects and images of treated defects acquired at 2 weeks. The reduced ability to detect contrast agent in BG-treated defects suggested greater attenuation of ultrasound signal due to early bone formation. Micro-computed tomography imaging at 12 weeks demonstrated significantly greater bone volume fraction within BG-treated defects than in controls. These results suggest that neovascularization induced by localized BG delivery promotes bone regeneration in this highly compromised model of bone healing and may offer an alternative approach to costly growth factors and their potential side-effects.

  View details for PubMedID 18795867

• Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering PHYSICS IN MEDICINE AND BIOLOGY Qin, S., Caskey, C. F., Ferrara, K. W. 2009; 54 (6): R27–R57

  Abstract

  Microbubble contrast agents and the associated imaging systems have developed over the past 25 years, originating with manually-agitated fluids introduced for intra-coronary injection. Over this period, stabilizing shells and low diffusivity gas materials have been incorporated in microbubbles, extending stability in vitro and in vivo. Simultaneously, the interaction of these small gas bubbles with ultrasonic waves has been extensively studied, resulting in models for oscillation and increasingly sophisticated imaging strategies. Early studies recognized that echoes from microbubbles contained frequencies that are multiples of the microbubble resonance frequency. Although individual microbubble contrast agents cannot be resolved-given that their diameter is on the order of microns-nonlinear echoes from these agents are used to map regions of perfused tissue and to estimate the local microvascular flow rate. Such strategies overcome a fundamental limitation of previous ultrasound blood flow strategies; the previous Doppler-based strategies are insensitive to capillary flow. Further, the insonation of resonant bubbles results in interesting physical phenomena that have been widely studied for use in drug and gene delivery. Ultrasound pressure can enhance gas diffusion, rapidly fragment the agent into a set of smaller bubbles or displace the microbubble to a blood vessel wall. Insonation of a microbubble can also produce liquid jets and local shear stress that alter biological membranes and facilitate transport. In this review, we focus on the physical aspects of these agents, exploring microbubble imaging modes, models for microbubble oscillation and the interaction of the microbubble with the endothelium.

  View details for PubMedID 19229096

  View details for PubMedCentralID PMC2818980

• Imaging nanoparticle stability and activation in vivo Ferrara, K. W., Seo, J. W., Zhang, H., IEEE IEEE. 2009: 4580-+

  Abstract

  While liposomes and nanoparticles have been the subject of intense research for more than 40 years, few particles have been translated into clinical practice. Advantages of these particles include the potential to overcome the cardiac, renal or neural toxicity of systemic chemotherapy, the opportunities for multivalent targeting, the gradual yet significant accumulation within tumors due to leaky blood vessels and the myriad of new approaches to locally alter the properties of the particle in the region of interest. Given the complexity of the design and co-optimization of the surface architecture, shell formulation and drug loading, methods to image the pharmacokinetics of nanoparticles in living systems are an essential part of an efficient research methodology. Here, we describe our efforts to label the shell and drug core of lipid-shelled particles with a goal of facilitating translation of activatable particles.

  View details for PubMedID 19963843

• Ultrasound Mediated Drug Delivery: The effect of microbubbles on a gel boundary Caskey, C. F., Qin, S., Ferrara, K. W., IEEE IEEE. 2009: 134-+

  Abstract

  When microbubble contrast agents are driven by ultrasound, the transport of drugs and particles across cell membranes and blood vessel walls is enhanced. While a wide range of acoustic parameters enhance delivery, the acoustic parameters that maximize delivery while simultaneously minimizing biological effects have not been fully characterized. Here, we use a gel phantom with a Young's modulus similar to that of tissue to directly observe bubble interaction with the gel surface during insonation. Using parameters relevant to diagnostic imaging and drug delivery, we observe fluid jets that impinge on the surface and tunnels that follow the sound beam axis.

  View details for DOI 10.1109/IEMBS.2009.5335240

  View details for Web of Science ID 000280543600035

  View details for PubMedID 19965123

• COPPER-64 LABELING OF LIPOSOMES: POST-LABELING METHOD USING BIFUNCTIONAL LIGAND (BFL) Seo, J., Meares, C. F., Ferrara, K. W. JOHN WILEY & SONS LTD. 2009: S100

  View details for Web of Science ID 000268724900101

• A Novel Method to Label Preformed Liposomes with (CU)-C-64 for Positron Emission Tomography (PET) Imaging BIOCONJUGATE CHEMISTRY Seo, J., Zhang, H., Kukis, D. L., Meares, C. F., Ferrara, K. W. 2008; 19 (12): 2577–84

  Abstract

  Radiolabeling of liposomes with 64Cu (t(1/2)=12.7 h) is attractive for molecular imaging and monitoring drug delivery. A simple chelation procedure, performed at a low temperature and under mild conditions, is required to radiolabel preloaded liposomes without lipid hydrolysis or the release of the encapsulated contents. Here, we report a 64Cu postlabeling method for liposomes. A 64Cu-specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT), was conjugated with an artificial lipid to form a BAT-PEG-lipid. After incorporation of 0.5% (mol/mol) BAT-PEG-lipid during liposome formulation, liposomes were successfully labeled with 64Cu in 0.1 M NH4OAc pH 5 buffer at 35 degrees C for 30-40 min with an incorporation yield as high as 95%. After 48 h of incubation of 64Cu-liposomes in 50/50 serum/PBS solution, more than 88% of the 64Cu label was still associated with liposomes. After injection of liposomal 64Cu in a mouse model, 44+/-6.9, 21+/-2.7, 15+/-2.5, and 7.4+/-1.1 (n=4) % of the injected dose per cubic centimeter remained within the blood pool at 30 min, 18, 28, and 48 h, respectively. The biodistribution at 48 h after injection verified that 7.0+/-0.47 (n=4) and 1.4+/-0.58 (n=3) % of the injected dose per gram of liposomal 64Cu and free 64Cu remained in the blood pool, respectively. Our results suggest that this fast and easy 64Cu labeling of liposomes could be exploited in tracking liposomes in vivo for medical imaging and targeted delivery.

  View details for PubMedID 18991368

• Short-duration-focused ultrasound stimulation of Hsp70 expression in vivo (vol 53, pg 3641, 2008) PHYSICS IN MEDICINE AND BIOLOGY Kruse, D. E., Mackanos, M. A., O'Connell-Rodwell, C. E., Contag, C. H., Ferrara, K. W. 2008; 53 (22): 6639

  View details for DOI 10.1088/0031-9155/53/22/C01

  View details for Web of Science ID 000260758400022

• Dynamic microPET imaging of ultrasound contrast agents and lipid delivery JOURNAL OF CONTROLLED RELEASE Tartis, M. S., Kruse, D. E., Zheng, H., Zhang, H., Kheirolomoom, A., Marik, J., Ferrara, K. W. 2008; 131 (3): 160–66

  Abstract

  Interest in ultrasound contrast agents (lipid-shelled microbubbles) as delivery vehicles is increasing; however, the biodistribution of these agents remains uncharacterized, both with and without ultrasound. In this study, an (18)F-labeled lipid ([(18)F]fluorodipalmitin), incorporated in microbubble shells, was used as a dynamic microPET probe for quantitative 90-minute biodistribution measurements in male Fischer 344 rats (n=2). The spleen retained the highest concentration of radioactive lipid at approximately 2.6%-injected dose per cubic centimeter (% ID/cc) and the liver demonstrated the largest total accumulation (approximately 17% ID). The microbubble pharmacokinetic profile differed from free lipid, which is rapidly cleared from blood, and liposomes, which remain in circulation. Additionally, region of interest (ROI) analysis over 60 minutes (post-ultrasound treatment) quantified the delivery of lipid by therapeutic ultrasound from microbubbles to kidney tissue (n=8). The ultrasound sequence consisted of a 200 kPa, 5.3 MHz radiation force pulse followed by a 1.6 MPa, 1.4 MHz fragmentation pulse and was applied to one kidney, while the contralateral kidney served as a control. ROI-estimated activity in treated kidneys was slightly but significantly greater at 0 and 60 min than in untreated kidneys (p=0.0012 and 0.0035, respectively). This effect increased with the number of microbubbles injected (p=0.006). In summary, [(18)F]fluorodipalmitin was used to characterize the biodistribution of contrast microbubble shells and the deposition of lipid was shown to be locally increased after insonation.

  View details for PubMedID 18718854

• Efficient array design for sonotherapy PHYSICS IN MEDICINE AND BIOLOGY Stephens, D. N., Kruse, D. E., Ergun, A. S., Barnes, S., Lu, X., Ferrara, K. W. 2008; 53 (14): 3943–69

  Abstract

  New linear multi-row, multi-frequency arrays have been designed, constructed and tested as fully operational ultrasound probes to produce confocal imaging and therapeutic acoustic intensities with a standard commercial ultrasound imaging system. The triple-array probes and imaging system produce high quality B-mode images with a center row imaging array at 5.3 MHz and sufficient acoustic power with dual therapeutic arrays to produce mild hyperthermia at 1.54 MHz. The therapeutic array pair in the first probe design (termed G3) utilizes a high bandwidth and peak pressure, suitable for mechanical therapies. The second multi-array design (termed G4) has a redesigned therapeutic array pair which is optimized for a high time-averaged power output suitable for mild hyperthermia applications. The 'thermal therapy' design produces more than 4 W of acoustic power from the low-frequency arrays with only a 10.5 degrees C internal rise in temperature after 100 s of continuous use with an unmodified conventional imaging system or substantially longer operation at lower acoustic power. The low-frequency arrays in both probe designs were examined and contrasted for real power transfer efficiency with a KLM model which includes all lossy contributions in the power delivery path from system transmitters to the tissue load. Laboratory verification was successfully performed for the KLM-derived estimates of transducer parallel model acoustic resistance and dissipation resistance, which are the critical design factors for acoustic power output and undesired internal heating, respectively.

  View details for PubMedID 18591737

• Short-duration-focused ultrasound stimulation of Hsp70 expression in vivo PHYSICS IN MEDICINE AND BIOLOGY Kruse, D. E., Mackanos, M. A., O'Connell-Rodwell, C. E., Contag, C. H., Ferrara, K. W. 2008; 53 (13): 3641-3660

  Abstract

  The development of transgenic reporter mice and advances in in vivo optical imaging have created unique opportunities to assess and analyze biological responses to thermal therapy directly in living tissues. Reporter mice incorporating the regulatory regions from the genes encoding the 70 kDa heat-shock proteins (Hsp70) and firefly luciferase (luc) as reporter genes can be used to non-invasively reveal gene activation in living tissues in response to thermal stress. High-intensity-focused ultrasound (HIFU) can deliver measured doses of acoustic energy to highly localized regions of tissue at intensities that are sufficient to stimulate Hsp70 expression. We report activation of Hsp70-luc expression using 1 s duration HIFU heating to stimulate gene expression in the skin of the transgenic reporter mouse. Hsp70 expression was tracked for 96 h following the application of 1.5 MHz continuous-wave ultrasound with spatial peak intensities ranging from 53 W cm(-2) up to 352 W cm(-2). The results indicated that peak Hsp70 expression is observed 6-48 h post-heating, with significant activity remaining at 96 h. Exposure durations were simulated using a finite-element model, and the predicted temperatures were found to be consistent with the observed Hsp70 expression patterns. Histological evaluation revealed that the thermal damage starts at the stratum corneum and extends deeper with increasing intensity. These results indicated that short-duration HIFU may be useful for inducing heat-shock expression, and that the period between treatments needs to be greater than 96 h due to the protective properties of Hsp70.

  View details for DOI 10.1088/0031-9155/53/13/017

  View details for PubMedID 18562783

• Driving delivery vehicles with ultrasound ADVANCED DRUG DELIVERY REVIEWS Ferrara, K. W. 2008; 60 (10): 1097–1102

  Abstract

  Therapeutic applications of ultrasound have been considered for over 40 years, with the mild hyperthermia and associated increases in perfusion produced by ultrasound harnessed in many of the earliest treatments. More recently, new mechanisms for ultrasound-based or ultrasound-enhanced therapies have been described, and there is now great momentum and enthusiasm for the clinical translation of these techniques. This dedicated issue of Advanced Drug Delivery Reviews, entitled "Ultrasound for Drug and Gene Delivery," addresses the mechanisms by which ultrasound can enhance local drug and gene delivery and the applications that have been demonstrated at this time. In this commentary, the identified mechanisms, delivery vehicles, applications and current bottlenecks for translation of these techniques are summarized.

  View details for PubMedID 18479775

  View details for PubMedCentralID PMC2811088

• Transmitted ultrasound pressure variation in micro blood vessel phantoms ULTRASOUND IN MEDICINE AND BIOLOGY Qin, S., Kruse, D. E., Ferrara, K. W. 2008; 34 (6): 1014–20

  Abstract

  Silica, cellulose and polymethylmethacrylate tubes with inner diameters of ten to a few hundred microns are commonly used as blood vessel phantoms in in vitro studies of microbubble or nanodroplet behavior during insonation. However, a detailed investigation of the ultrasonic fields within these micro-tubes has not yet been performed. This work provides a theoretical analysis of the ultrasonic fields within micro-tubes. Numerical results show that for the same tube material, the interaction between the micro-tube and megaHertz-frequency ultrasound may vary drastically with incident frequency, tube diameter and wall thickness. For 10 MHz ultrasonic insonation of a polymethylmethacrylate (PMMA) tube with an inner diameter of 195 microm and an outer diameter of 260 microm, the peak pressure within the tube can be up to 300% of incident pressure amplitude. However, using 1 MHz ultrasound and a silica tube with an inner diameter of 12 microm and an outer diameter of 50 microm, the peak pressure within the tube is only 12% of the incident pressure amplitude and correspondingly, the spatial-average-time-average intensity within the tube is only 1% of the incident intensity.

  View details for PubMedID 18395962

• Dynamic imaging of arginine-rich heart-targeted vehicles in a mouse model BIOMATERIALS Zhang, H., Kusunose, J., Kheirolomoom, A., Seo, J. W., Qi, J., Watson, K. D., Lindfors, H. A., Ruoslahti, E., Sutcliffe, J. L., Ferrara, K. W. 2008; 29 (12): 1976–88

  Abstract

  Efficacious delivery of drugs and genes to the heart is an important goal. Here, a radiolabeled peptide-targeted liposome was engineered to bind to the heart, and the biodistribution and pharmacokinetics were determined by dynamic positron emission tomography in the FVB mouse. Efficient targeting occurred only with an exposed ligand and a dense concentration of peptide (6000 peptides/particles). Liposomes targeted with CRPPR or other arginine-rich peptides with an exposed guanidine moiety bound within 100 s after intravenous injection and remained stably bound. With CRPPR-targeted particles, the radioisotope density in the heart averaged 44 +/- 9% injected dose/gram of tissue, more than 30-fold higher than in skeletal muscle. The rapid and efficient targeting of these particles can be exploited in drug and gene delivery systems and with dynamic positron emission tomography provides a model system to optimize targeting of engineered particles.

  View details for PubMedID 18255141

• A stimulus-responsive contrast agent for ultrasound molecular imaging BIOMATERIALS Borden, M. A., Zhang, H., Gillies, R. J., Dayton, P. A., Ferrara, K. W. 2008; 29 (5): 597–606

  Abstract

  Complement activation by targeting ligands is an important issue that governs the fate of targeted colloidal contrast agents for molecular imaging. Here, we extend previous work on a stimulus-responsive microbubble construct, in which the ligand is normally buried by a polymeric overbrush and transiently revealed by ultrasound radiation force, to show reduced complement activation and focused adhesion to cells using a physiological peptide ligand. Attachment of C3/C3b in vitro and production of soluble C3a anaphylotoxin in vitro and in vivo decreased significantly for the buried-ligand architecture vs. the conventional exposed-ligand motif and no-ligand control. Additionally, the buried-ligand architecture prevented adhesion of Arg-Gly-Tyr (RGD)-bearing microbubbles to integrin-expressing human umbilical vein endothelial cells (HUVEC) when driven by buoyancy in a static chamber, but it did not affect adhesion efficiency when activated by ultrasound radiation force pulses. These results show, for the first time, the molecular mechanism for reduced immunogenicity for the buried-ligand architecture and feasibility of targeting with this motif using a physiological ligand-receptor pair.

  View details for PubMedID 17977595

  View details for PubMedCentralID PMC2266628

• Parameter space for microbubble wall interaction estimated from gel phantom Caskey, C. F., Qin, S., Dayton, P. A., Ferrara, K. W., IEEE IEEE. 2008: 341-+

  View details for DOI 10.1109/ULTSYM.2008.0084

  View details for Web of Science ID 000268845800084

• Thermal Efficiency in Sonotherapy Array Design Stephens, D. N., Kruse, D. E., Lai, C., Ergun, A. S., Barnes, S., Ferrara, K. W., IEEE IEEE. 2008: 666-+

  View details for DOI 10.1109/ULTSYM.2008.0160

  View details for Web of Science ID 000268845800160

• Imaging of angiogenesis using Cadence (TM) contrast pulse sequencing and targeted contrast agents CONTRAST MEDIA & MOLECULAR IMAGING Stieger, S. M., Dayton, P. A., Borden, M. A., Caskey, C. F., Griffey, S. M., Wisner, E. R., Ferrara, K. W. 2008; 3 (1): 9–18

  Abstract

  Low-power multipulse contrast ultrasound imaging provides a promising tool to quantify angiogenesis noninvasively when used with contrast agents targeted to vascular markers expressed by the angiogenic endothelium. Targeted ultrasound contrast agents, with a diameter on the order of micrometers, cannot extravasate and therefore are targeted solely to receptors expressed by the vascular endothelium. The aim of this study was to evaluate the potential of a low-power multipulse imaging sequence, Cadence(TM) contrast pulse sequencing (CPS), combined with targeted contrast agents to quantify angiogenesis.Targeted microbubbles were prepared by conjugating echistatin via biotin-avidin linkage to the surface of a phospholipid microbubble shell. The density of echistatin present on the shell was confirmed with flow-cytometry and quantified by total fluorescence. The binding of targeted microbubbles was evaluated in vitro by quantifying the adherence of targeted microbubbles to rat aortic endothelial cells, compared with control (nontargeted) microbubbles. The circulation time and adherence of targeted microbubbles was evaluated in vivo in a Matrigel model in rats and compared with control microbubbles using CPS in addition to a destructive ultrasound pulse.Using only the low-power CPS pulse, the echo intensity produced in the neovasculature of the Matrigel pellet was significantly greater with targeted microbubbles than with the control contrast agent (p < 0.001). Combining CPS with the destructive pulse, the processed image was significantly different in intensity (p < 0.001) and spatial extent between targeted and control agents (p < 0.001). When the morphology of the histological sample and ultrasound image correlated, the microvessel density count and the percentage of the circular area enhanced by ultrasound were correlated (p < 0.05).Low-power multipulse imaging in combination with targeted echistatin-bearing microbubbles facilitated a noninvasive, quantitative evaluation of early angiogenesis during real-time imaging. The addition of high-intensity destructive pulses facilitated estimation of the spatial extent of angiogenesis.

  View details for PubMedID 18335479

• A Sensitive Ultrasonic Imaging method for Targeted Contrast Microbubble Detection Zheng, H., Kruse, D. E., Stephens, D. N., Ferrara, K. W., Sutcliffe, P., Gardner, E., IEEE IEEE. 2008: 5290-+

  Abstract

  We have recently developed a targeted imaging technique for selective and sensitive ultrasound molecular imaging by taking advantage of wideband transient high frequency acoustic emission from ultrasound contrast agents. The imaging modality makes use of a novel multi-frequency co-linear array (two outer 1.4 MHz and one center 5.3 MHz arrays) transducer integrated with the Siemens AntaresSystem. The imaging sequence includes a B-mode imaging pulse sequence in which a short pulse is transmitted with the outer low frequency arrays and received with the inner high frequency array (TLRH: transmit at low frequency and receive at high frequency), followed by a long radiation force pulse to induce immediate bubble adhesion using the center array, and a second B-mode imaging pulse sequence. The RF data obtained from the second B-mode pulse sequence are averaged and then subtracted from the first B-mode sequence. The imaging technique was tested in a targeted imaging phantom, where lipid-shelled biotin microbubbles flow within an avidin coated-cellulose. Results showed that tissue signals were suppressed up to 33 dB and a targeted bubble contrast-to-free bubble signal ratio of up to 23 dB was obtained from the composite sequence imaging.

  View details for PubMedID 19163911

• Ultrasound-driven microbubble oscillation and translation within small phantom vessels ULTRASOUND IN MEDICINE AND BIOLOGY Zheng, H., Dayton, P. A., Caskey, C., Zhao, S., Qin, S., Ferrara, K. W. 2007; 33 (12): 1978–87

  Abstract

  The use of ultrasound radiation force to manipulate microbubbles in blood vessels has attracted recent interest as a method to increase the efficiency of ultrasonic molecular imaging and drug delivery. However, recent studies indicate that microbubble oscillation is diminished within small blood vessels, and therefore we investigate microbubble oscillation and translation within 12 microm vessels using high-speed photography. With each 0.1- to 1-MPa ultrasound pulse, microbubbles (radius of 1, 1.5 and 2 microm) within 12 microm tubes translate 5 to 10 times less than those within 200 microm tubes. Application of a pulse train with a high pulse repetition frequency displaces bubbles to the wall of 12- and 200-microm tubes within an interval ( approximately 1 s) that is reasonable for clinical translation. Modeling of coupled oscillation and translation for unconstrained microbubbles, based on a modified Rayleigh-Plesset (RP) and the trajectory equations, is compared with experimental observations and demonstrates agreement for the larger displacements observed within the 200 microm tubes. This study has implications for contrast-assisted ultrasound applications, aiding the manipulation of targeted microbubbles and for further theoretical understanding of the complex bubble dynamics within constrained vessel.

  View details for PubMedID 17900793

• Cholesterol transport from liposomal delivery vehicles BIOMATERIALS Kheirolomoom, A., Ferrara, K. W. 2007; 28 (29): 4311-4320

  Abstract

  Rapid internalization of drugs from delivery vehicles via non-endocytotic pathways is an important goal. The transport of imaging probes attached to cholesterol and introduced via a liposomal formulation is considered here, in order to evaluate the intracellular distribution and kinetics of small molecular cargo that might be attached to cholesterol or phospholipids. The internalization efficiencies of two fluorescent cholesterol analogues, one carrying a fluorophore on the head of the cholesterol molecule 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (BODIPY)-cholesteryl ester (CE) (BODIPY-CE) and the other on the tail (25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl]amino]-27-norcholesterol (NBD-cholesterol)), were compared with those of other phospholipid molecules (NBD-phosphatidylcholine (PC) and NBD-phosphatidylethanolamine (PE)) using a liposomal formulation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 85.5 M%; 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2k), 9.5 M%; fluorescent analogue, 5 M%). The rate and transfer efficiency were NBD-cholesterol>BODIPY-CE>NBD-PC>NBD-PE. NBD-cholesterol, delivered by liposomes with an average diameter of 100 nm, localized in the perinuclear region and lipid storage droplets, with transfer observed in as little as 5 min. NBD-cholesterol transport was approximately constant with time, suggesting a unidirectional mode of entry. In the absence of PEG within the liposome, the transfer rate decreased. Filipin, a caveolae-blocking agent, caused 70% inhibition of cholesterol internalization in treated cells, suggesting that cholesterol internalization follows a caveolae-mediated pathway.

  View details for DOI 10.1016/j.biomaterials.2007.06.008

  View details for PubMedID 17610949

• Molecular imaging update - ultrasound molecular imaging: on the move JOURNAL OF NUCLEAR MEDICINE Ferrara, K. 2007; 48 (9): 22N

  View details for Web of Science ID 000252894700002

• DNA and polylysine adsorption and multilayer construction onto cationic lipid-coated microbubbles LANGMUIR Borden, M. A., Caskey, C. F., Little, E., Gillies, R. J., Ferrara, K. W. 2007; 23 (18): 9401-9408

  Abstract

  We report on a novel application of the layer-by-layer (LbL) assembly technique to attach multiple layers of DNA and poly-l-lysine (PLL) onto preformed lipid-coated microbubbles to increase the DNA loading capacity. We first measured the effects of the cationic lipid fraction and salt concentration on the microbubble stability. Microbubble production and stability were robust up to a cationic lipid fraction of 40 mol % in 10 mM NaCl. DNA adsorption was heterogeneous over the microbubble shell and occurred primarily on the condensed phase domains. The amount of adsorbed DNA, and subsequently adsorbed PLL, increased linearly with the fraction of cationic lipid in the shell. DNA loading was further enhanced by the LbL assembly method to construct polyelectrolyte multilayers (PEMs) of DNA and PLL. PEM buildup was demonstrated by experimental results from zeta potential analysis, fluorescence microscopy, UV spectroscopy, and flow cytometry. The PEMs exhibited two growth stages and were heterogeneously distributed over the microbubble surface. The DNA loading capacity onto the microbubbles was enhanced by over 10-fold by using five paired layers. However, the PEM shell did not prevent oscillation or destruction during ultrasound insonification. These results suggest that the surface can be compartmentalized to make multifunctional, high-payload ultrasound contrast agents for targeted gene therapy.

  View details for DOI 10.1021/la7009034

  View details for PubMedID 17665937

• Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Caskey, C. F., Stieger, S. M., Qin, S., Dayton, P. A., Ferrara, K. W. 2007; 122 (2): 1191–1200

  Abstract

  Many thousands of contrast ultrasound studies have been conducted in clinics around the world. In addition, the microbubbles employed in these examinations are being widely investigated to deliver drugs and genes. Here, for the first time, the oscillation of these microbubbles in small vessels is directly observed and shown to be substantially different than that predicted by previous models and imaged within large fluid volumes. Using pulsed ultrasound with a center frequency of 1 MHz and peak rarefactional pressure of 0.8 or 2.0 MPa, microbubble expansion was significantly reduced when microbubbles were constrained within small vessels in the rat cecum (p<0.05). A model for microbubble oscillation within compliant vessels is presented that accurately predicts oscillation and vessel displacement within small vessels. As a result of the decreased oscillation in small vessels, a large resting microbubble diameter resulting from agent fusion or a high mechanical index was required to bring the agent shell into contact with the endothelium. Also, contact with the endothelium was observed during asymmetrical collapse, not during expansion. These results will be used to improve the design of drug delivery techniques using microbubbles.

  View details for PubMedID 17672665

• The natural frequency of nonlinear oscillation of ultrasound contrast agents in microvessels ULTRASOUND IN MEDICINE AND BIOLOGY Qin, S., Ferrara, K. W. 2007; 33 (7): 1140-1148

  Abstract

  Ultrasound contrast agents (UCAs) are under intensive investigation for their applications in physiological and molecular imaging and drug delivery. Prediction of the natural frequency of the oscillation of UCAs in microvessels has drawn increasing attention. To our knowledge, the existing models to predict the natural frequency of oscillation of UCAs in microvessels all apply the linear approximation and treat the blood vessel wall as a rigid boundary. In the potential applications of ultrasound imaging drug and gene delivery, the compliance of small vessels may play an important role in the bubble's oscillation. The goal of this work is to provide a lumped-parameter model to study the natural frequency of nonlinear oscillation of UCAs in microvessels. Three types of the blood vessel conditions have been considered: i.e., rigid vessels, normal compliable vessels and vessels with increasing stiffness that could correspond to tumor vasculature. The corresponding bubble oscillation frequencies in vessels with a radius less than 100 microm are examined in detail. When a bubble with a radius of 4 microm is confined in a compliable vessel (inner radius 5 microm and length 100 microm), the natural frequency of bubble oscillation increases by a factor of 1.7 compared with a bubble in an unbounded field. The natural frequency of oscillation of a bubble in a compliable vessel increases with decreasing vessel size while decreasing with increasing values of vessel rigidity. This model suggests that contrast agent size, blood vessel size distribution and the type of vasculature should comprehensively be considered for choosing the transmitted frequency in ultrasound contrast imaging and drug delivery.

  View details for DOI 10.1016/j.ultrasmedbio.2006.12.009

  View details for PubMedID 17478030

• Acoustically-active microbubbles conjugated to liposomes: Characterization of a proposed drug delivery vehicle JOURNAL OF CONTROLLED RELEASE Kheirolomoom, A., Dayton, P. A., Lum, A. F., Little, E., Paoli, E. E., Zheng, H., Ferrara, K. W. 2007; 118 (3): 275-284

  Abstract

  A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes, each containing 5% DSPE-PEG2kBiotin, was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. High-speed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse in a manner similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37 degrees C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.

  View details for PubMedID 17300849

• Selective imaging of adherent targeted ultrasound contrast agents PHYSICS IN MEDICINE AND BIOLOGY Zhao, S., Kruse, D. E., Ferrara, K. W., Dayton, P. A. 2007; 52 (8): 2055-2072

  Abstract

  The goal of ultrasonic molecular imaging is the detection of targeted contrast agents bound to receptors on endothelial cells. We propose imaging methods that can distinguish adherent microbubbles from tissue and from freely circulating microbubbles, each of which would otherwise obscure signal from molecularly targeted adherent agents. The methods are based on a harmonic signal model of the returned echoes over a train of pulses. The first method utilizes an 'image-push-image' pulse sequence where adhesion of contrast agents is rapidly promoted by acoustic radiation force and the presence of adherent agents is detected by the signal change due to targeted microbubble adhesion. The second method rejects tissue echoes using a spectral high-pass filter. Free agent signal is suppressed by a pulse-to-pulse low-pass filter in both methods. An overlay of the adherent and/or flowing contrast agents on B-mode images can be readily created for anatomical reference. Contrast-to-tissue ratios from adherent microbubbles exceeding 30 dB and 20 dB were achieved for the two methods proposed, respectively. The performance of these algorithms is compared, emphasizing the significance and potential applications in ultrasonic molecular imaging.

  View details for DOI 10.1088/0031-9155/52/8/002

  View details for PubMedID 17404455

• Enhancement of vascular permeability with low-frequency contrast-enhanced ultrasound in the chorioallantoic membrane model RADIOLOGY Stieger, S. M., Caskey, C. F., Adamson, R. H., Qin, S., Curry, F. E., Wisner, E. R., Ferrara, K. W. 2007; 243 (1): 112-121

  Abstract

  To characterize the effect of low-frequency contrast material-enhanced ultrasound on the vascular endothelium and to determine the parameters and techniques required to deliver a therapeutic agent by using the chorioallantoic membrane (CAM) model.All in vivo animal procedures were conducted with institutional Animal Care and Use Committee approval. Extravasation of 8.5-nm-diameter fluorescein isothiocyanate-labeled dextran was evaluated in the vasculature of a chick CAM model. Intravital microscopy was performed during contrast-enhanced ultrasound exposure (1.00 or 2.25 MHz); results were compared with results of electron microscopy of the insonated regions. Data acquired after insonation with greater mechanical stress (n = 30 animals) (mechanical index [MI] > 1.3) and with lower mechanical stress (n = 86 animals) (MI < 1.13) were compared with measurements in control conditions (n = 46 animals). The diameter of affected vessels; number of extravasation sites; extravasation rate, area, and location; and changes in endothelial cells and basement membrane were evaluated. Differences were tested with analysis of variance or the Student t test.After ultrasound application, convective transport of the model drug was observed through micron-sized openings with a mean fluid velocity of 188.6 microm/sec in the low-stress class and 362.5 microm/sec in the high-stress class. Electron microscopy revealed micron-sized focal endothelial gaps and disseminated blebs, vacuoles, and filopodia extending across tens of microns. The threshold pressure for extravasation was 0.5 MPa for a transmitted center frequency of 1.00 MHz (MI = 0.5) and 1.6 MPa for a frequency of 2.25 MHz (MI = 1.06); thus, the frequency dependence of the threshold was not predicted simply by the MI.Low-frequency contrast-enhanced ultrasound can increase vascular permeability and result in convective extravasation of an 8.5-nm-diameter model drug.

  View details for DOI 10.1148/radiol.2431060167

  View details for PubMedID 17392250

• Modulation of ATP/ADP concentration at the endothelial surface by shear stress: Effect of flow recirculation ANNALS OF BIOMEDICAL ENGINEERING Choi, H. W., Ferrara, K. W., Barakat, A. I. 2007; 35 (4): 505-516

  Abstract

  The extracellular presence of the adenine nucleotides ATP and ADP induces calcium mobilization in vascular endothelial cells (ECs). ATP/ADP concentration at the EC surface is determined by a balance of convective-diffusive transport to and from the EC surface, hydrolysis by ectonucleotidases at the cell surface, and flow-induced ATP release from ECs. Our previous numerical simulations in a parallel plate geometry had demonstrated that flow-induced ATP release has a profound effect on nucleotide concentration at the EC surface. In the present study, we have extended the modeling to probe the impact of flow separation and recirculation downstream of a backward facing step (BFS) on ATP/ADP concentration at the EC surface. The results show that for both steady and pulsatile flow over a wide range of wall shear stresses, the ATP+ADP concentration at the EC surface is considerably lower within the flow recirculation region than in areas of undisturbed flow outside the recirculation zone. Pulsatile flow also leads to sharp temporal gradients in nucleotide concentration. If confirmed experimentally, the present findings suggest that disturbed and undisturbed flow may affect EC calcium mobilization differently. Such differences might, in turn, contribute to the observed endothelial dysfunction in regions of disturbed flow.

  View details for DOI 10.1007/s10439-006-9247-9

  View details for PubMedID 17253149

• Contrast imaging with chirped excitation IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Sun, Y., Kruse, D. E., Ferrara, K. W. 2007; 54 (3): 520-529

  Abstract

  Coded excitation has been successfully used in imaging to increase the signal-to-noise ratio (SNR) and penetration depth. With a contrast agent, wideband signals have been hypothesized to increase the contrast-to-tissue ratio (CTR). However, nonlinear properties of contrast agents make decoding difficult when applying coded excitation to contrast imaging. We propose two chirped excitation methods to image contrast agents, with a mechanical index (MI) ranging from 0.05 to 0.34. In the single chirp method, one chirp is transmitted, followed by a clutter filter to reject tissue echoes, then a matched filter is used to recover range resolution. In the chirp sequence method, an increasing and decreasing chirp sequence is transmitted followed by subtraction of the compressed echoes to reject tissue echoes (assuming tissue is a linear scatterer at low MI). Ten independent acoustic experiments were performed to evaluate the CTR for chirp and tone burst insonation, with the same spatial peak temporal averaged intensity (I(SPTA)). A significant increase in CTR, ranging from 4 dB to 8 dB, is observed for chirped excitation as compared with tone burst insonation, at an I(SPTA) of 0.1 and 0.3 mW/cm2 (P < or = 5e-3). To achieve the same CTR of 15 dB, the spatial peak pulse averaged intensity (I(SPPA)) can be decreased by 6 dB for chirp insonation as compared with tone burst insonation (P < 1e-5). Additionally, an increase of more than 10 dB in tissue rejection ratio (TRR) is observed for a chirp sequence insonation compared to tone burst phase inversion for this set of parameters (P < or = 1e-9). Deconvolution of the linear microbubble response from the received echoes is proposed as a method to recover spatial resolution. The difference in the axial resolution resulting from chirp and three-cycle tone burst insonation is approximately 220 microm. The difference in the mainlobe width between experimental and predicted compressed echoes is less than 20%. The side-lobe amplitude is 9 dB to 16 dB below the mainlobe with a transmitted I(SPTA) from 0.1 to 6.6 mW/cm2.

  View details for DOI 10.1109/TUFFC.2007.275

  View details for PubMedID 17375821

• Long-circulating liposomes radiolabeled with [F-18]fluorodipalmitin ([F-18]FDP) NUCLEAR MEDICINE AND BIOLOGY Marik, J., Tartis, M. S., Zhang, H., Fung, J. Y., Kheirolomoom, A., Sutcliffe, J. L., Ferrara, K. W. 2007; 34 (2): 165-171

  Abstract

  Synthesis of a radiolabeled diglyceride, 3-[(18)F]fluoro-1,2-dipalmitoylglycerol [[(18)F]fluorodipalmitin ([(18)F]FDP)], and its potential as a reagent for radiolabeling long-circulating liposomes were investigated. The incorporation of (18)F into the lipid molecule was accomplished by nucleophilic substitution of the p-toluenesulfonyl moiety with a decay-corrected yield of 43+/-10% (n=12). Radiolabeled, long-circulating polyethylene-glycol-coated liposomes were prepared using a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] ammonium salt (61:30:9) and [(18)F]FDP with a decay-corrected yield of 70+/-8% (n=4). PET imaging and biodistribution studies were performed with free [(18)F]FDP and liposome-incorporated [(18)F]FDP. Freely injected [(18)F]FDP had the highest uptake in the liver, spleen and lungs. Liposomal [(18)F]FDP remained in blood circulation at near-constant levels for at least 90 min, with a peak concentration near 2.5%ID/cc. Since [(18)F]FDP was incorporated into the phospholipid bilayer, it could potentially be used for radiolabeling a variety of lipid-based drug carriers.

  View details for DOI 10.1016/j.nucmedbio.2006.12.004

  View details for PubMedID 17307124

• Quantitative contrast enhanced ultrasound and CT assessment of tumor response to antiangiogenic therapy in rats ULTRASOUND IN MEDICINE AND BIOLOGY Pollard, R. E., Broumas, A. R., Wisner, E. R., Vekich, S. V., Ferrara, K. W. 2007; 33 (2): 235-245

  Abstract

  Contrast-enhanced computed tomography (CECT) and contrast-enhanced destruction-replenishment subharmonic ultrasound (CEDRSU) were used to quantify blood flow and tumor viability during antiangiogenic therapy. SU11657 or placebo was administered to R3230AC tumor-baring rats over a two-week period. CEDRSU vascular volume (ASI) and volume flow (VF) and CECT perfusion (PR) and permeability (PM) measurements were made on day 0, 7 and 14. The percent change in imaging parameters was calculated between day 0 and 7 and 14. Serum drug level (SDL) was compared with imaging parameters. Imaging estimates of tumor viability were compared with histology images on day 14. The percent change in imaging measures for control and treated groups were significantly different on day 7(ASI, p = 0.02; VF, p = 0.008, PR, p = 0.0007; PM, p = 0.003) and 14 (ASI, p = 0.0004; VF, p = 0.002, PR, p = 0.003; PM, p = 0.005). Imaging identified animals with lower SDLs as having higher tumor vascularity and flow. Spatial estimates of tumor viability correlated with histology (CEDRSU, r(2) = 0.92, p < 0.001; CT, r(2) = 0.86, p < 0.001). CEDRSU and CECT provide measures of blood flow and viability in tumors during antiangiogenic therapy. Tumors with higher flow were identified in animals with lower SDL. SU11657 treatment results in decreased tumor flow and viability.

  View details for DOI 10.1016/j.ultrasmedbio.2006.07.036

  View details for PubMedID 17306694

• Workshop on imaging science development for cancer prevention and preemption. Cancer biomarkers Kelloff, G. J., Sullivan, D. C., Baker, H., Clarke, L. P., Nordstrom, R., Tatum, J. L., Dorfman, G. S., Jacobs, P., Berg, C. D., Pomper, M. G., Birrer, M. J., Tempero, M., Higley, H. R., Petty, B. G., Sigman, C. C., Maley, C., Sharma, P., Wax, A., Ginsberg, G. G., Dannenberg, A. J., Hawk, E. T., Messing, E. M., Grossman, H. B., Harisinghani, M., Bigio, I. J., Griebel, D., Henson, D. E., Fabian, C. J., Ferrara, K., Fantini, S., Schnall, M. D., Zujewski, J. A., Hayes, W., Klein, E. A., Demarzo, A., Ocak, I., Ketterling, J. A., Tempany, C., Shtern, F., Parnes, H. L., Gomez, J., Srivastava, S., Szabo, E., Lam, S., Seibel, E. J., Massion, P., McLennan, G., Cleary, K., Suh, R., Burt, R. W., Pfeiffer, R. M., Hoffman, J. M., Roy, H. K., Wang, T., Limburg, P. J., El-Deiry, W. S., Papadimitrakopoulou, V., Hittelman, W. N., MacAulay, C., Veltri, R. W., Solomon, D., Jeronimo, J., Richards-Kortum, R., Johnson, K. A., Viner, J. L., Stratton, S. P., Rajadhyaksha, M., Dhawan, A. 2007; 3 (1): 1-33

  Abstract

  The concept of intraepithelial neoplasm (IEN) as a near-obligate precursor of cancers has generated opportunities to examine drug or device intervention strategies that may reverse or retard the sometimes lengthy process of carcinogenesis. Chemopreventive agents with high therapeutic indices, well-monitored for efficacy and safety, are greatly needed, as is development of less invasive or minimally disruptive visualization and assessment methods to safely screen nominally healthy but at-risk patients, often for extended periods of time and at repeated intervals. Imaging devices, alone or in combination with anticancer drugs, may also provide novel interventions to treat or prevent precancer.

  View details for PubMedID 17655039

• Pharmacokinetics of encapsulated paclitaxel: Multi-probe analysis with pet Tartis, M. S., Marik, J., Kheirolomoom, A., Pollard, R. E., Zhang, H., Qi, J., Sutcliffe, J. L., Ferrara, K. W., ASME AMER SOC MECHANICAL ENGINEERS. 2007: 113–14

  View details for Web of Science ID 000252105700057

• Efficient array design for sonotherapy enhanced drug delivery Stephens, D. N., Kruse, D. E., Barnes, S., Clary, T., Lu, X., Nock, L., Ferrara, K., IEEE IEEE. 2007: 108-+

  View details for DOI 10.1109/ULTSYM.2007.40

  View details for Web of Science ID 000254281800025

• Spatial and temporal controlled tissue heating on a modified clinical ultrasound scanner for generating mild hyperthermia in tumors Kruse, D. E., Stephens, D. N., Paoli, E. E., Barnes, S. H., Ferrara, K. W., IEEE IEEE. 2007: 313-+

  View details for DOI 10.1109/ULTSYM.2007.89

  View details for Web of Science ID 000254281800074

• Microbubble oscillations in get phantom and ex vivo preparation validate proposed mechanisms for contrast-based drug delivery Caskey, C. F., Stieger, S. M., Qin, S., Dayton, P. A., Ferrara, K. W., IEEE IEEE. 2007: 769–72

  View details for DOI 10.1109/ULTSYM.2007.197

  View details for Web of Science ID 000254281800182

• A novel sensitive targeted imaging technique for ultrasonic molecular imaging Zheng, H., Kruse, D. E., Stephens, D. N., Ferrara, K. W., IEEE IEEE. 2007: 957–60

  View details for DOI 10.1109/ULTSYM.2007.244

  View details for Web of Science ID 000254281800229

• Dynamics of ultrasound contrast agents within rat cecum vessels Qin, S., Caskey, C. F., Ferrara, K. W., IEEE IEEE. 2007: 1981–84

  View details for DOI 10.1109/ULTSYM.2007.498

  View details for Web of Science ID 000254281801246

• Ultrasound microbubble contrast agents: Fundamentals and application to gene and drug delivery ANNUAL REVIEW OF BIOMEDICAL ENGINEERING Ferrara, K., Pollard, R., Borden, M. 2007; 9: 415-447

  Abstract

  This review offers a critical analysis of the state of the art of medical microbubbles and their application in therapeutic delivery and monitoring. When driven by an ultrasonic pulse, these small gas bubbles oscillate with a wall velocity on the order of tens to hundreds of meters per second and can be deflected to a vessel wall or fragmented into particles on the order of nanometers. While single-session molecular imaging of multiple targets is difficult with affinity-based strategies employed in some other imaging modalities, microbubble fragmentation facilitates such studies. Similarly, a focused ultrasound beam can be used to disrupt delivery vehicles and blood vessel walls, offering the opportunity to locally deliver a drug or gene. Clinical translation of these vehicles will require that current challenges be overcome, where these challenges include rapid clearance and low payload. The technology, early successes with drug and gene delivery, and potential clinical applications are reviewed.

  View details for DOI 10.1146/annurev.bioeng.8.061505.095852

  View details for PubMedID 17651012

• Acoustic response from adherent targeted contrast agents JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Zhao, S., Kruse, D. E., Ferrara, K. W., Dayton, P. A. 2006; 120 (6): EL63-EL69

  Abstract

  In ultrasonic molecular imaging, encapsulated micron-sized gas bubbles are tethered to a blood vessel wall by targeting ligands. A challenging problem is to detect the echoes from adherent microbubbles and distinguish them from echoes from nonadherent agents and tissue. Echoes from adherent contrast agents are observed to include a high amplitude at the fundamental frequency, and significantly different spectral shape compared with free agents (p <0.0003). Mechanisms for the observed acoustical difference and potential techniques to utilize these differences for molecular imaging are proposed.

  View details for DOI 10.1121/1.2364303

  View details for Web of Science ID 000242959400064

  View details for PubMedID 17225437

  View details for PubMedCentralID PMC1780256

• Therapeutic effects of paclitaxel-containing ultrasound contrast agents ULTRASOUND IN MEDICINE AND BIOLOGY Tartis, M. S., McCallan, J., Lum, A. F., LaBell, R., Stieger, S. M., Matsunaga, T. O., Ferrara, K. W. 2006; 32 (11): 1771-1780

  Abstract

  Drug delivery vehicles that combine ultrasonic and molecular targeting are shown to locally concentrate a drug in a region-of-interest. The drug delivery vehicles, referred to as acoustically active lipospheres (AALs), are microbubbles surrounded by a shell of oil and lipid. In a region limited to the focal area of ultrasound application, circulating AALs are deflected by radiation force to a vessel wall and can subsequently be fragmented. Ligands targeting the alphavbeta3 integrin are conjugated to the AAL shell and increase in vitro binding by 26.5-fold over nontargeted agents. Toxicity assays demonstrate that paclitaxel-containing AALs exert a greater antiproliferative effect after insonation than free paclitaxel at an equivalent concentration. Lastly, ultrasound and molecular targeting are combined to deliver a model drug to the endothelium and interstitium of chorioallantoic membrane vasculature in vivo.

  View details for DOI 10.1016/j.ultrasmedbio.2006.03.017

  View details for PubMedID 17112963

• Acoustic response of compliable microvessels containing ultrasound contrast agents PHYSICS IN MEDICINE AND BIOLOGY Qin, S., Ferrara, K. W. 2006; 51 (20): 5065-5088

  Abstract

  The existing models of the dynamics of ultrasound contrast agents (UCAs) have largely been focused on an UCA surrounded by an infinite liquid. Preliminary investigations of a microbubble's oscillation in a rigid tube have been performed using linear perturbation, under the assumption that the tube diameter is significantly larger than the UCA diameter. In the potential application of drug and gene delivery, it may be desirable to fragment the agent shell within small blood vessels and in some cases to rupture the vessel wall, releasing drugs and genes at the site. The effect of a compliant small blood vessel on the UCA's oscillation and the microvessel's acoustic response are unknown. The aim of this work is to propose a lumped-parameter model to study the interaction of a microbubble oscillation and compliable microvessels. Numerical results demonstrate that in the presence of UCAs, the transmural pressure through the blood vessel substantially increases and thus the vascular permeability is predicted to be enhanced. For a microbubble within an 8 to 40 microm vessel with a peak negative pressure of 0.1 MPa and a centre frequency of 1 MHz, small changes in the microbubble oscillation frequency and maximum diameter are observed. When the ultrasound pressure increases, strong nonlinear oscillation occurs, with an increased circumferential stress on the vessel. For a compliable vessel with a diameter equal to or greater than 8 microm, 0.2 MPa PNP at 1 MHz is predicted to be sufficient for microbubble fragmentation regardless of the vessel diameter; however, for a rigid vessel 0.5 MPa PNP at 1 MHz may not be sufficient to fragment the bubbles. For a centre frequency of 1 MHz, a peak negative pressure of 0.5 MPa is predicted to be sufficient to exceed the stress threshold for vascular rupture in a small (diameter less than 15 microm) compliant vessel. As the vessel or surrounding tissue becomes more rigid, the UCA oscillation and vessel dilation decrease; however the circumferential stress is predicted to increase. Decreasing the vessel size or the centre frequency increases the circumferential stress. For the two frequencies considered in this work, the circumferential stress does not scale as the inverse of the square root of the acoustic frequency va as in the mechanical index, but rather has a stronger frequency dependence, 1/va.

  View details for DOI 10.1088/0031-9155/51/20/001

  View details for PubMedID 17019026

• Application of ultrasound to selectively localize nanodroplets for targeted imaging and therapy MOLECULAR IMAGING Dayton, P. A., Zhao, S., Bloch, S. H., Schumann, P., Penrose, K., Matsunaga, T. O., Zutshi, R., Doinikov, A., Ferrara, K. W. 2006; 5 (3): 160-174

  Abstract

  Lipid-coated perfluorocarbon nanodroplets are submicrometer-diameter liquid-filled droplets with proposed applications in molecularly targeted therapeutics and ultrasound (US) imaging. Ultrasonic molecular imaging is unique in that the optimal application of these agents depends not only on the surface chemistry, but also on the applied US field, which can increase receptor-ligand binding and membrane fusion. Theory and experiments are combined to demonstrate the displacement of perfluorocarbon nanoparticles in the direction of US propagation, where a traveling US wave with a peak pressure on the order of megapascals and frequency in the megahertz range produces a particle translational velocity that is proportional to acoustic intensity and increases with increasing center frequency. Within a vessel with a diameter on the order of hundreds of micrometers or larger, particle velocity on the order of hundreds of micrometers per second is produced and the dominant mechanism for droplet displacement is shown to be bulk fluid streaming. A model for radiation force displacement of particles is developed and demonstrates that effective particle displacement should be feasible in the microvasculature. In a flowing system, acoustic manipulation of targeted droplets increases droplet retention. Additionally, we demonstrate the feasibility of US-enhanced particle internalization and therapeutic delivery.

  View details for DOI 10.2310/7290.2006.00019

  View details for PubMedID 16954031

• Ultrasound radiation force modulates ligand availability on targeted contrast agents MOLECULAR IMAGING Borden, M. A., Sarantos, M. R., Stieger, S. M., Simon, S. I., Ferrara, K. W., Dayton, P. A. 2006; 5 (3): 139-147

  Abstract

  Radiation force produced by low-amplitude ultrasound at clinically relevant frequencies remotely translates freely flowing microbubble ultrasound contrast agents over distances up to centimeters from the luminal space to the vessel wall in order to enhance ligand-receptor contact in targeting applications. The question arises as to how the microbubble shell might be designed at the molecular level to fully take advantage of such physical forces in targeted adhesion for molecular imaging and controlled therapeutic release. Herein, we report on a novel surface architecture in which the tethered ligand is buried in a polymeric overbrush. Our results, with biotin-avidin as the model ligand-receptor pair, show that the overbrush conceals the ligand, thereby reducing immune cell binding and increasing circulation persistence. Targeted adhesion is achieved through application of ultrasound radiation force to instantly reveal the ligand within a well-defined focal zone and simultaneously bind the ligand and receptor. Our data illustrate how the adhesive properties of the contrast agent surface can be reversibly changed, from stealth to sticky, through the physical effects of ultrasound. This technique can be combined with any ligand-receptor pair to optimize targeted adhesion for ultrasonic molecular imaging.

  View details for DOI 10.2310/7290.2006.00016

  View details for PubMedID 16954028

• Observation of contrast agent response to chirp insonation with a simultaneous optical-acoustical system IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Sun, Y., Zhao, S., Dayton, P. A., Ferrara, K. W. 2006; 53 (6): 1130-1137

  Abstract

  Rayleigh-Plesset analysis, ultra-high speed photography, and single bubble acoustical recordings previously were applied independently to characterize the radial oscillation and resulting echoes from a microbubble in response to an ultrasonic pulse. In addition, high-speed photography has shown that microbubbles are destroyed over a single pulse or pulse train by diffusion and fragmentation. In order to develop a single model to characterize microbubble echoes based on oscillatory and destructive characteristics, an optical-acoustical system was developed to simultaneously record the optical image and backscattered echo from each microbubble. Combined observation provides the opportunity to compare predictions for oscillation and echoes with experimental results and identify discrepancies due to diffusion or fragmentation. Optimization of agents and insonating pulse parameters may be facilitated with this system. The mean correlation of the predicted and experimental radius-time curves and echoes exceeds 0.7 for the parameters studied here. An important application of this new system is to record and analyze microbubble response to a long pulse in which diffusion is shown to occur over the pulse duration. The microbubble response to an increasing or decreasing chirp is evaluated using this new tool. For chirp insonation beginning with the lower center frequency, low-frequency modulation of the oscillation envelope was obvious. However, low-frequency modulation was not observed in the radial oscillation produced by decreasing chirp insonation. Comparison of the echoes from similar sized microbubbles following increasing and decreasing chirp insonation demonstrated that the echoes were not time-reversed replicas. Using a transmission pressure of 620 kPa, the -6 dB echo length was 0.9 and 1.1 micros for increasing and decreasing chirp insonation, respectively (P = 0.02). The mean power in the low-frequency portion of the echoes was 8 (mV)2 and 13 (mV)2 for increasing and decreasing chirp insonation, respectively (P = 0.01).

  View details for PubMedID 16846145

• Ultrasound detection and characterization of polycystic kidney disease in a mouse model COMPARATIVE MEDICINE Pollard, R., Yunis, R., Kultz, D., Martin, P., Griffey, S., Ferrara, K. 2006; 56 (3): 215-221

  Abstract

  We sought to use ultrasonography to quantify renal size and echogenicity in a mouse model of polycystic kidney disease. We imaged 36 wild-type (WT) and juvenile cystic kidney (jck) mice by using a standard ultrasound unit and 10-5 MHz linear transducer. Mice were imaged at 3 (6 WT, 7 jck), 6 (7 WT, 5 jck), and 9 (6 WT, 5 jck) wk of age. Kidney length, width, and height were recorded for volume calculation. Sagittal images of both kidneys were recorded for assessment of intensity. Quantitative values were obtained from areas of similar depth and gain settings. Kidney and liver intensities were determined for calculation of their ratio. Representative histologic kidney sections were stained with hematoxylin and eosin and digitized for calculation of cyst number, mean cyst area, and percentage cystic area. We found that renal volume was greater in jck than WT mice at 3 (P < 0.0001), 6 (P < 0.0001), and 9 (P < 0.0001) wk of age. In addition, kidney intensity and kidney:liver ratio were higher in jck than WT mice at 3 (P < 0.002 for both parameters), 6 (P < 0.04), and 9 wk (P < 0.008). Kidneys with smaller mean cyst size and less percentage cystic space had higher intensity values. We therefore conclude that ultrasound measures of renal volume and intensity can noninvasively identify jck-affected mice as early as 3 wk of age. Cortical intensity is greater in jck versus WT mice and appears affected by percentage cyst area and mean cyst size.

  View details for PubMedID 16774131

• Ultrasound assessment of angiogenesis in a matrigel model in rats ULTRASOUND IN MEDICINE AND BIOLOGY Stieger, S. M., Bloch, S. H., Foreman, O., Wisner, E. R., Ferrara, K. W., Dayton, P. A. 2006; 32 (5): 673-681

  Abstract

  Matrigel, a basement membrane extract, has been extensively used in in vivo angiogenesis. Contrast ultrasound imaging (CUI) of implanted Matrigel plugs with (+bFGF) and without basic fibroblast growth factor (-bFGF) was performed 7 and 14 d after implantation, followed by histologic analysis. Statistically significant differences between +bFGF and -bFGF plugs were apparent at d 7 in both plug size and contrast enhancement (both p < 0.05). Histopathology revealed differences in microvessel density (MVD) between +bFGF and -bFGF at d 7 and d 14. A significant correlation between MVD and both power Doppler contrast-enhanced area (r = 0.65, p < 0.05) and fraction of plug enhanced (r = 0.59, p < 0.05) was present. CUI of Matrigel plugs was shown to be a robust method for distinguishing between two different angiogenic states. Ultrasound measurements of blood flow in the plugs correlated with MVD, a histologic technique used to quantify tumor angiogenesis.

  View details for DOI 10.1016/j.ultrasmedbio.2005.12.008

  View details for PubMedID 16677927

• Lateral phase separation in lipid-coated microbubbles LANGMUIR Borden, M. A., Martinez, G. V., Ricker, J., Tsvetkova, N., Longo, M., Gillies, R. J., Dayton, P. A., Ferrara, K. W. 2006; 22 (9): 4291-4297

  Abstract

  In the design of lipid-coated microbubble ultrasound contrast agents for molecular imaging and targeted drug delivery, the surface distribution of the shell species is important because it dictates such properties as ligand location, brush coverage, and amount of drug loading. We used a combination of spectroscopy and microscopy techniques to test the prevailing notion that the main phosphatidyl choline (PC) and lipopolymer species are completely miscible within the monolayer shell. NMR spectroscopy showed that the shell composition is roughly equivalent to the bulk lipid ratio. FTIR spectroscopy showed a sharp melting peak corresponding to the main phase-transition temperature of the main PC species, with no observed pretransitions while scanning from room temperature, indicating a single PC-rich ordered phase. Electron and fluorescence microscopy showed a heterogeneous microstructure with dark (ordered) domains and bright (disordered) regions. Domain formation was thermotropic and reversible. Fluorescent labeling of the lipopolymer following shell formation showed that it partitions preferentially into the disordered interdomain regions. The ordered domains, therefore, are composed primarily of PC, and the disordered interdomain regions are enriched in lipopolymer. Phase heterogeneity was observed at all lipopolymer concentrations (0.5 to 20 mol %), and the degree of phase separation increased with lipopolymer content. The composition and temperature dependence of the microstructure indicates that phase separation is driven thermodynamically rather than being a kinetically trapped relic of the shell-formation process. The overall high variation in microstructure, including the existence of anomalous three-phase coexistence, highlights the nonequilibrium (history-dependent) nature of the monolayer shell.

  View details for DOI 10.1021/la052841v

  View details for PubMedID 16618177

• Ultrasound radiation force enables targeted deposition of model drug carriers loaded on microbubbles JOURNAL OF CONTROLLED RELEASE Lum, A. F., Borden, M. A., Dayton, P. A., Kruse, D. E., Simon, S. I., Ferrara, K. W. 2006; 111 (1-2): 128-134

  Abstract

  A novel drug delivery vehicle that specifically targets using ultrasound radiation force (USRF) and biotin-avidin interactions is presented. Model vehicles consist of avidinated fluorescent nanobeads bound directly to the biotinylated lipid shells of preformed microbubbles. USRF was used to deflect the vehicle from the center of flow to a tube surface in order to facilitate receptor-ligand mediated adhesion. At wall shear stress levels commensurate with venous and arterial flow, USRF was used to direct the vehicles to a biotinylated tube surface. Subsequent high-pressure pulses fragmented the carrier, and molecular interactions induced deposition of the nanobeads on the wall. Targeting of nanobeads to the tube was molecularly specific and dependent on, in order of importance, vehicle concentration, wall shear stress, nanobead size, and insonation time. The observation that portions of the microbubble lipid monolayer shell remain attached to adherent nanobeads is important for future consideration of drug transport mechanisms. This versatile method of delivery is shown to enable targeted deposition of nanoparticles in shear flow and could be modified to carry therapeutic agents for controlled release in targeted delivery applications.

  View details for DOI 10.1016/j.jconrel.2005.11.006

  View details for PubMedID 16380187

• Biomedical imaging graduate curricula and courses: Report from the 2005 Whitaker Biomedical Engineering Educational Summit ANNALS OF BIOMEDICAL ENGINEERING Louie, A., Izatt, J., Ferrara, K. 2006; 34 (2): 239-247

  Abstract

  We present an overview of graduate programs in biomedical imaging that are currently available in the US. Special attention is given to the emerging technologies of molecular imaging and biophotonics. Discussions from the workshop on Graduate Imaging at the 2005 Whitaker Educational Summit meeting are summarized.

  View details for DOI 10.1007/s10439-005-9033-0

  View details for PubMedID 16482417

• Microbubble oscillation in tubes with diameters of 12, 25, and 195 microns APPLIED PHYSICS LETTERS Caskey, C. F., Kruse, D. E., Dayton, P. A., Kitano, T. K., Ferrara, K. W. 2006; 88 (3)

  View details for DOI 10.1063/1.2164392

  View details for Web of Science ID 000234757100083

• Detection of Echoes from Adherent Targeted Microbubbles Zhao, S., Kruse, D. E., Ferrara, K. W., Dayton, P. A., IEEE IEEE. 2006: 525–28

  View details for Web of Science ID 000260407800125

• Multi-frequency Array Development for Drug Delivery Therapies Stephens, D. N., Lu, X., Proulx, T., Walters, W., Dayton, P., Tartis, M., Kruse, D. E., Lum, A. H., Kitano, T., Stieger, S. M., Ferrara, K., IEEE IEEE. 2006: 66-+

  View details for DOI 10.1109/ULTSYM.2006.30

  View details for Web of Science ID 000260407800016

• Ultrasound Radiation Force Enables Targeted Deposition of Molecularly Targeted Nanoparticles Loaded on Microbubbles Under Flow Conditions Lum, A. H., Borden, M. A., Dayton, P. A., Peng, L., Kruse, D. E., Simon, S. I., Lam, K. S., Ferrara, K. W., IEEE IEEE. 2006: 108-+

  View details for Web of Science ID 000260407800026

• Contrast Imaging with Chirped Excitation Sun, Y., Kruse, D. E., Ferrara, K. W., IEEE IEEE. 2006: 220–23

  View details for Web of Science ID 000260407800054

• Dynamics of ultrasound contrast agents and microvessels with MHz-frequency ultrasound Qin, S., Ferrara, K. W., IEEE IEEE. 2006: 420–23

  View details for Web of Science ID 000260407800099

• Acoustic localization of sub-micron droplets for targeted imaging and therapy Dayton, P. A., Zhao, S., Zheng, H., Zutshi, R., Schumann, P., Penrose, K., Matsunaga, T. O., Ferrara, K. W., IEEE IEEE. 2006: 521-+

  View details for DOI 10.1109/ULTSYM.2006.146

  View details for Web of Science ID 000260407800124

• Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Borden, M. A., Kruse, D. E., Caskey, C. F., Zhao, S. K., Dayton, P. A., Ferrara, K. W. 2005; 52 (11): 1992-2002

  Abstract

  We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 microm were isolated and recorded during pulsing with bright-field and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.

  View details for PubMedID 16422411

• High-frequency dynamics of ultrasound contrast agents IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Sun, Y., Kruse, D. E., Dayton, P. A., Ferrara, K. W. 2005; 52 (11): 1981-1991

  Abstract

  Ultrasound contrast agents enhance echoes from the microvasculature and enable the visualization of flow in smaller vessels. Here, we optically and acoustically investigate microbubble oscillation and echoes following insonation with a 10 MHz center frequency pulse. A high-speed camera system with a temporal resolution of 10 ns, which provides two-dimensional (2-D) frame images and streak images, is used in optical experiments. Two confocally aligned transducers, transmitting at 10 MHz and receiving at 5 MHz, are used in acoustical experiments in order to detect subharmonic components. Results of a numerical evaluation of the modified Rayleigh-Plesset equation are used to predict the dynamics of a microbubble and are compared to results of in vitro experiments. From the optical observations of a single microbubble, nonlinear oscillation, destruction, and radiation force are observed. The maximum bubble expansion, resulting from insonation with a 20-cycle, 10-MHz linear chirp with a peak negative pressure of 3.5 MPa, has been evaluated. For an initial diameter ranging from 1.5 to 5 microm, a maximum diameter less than 8 microm is produced during insonation. Optical and acoustical experiments provide insight into the mechanisms of destruction, including fragmentation and active diffusion. High-frequency pulse transmission may provide the opportunity to detect contrast echoes resulting from a single pulse, may be robust in the presence of tissue motion, and may provide the opportunity to incorporate high-frequency ultrasound into destruction-replenishment techniques.

  View details for PubMedID 16422410

• Asymmetric oscillation of adherent targeted ultrasound contrast agents APPLIED PHYSICS LETTERS Zhao, S. K., Ferrara, K. W., Dayton, P. A. 2005; 87 (13)

  Abstract

  With a lipid shell containing biotin, micron-sized bubbles bound to avidin on a porous and flexible cellulose boundary were insonified by ultrasound. The oscillation of these targeted microbubbles was observed by high-speed photography and compared to the oscillation of free-floating microbubbles. Adherent microbubbles were observed to oscillate asymmetrically in the plane normal to the boundary, and nearly symmetrically in the plane parallel to the boundary, with a significantly smaller maximum expansion in each dimension for bound than free bubbles. With sufficient transmitted pressure, a jet was produced traveling toward the boundary.

  View details for DOI 10.1063/1.2061872

  View details for PubMedID 16755307

• Phase behavior and transport properties of the lipid-monolayer shell of a microbubble Borden, M. A., Pu, G., Longo, M. L., Dayton, P. A., Ferrara, K. W. AMER CHEMICAL SOC. 2005: U1072–U1073

  View details for Web of Science ID 000236797302153

• A new imaging strategy using wideband transient response of ultrasound contrast agents IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Kruse, D. E., Ferrara, K. W. 2005; 52 (8): 1320-1329

  Abstract

  High-resolution clinical systems operating near 15 MHz are becoming more available; however, they lack sensitive harmonic imaging modes for ultrasound contrast agent (UCA) detection, primarily due to limited bandwidth. When an UCA is driven to nonlinear oscillation, a very wideband acoustic transient response is produced that extends beyond 15 MHz. We propose a novel strategy using two separate transducers at widely separated frequencies and arranged confocally to simultaneously excite and receive acoustic transients from UCAs. Experiments were performed to demonstrate that this new mode shows similar resolution, higher echo amplitudes, and greatly reduced attenuation compared to transmission at a higher frequency, and superior resolution compared to transmission and reception at a lower frequency. The proposed method is shown to resolve two 200 microm tubes with centers separated by 400 microm. Strong acoustic transients were detected for rarefaction-first 1-cycle pulses with peak-negative pressures above 300 kPa. The results of this work may lead to uses in flow and/or targeted imaging in applications requiring very high sensitivity to contrast agents.

  View details for PubMedID 16245601

• The effect of size on the acoustic response of polymer-shelled contrast agents ULTRASOUND IN MEDICINE AND BIOLOGY Bloch, S. H., Short, R. E., Ferrara, K. W., Wisner, E. R. 2005; 31 (3): 439-444

  Abstract

  In this technical note, we study three polymer-shelled microbubble contrast agents manufactured by POINT Biomedical Corporation that have identical shell composition and mean volumetric diameters of 0.74 microm, 0.91 microm and 1.33 microm. We investigate the effect of agent size on the amplitude, frequency and probability of acoustic echoes received in response to five-cycle, 2.25-MHz pulses of varying pressure. We find that the amplitude and frequency response from the three agents is not significantly different. However, significant differences among the agents do exist in the probability of response to acoustic interrogation: at a pressure of 1.06 MPa, an echo from the 1.33 microm agent is 5 times as likely as an echo from the 0.91 microm agent and 18 times as likely as an echo from the 0.74 microm agent. We hypothesize that there exists an effective pressure-dependent threshold diameter above which single polymer-shelled agents respond to acoustic interrogation.

  View details for DOI 10.1016/j.ultrasmedbio.2004.12.016

  View details for PubMedID 15749568

• Asymmetric oscillation of cavitation bubbles in a microvessel and its implications upon mechanisms of clinical vessel injury in shock-wave lithotripsy INTERNATIONAL JOURNAL OF NON-LINEAR MECHANICS Hu, Y. T., Qin, S. P., Hu, T., Ferrara, K. W., Jiang, Q. 2005; 40 (2-3): 341–50

  View details for DOI 10.1016/j.ijnonlinmec.2004.006.007

  View details for Web of Science ID 000224925400015

• Contrast-enhanced computed tomography and ultrasound for the evaluation of tumor blood flow INVESTIGATIVE RADIOLOGY Broumas, A. R., Pollard, R. E., Bloch, S. H., Wisner, E. R., Griffey, S., Ferrara, K. W. 2005; 40 (3): 134-147

  Abstract

  We evaluated implanted rat mammary adenocarcinoma tumors during a 5-week period using ultrasound, computed tomography (CT), and histology.Contrast-enhanced ultrasound with a destruction-replenishment imaging scheme was used to derive estimates of blood volume and flow. These ultrasound-derived measures of microvascular physiology were compared with contrast-enhanced CT-derived measures of perfusion and vascular volume made by the Mullani-Gould formula and Patlak analysis, respectively.The tumor cross-sectional area and necrotic core cross-sectional area determined by the 3 methods were correlated (r>0.8, P<0.001, n=15). The spatial integral of perfusion estimated by CT correlated with the spatial integral of flow from ultrasound (P<0.05). The contrast-enhanced tumor area calculated from the ultrasound analysis was highly correlated with the contrast-enhanced area estimated by CT images (r=0.89, P<0.001, n=15). However, the fraction of the tumor area enhanced by the CT contrast agent was significantly larger than either the fraction enhanced by ultrasound contrast agent or than the viable area as estimated from histology slides.Destruction-replenishment ultrasound provides valuable information about the spatial distribution of blood flow and vascular volume in tumors and ultrasound analysis compares favorably with a validated contrast-enhanced CT method.

  View details for PubMedID 15714088

• Targeted chemotherapy delivery with ultrasound Shortencarier, M., Dayton, P., Ferrara, K., Matsunaga, T., LaBell, R., Schumann, P., IEEE IEEE. 2005: 265–68

  View details for Web of Science ID 000236090700065

• Sonothrombolysis with phospholipid-coated perfluoropropane microbubbles Unger, E., Matsunaga, T. O., Pharm, D., Zutshi, R., Labell, R., Porter, T. R., Lof, J., Xie, F., Ferrara, K., Dayton, P., Bloch, S., TerHaar, G. R., Rivens AMER INST PHYSICS. 2005: 58-61

  View details for Web of Science ID 000228806700014

• On the oscillations of microbubbles in tubes with diameters as small as 12 microns Caskey, C. F., Kruse, D. E., Dayton, P. A., Ferrara, K. W., IEEE IEEE. 2005: 854–57

  View details for Web of Science ID 000236090701034

• Contrast echo processing for very wideband systems using higher order statistics Kruse, D. E., Sun, Y., Ferrara, K. W., IEEE IEEE. 2005: 1695–99

  View details for Web of Science ID 000236090702069

• High-resolution functional vascular assessment with ultrasound IEEE TRANSACTIONS ON MEDICAL IMAGING Yeh, C. K., Ferrara, K. W., Kruse, D. E. 2004; 23 (10): 1263-1275

  Abstract

  In order to improve the resolution of contrast-assisted imaging systems, we have created a high-frequency destruction/contrast replenishment imaging system with a spatial resolution of 160 microm x 160 microm. The system utilizes a 1-MHz cylindrically focused transducer for destruction and a 25-MHz spherically focused transducer for pulse/echo imaging. Speckle tracking and a clutter filter are applied across frames to remove the challenging physiologic motion artifacts that are obtained when imaging with a mechanically scanned transducer. Using a new estimation technique, flow constants proportional to absolute flow rate were estimated from B-mode time-intensity curves (TICs). The in vitro results indicate a correlation between the actual flow velocity and the estimated rate constant. In vivo images are presented showing blood perfusion in the ciliary processes and iris of the rabbit eye. The regions of interest (ROIs) from the ciliary processes yielded slower perfusion compared with the iris, as expected from vascular casts of the microcirculation in this region. Potential applications of this system include high-resolution perfusion assessment in small animals.

  View details for DOI 10.1109/TMI.2004.834614

  View details for Web of Science ID 000224214000008

  View details for PubMedID 15493694

• Targeted imaging using ultrasound contrast agents. Progess and opportunities for clinical and research applications. IEEE engineering in medicine and biology magazine Bloch, S. H., Dayton, P. A., Ferrara, K. W. 2004; 23 (5): 18-29

  View details for PubMedID 15565796

• Radiation-force assisted targeting facilitates ultrasonic molecular imaging. Molecular imaging Zhao, S., Borden, M., Bloch, S. H., Kruse, D., Ferrara, K. W., Dayton, P. A. 2004; 3 (3): 135-148

  Abstract

  Ultrasonic molecular imaging employs contrast agents, such as microbubbles, nanoparticles, or liposomes, coated with ligands specific for receptors expressed on cells at sites of angiogenesis, inflammation, or thrombus. Concentration of these highly echogenic contrast agents at a target site enhances the ultrasound signal received from that site, promoting ultrasonic detection and analysis of disease states. In this article, we show that acoustic radiation force can be used to displace targeted contrast agents to a vessel wall, greatly increasing the number of agents binding to available surface receptors. We provide a theoretical evaluation of the magnitude of acoustic radiation force and show that it is possible to displace micron-sized agents physiologically relevant distances. Following this, we show in a series of experiments that acoustic radiation force can enhance the binding of targeted agents: The number of biotinylated microbubbles adherent to a synthetic vessel coated with avidin increases as much as 20-fold when acoustic radiation force is applied; the adhesion of contrast agents targeted to alpha(v)beta3 expressed on human umbilical vein endothelial cells increases 27-fold within a mimetic vessel when radiation force is applied; and finally, the image signal-to-noise ratio in a phantom vessel increases up to 25 dB using a combination of radiation force and a targeted contrast agent, over use of a targeted contrast agent alone.

  View details for PubMedID 15530249

• A method for radiation-force localized drug delivery using gas-filled lipospheres IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Shortencarier, M. J., Dayton, P. A., Bloch, S. H., Schumann, P. A., Matsunaga, T. O., Ferrara, K. W. 2004; 51 (7): 822-831

  Abstract

  We have developed a method using ultrasound and acoustically active lipospheres (AALs) that might be used to deliver bioactive substances to the vascular endothelium. The AALs consist of a small gas bubble surrounded by a thick oil shell and enclosed by an outermost lipid layer. The AALs are similar to ultrasound contrast agents: they can be nondestructively deflected using ultrasound radiation force, and fragmented with high-intensity ultrasound pulses. The lipid-oil complex might be used to carry bioactive substances at high concentrations. An optimized sequence of ultrasound pulses can deflect the AALs toward a vessel wall then disrupt them, painting their contents across the vascular endothelium. This paper presents results from a series of in vitro and ex vivo experiments demonstrating localization of a fluorescent model drug. In experiments using a human melanoma cell (A2085) monolayer, a specific radiation force-fragmentation ultrasound pulse sequence increased cell fluorescence more than 10-fold over no ultrasound or fragmentation pulses alone, and by 50% over radiation force pulses alone. We observe that dye transfer is limited to cells that are in the region of ultrasonic focus, indicating that the application of radiation force pulses to bring the delivery vehicle into proximity with the cell is required for successful adhesion of the vehicle fragments to the cell membrane. We also demonstrate dye transfer from flowing AALs, both in a mimetic vessel and in excised rat cecum. We believe that this method could be successfully used for drug delivery in vivo.

  View details for PubMedID 15301001

• Quantitative evaluation of perfusion and permeability of peripheral tumors using contrast-enhanced computed tomography INVESTIGATIVE RADIOLOGY Pollard, R. E., Garcia, T. C., Stieger, S. M., Ferrara, K. W., Sadlowski, A. R., Wisner, E. R. 2004; 39 (6): 340-349

  Abstract

  Our purpose was to validate contrast-enhanced computed tomography (CECT)-derived quantitative measures of perfusion and permeability against gold standard techniques of fluorescent microspheres and Evan's Blue dye, respectively.Normal and tumor-bearing (R3230AC) Fischer 344 rats were used. CECT perfusion measurements of normal and tumor tissue were compared with quantitative fluorescent microsphere perfusion measures. CECT permeability measurements from tumors were compared with semiquantitative Evan's Blue Dye permeability estimates. CT images were obtained precontrast and an imaging plane was selected. Serial, stationary images were obtained every 2 seconds for 2 minutes after intravenous bolus of iodinated contrast. Permeability and perfusion were measured by applying Patlak analysis to time-density data from normal tissue or tumor and femoral artery.There was good correlation between fluorescent microsphere and CECT measurements of perfusion (r2 = 0.681, P < 0.001) and between Evan's Blue Dye and CECT measurements of permeability (r2 = 0.873, P = 0.0007).CECT provides useful, quantifiable measures of perfusion and permeability in peripheral tumors.

  View details for DOI 10.1097/01rli.0000124456.82985.35

  View details for PubMedID 15167100

• Ultrasonic Analysis of Peptide-and Antibody-Targeted Microbubble Contrast Agents for Molecular Imaging of alpha(V)beta(3)-Expressing Cells MOLECULAR IMAGING Dayton, P. A., Pearson, D., Clark, J., Simon, S., Schumann, P. A., Zutshi, R., Matsunaga, T. O., Ferrara, K. W. 2004; 3 (2): 125-134

  Abstract

  The goal of targeted ultrasound contrast agents is to significantly and selectively enhance the detection of a targeted vascular site. In this manuscript, three distinct contrast agents targeted to the alphavbeta3 integrin are examined. The alphavbeta3 integrin has been shown to be highly expressed on metastatic tumors and endothelial cells during neovascularization, and its expression has been shown to correlate with tumor grade. Specific adhesion of these contrast agents to alphavbeta3-expressing cell monolayers is demonstrated in vitro, and compared with that of nontargeted agents. Acoustic studies illustrate a backscatter amplitude increase from monolayers exposed to the targeted contrast agents of up to 13-fold (22 dB) relative to enhancement due to control bubbles. A linear dependence between the echo amplitude and bubble concentration was observed for bound agents. The decorrelation of the echo from adherent targeted agents is observed over successive pulses as a function of acoustic pressure and bubble density. Frequency-domain analysis demonstrates that adherent targeted bubbles exhibit high-amplitude narrowband echo components, in contrast to the primarily wideband response from free microbubbles. Results suggest that adherent targeted contrast agents are differentiable from free-floating microbubbles, that targeted contrast agents provide higher sensitivity in the detection of angiogenesis, and that conventional ultrasound imaging techniques such as signal subtraction or decorrelation detection can be used to detect integrin-expressing vasculature with sufficient signal-to-noise.

  View details for PubMedID 15296677

• Acoustic signatures of submicron contrast agents IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Patel, D. N., Bloch, S. H., Dayton, P. A., Ferrara, K. W. 2004; 51 (3): 293-301

  Abstract

  Previous studies have revealed that hard-shelled submicron contrast agents exhibit large relative expansions and strong acoustical echoes that can be observed experimentally, and predicted by theoretical simulations. In this paper, we study harmonic imaging and pulse-pair imaging techniques designed to assist in the differentiation of these contrast agents from tissue. For harmonic imaging, we apply a high-sensitivity, narrowband strategy that differentiates the microbubble from tissue based on the generation of strong harmonic echoes. For pulse-pair imaging, we apply high spatial resolution, wideband strategies using phase inversion, which relies on the frequency differences observed in response to phase-inverted pulses, and signal subtraction, which takes advantage of the amplitude differences in response to identical pulses. The bubble-to-phantom signal amplitude ratio in the absence of motion approaches 20 dB using phase inversion and 30 dB using signal subtraction; both techniques are robust for up to 50 microm of simulated motion. With the experience gained in these studies, we hope to advance the development of multi-pulse or shaped-pulse techniques that are optimized for specific clinical applications.

  View details for PubMedID 15128216

• Optical observation of lipid- and polymer-shelled ultrasound microbubble contrast agents APPLIED PHYSICS LETTERS Bloch, S. H., Wan, M., Dayton, P. A., Ferrara, K. W. 2004; 84 (4): 631–33

  View details for DOI 10.1063/1.1643544

  View details for Web of Science ID 000188316500059

• Physico-chemical properties of the microbubble lipid shell - Composition, microstructure & properties of targeted ultrasound contrast agents Borden, M. A., Dayton, P., Zhao, S. K., Ferrara, K. W., Yuhas, M. P. IEEE. 2004: 20–23

  View details for Web of Science ID 000228557207006

• High frequency contrast imaging Sun, Y., Kruse, D. E., Dayton, P. A., Ferrara, K. W., Yuhas, M. P. IEEE. 2004: 990–93

  View details for Web of Science ID 000228557201045

• Increasing binding efficiency of ultrasound targeted agents with radiation force Zhao, S. K., Borden, M., Bloch, S. H., Kruse, D. E., Ferrara, K. W., Dayton, P. A., Yuhas, M. P. IEEE. 2004: 1114–17

  View details for Web of Science ID 000228557201075

• Contrast-enhanced US of microcirculation of superficially implanted tumors in rats RADIOLOGY Chomas, J. E., Pollard, R. E., Sadlowski, A. R., Griffey, S. M., Wisner, E. R., Ferrara, K. W. 2003; 229 (2): 439-446

  Abstract

  To determine the ability of contrast material-enhanced ultrasonography (US) to assess replenishment time in a rat kidney and adenocarcinoma tumor model.Mammary adenocarcinoma cells were implanted into the subcutaneous tissues of the flank of 11 rats. Resultant tumors were imaged serially with contrast-enhanced US and compared with images of the rat kidney, a highly perfused normal organ. The US acquisition and processing methods yield images of perfused tumor regions and the times required to achieve 80% replenishment. Findings at contrast-enhanced computed tomography (CT) and light microscopy of hematoxylin-eosin-stained tumor tissue were compared. Paired Student t test was performed to compare the accuracy of US with that of histologic examination and CT in the detection of viable tumor regions.Replenishment of the kidney cortex microvasculature requires 1-5 seconds compared with a replenishment time of 6-14 seconds in tumors. Over the time course of tumor growth, the mean perfusion time becomes progressively longer, and a wider range of perfusion times is detected. Comparison of findings at US, CT, and histologic examination suggested that all three methods yield correlated estimates of the percentage of viable perfused tumor cells. Results of the t test suggested that the viable tumor percentages observed at US are not significantly different from those observed at CT and histologic examination (US vs CT, P =.92; US vs histologic examination, P =.94).Repeated measurements of microvascular flow rate can be accomplished in a rat animal model with a minimally invasive technique.

  View details for PubMedID 14526091

• Effect of coupled oscillations on microbubble behavior JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Allen, J. S., Kruse, D. E., Dayton, P. A., Ferrara, K. W. 2003; 114 (3): 1678-1690

  Abstract

  Ultrasound contrast agents are encapsulated microbubbles whose nonlinear acoustic scattering signatures have been the foundation of their use in diagnostic imaging. The coupled oscillations of microbubbles along their lines of center are investigated theoretically using radial equations in the monopole approximation and an energy balance, which is obtained for the system. Coupled microbubble pairs of different initial radii are investigated numerically relative to the normal modes for the linearized system. For microbubble pairs of different size bubbles driven below the mode of the smaller bubble and above the mode of the larger bubble, it is shown that oscillations of the smaller agent are affected substantially more by the coupling than those of the larger one. For separation distances of 10 and 500 microns, a difference of approximately 10 dB occurs in the second harmonic output of a 1.0-micron radius agent coupled with a 2.2-micron radius agent forced at 2.0 MHz and 0.3 MPa. The subharmonic spectral peak is shown to decrease approximately 19 dB for the coupling of 1.5- and 2.2-micron radius agents at 10- and 500-micron distances under the same acoustic forcing conditions. These coupling effects on the radiated pressure and its spectral power are highlighted for contrast agent imaging applications.

  View details for DOI 10.1121/1.1600721

  View details for PubMedID 14514221

• Sentinel node detection using contrast-enhanced power Doppler ultrasound lymphography INVESTIGATIVE RADIOLOGY Wisner, E. R., Ferrara, K. W., Short, R. E., Ottoboni, T. B., Gabe, J. D., Patel, D. 2003; 38 (6): 358-365

  Abstract

  To establish the feasibility of using contrast-enhanced interstitial ultrasound (US) lymphography as an alternative to current sentinel node detection methods.Aqueous US contrast microbubble suspensions of varying diameter were evaluated in vitro to characterize response to insonation. Contrast media were then injected subcutaneously into the distal extremities of 11 normal dogs to target the cervical and popliteal lymph nodes (nodes, n = 40). First-order (sentinel) lymph nodes and second-order sublumbar nodes were imaged intermittently from 0 to at least 120 minutes following contrast injection using continuous power Doppler mode. Lymphoscintigraphy studies were performed on 4 dogs to verify lymphatic drainage patterns and sentinel lymph nodes.Contrast enhancement occurred in 34/40 (85%) sentinel nodes overall and in 30/32 (94%) nodes when submicron or near-micron diameter bubble formulations were used. In many instances, enhancement persisted throughout the imaging period. Contrast response was most pronounced using a high mechanical index and tissue artifact was reduced or eliminated when using a high pulse repetition frequency.Contrast-enhanced interstitial US lymphography could serve as an alternative to current sentinel node detection methods. Preliminary findings suggest that submicron or near-micron-diameter bubbles may be suitable for lymphatic imaging applications.

  View details for DOI 10.1097/01.rli.0000065423.05038.88

  View details for PubMedID 12908703

• Ultrasound as a low cost, practical method of functional imaging Ferrara, K., Dayton, P., Wisner, E., Pollard, R. AMER ASSOC PHYSICISTS MEDICINE AMER INST PHYSICS. 2003: 1374

  View details for Web of Science ID 000183658500243

• Ciliary body blood perfusion after cyclophotocoagulation using high frequency ultrasound imaging Lim, M. C., Kruse, D. E., Redline, D. E., Eisele, P. H., Ferrara, K. W. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2003: U25

  View details for Web of Science ID 000184606800121

• Visualization of blood-flow in the ciliary processes with high-frequency ultrasound Silverman, R. H., Kruse, D., Ferrara, K. W., Cannata, J., Shung, K. K., Chabi, A., Coleman, D. J. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2003: U286

  View details for Web of Science ID 000184607001071

• A new imaging strategy utilizing wideband transient response of ultrasound contrast agents Kruse, D. E., Yeh, C. K., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2003: 1081–85

  View details for Web of Science ID 000189492100243

• Detection of polymer-shelled ultrasound contrast agents using a signal subtraction method Bloch, S. H., Patel, D. N., Dayton, P. A., Wan, M., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2003: 416–19

  View details for Web of Science ID 000189492100091

• A new imaging strategy utilizing wideband transient response of ultrasound contrast agents Kruse, D. E., Yeh, C. K., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2003: 424–28

  View details for Web of Science ID 000189492100093

• A new high frequency destruction/reperfusion system Yeh, C. K., Kruse, D. E., Lim, M. C., Redline, D. E., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2003: 433–36

  View details for Web of Science ID 000189492100095

• Ultrasonic enhancement of alpha(v)beta(3) expressing-cells with targeted contrast agents Dayton, P. A., Pearson, D., Clark, J., Simon, S., Schumann, P., Zutshi, R., Matsunaga, T., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2003: 540–43

  View details for Web of Science ID 000189492100117

• Estimation of vascular density and blood velocity in tumors Sadlowski-Broumas, A., Pollard, R., Taylor, A., Griffey, S., Wisner, E., Ferrara, K., Yuhas, D. E., Schneider, S. C. IEEE. 2003: 645–48

  View details for Web of Science ID 000189492100140

• Contrast-assisted destruction-replenishment ultrasound for the assessment of tumor microvasculature in a rat model. Technology in cancer research & treatment Pollard, R. E., Sadlowski, A. R., Bloch, S. H., Murray, L., Wisner, E. R., Griffey, S., Ferrara, K. W. 2002; 1 (6): 459-470

  Abstract

  Angiogenesis, the development of new blood vessels, is necessary for tumor growth. Anti-angiogenic therapies have recently received attention as a possible cancer treatment. The purpose of this study was to monitor the vascularity of induced tumors in rats using contrast-enhanced ultrasound during anti-angiogenic therapy. Six rats with subcutaneously implanted R3230 murine mammary adenocarcinomas were treated with an orally administered anti-angiogenic agent (SU11657) beginning 28 days after tumor implantation (20 mg/kg BW once daily). Three additional tumor-bearing control rats were treated with an equivalent volume of vehicle alone. Sonographic evaluation of tumor blood flow was performed using a modified Siemens Sonoline Elegra equipped with a 5.0 MHz linear transducer prior to drug administration, during the first 51 hours following initial drug administration, and on days 8 and 15 after initiation of therapy. Tumor volumes were estimated at each time point using a prolate ellipsoid method from linear dimensions measured on the B-mode ultrasound image in the three major axes. A destruction-replenishment technique was used for tumor blood flow evaluation using a constant rate infusion of intravenously delivered ultrasound contrast media (Definity). A destructive pulse was fired first, followed by a chain of non-destructive pulses that allowed for visualization of vascular contrast agent replenishment. Parametric maps of the time required for contrast agent replenishment and the time-integrated intensity were generated for both the tumor and kidney. Following ultrasound examination, contrast-enhanced computed tomography of each tumor was performed in the same imaging plane as that used to acquire the ultrasound images. Fifteen days after the start of treatment, tumors were excised, preserved in 10% formalin, and sectioned in a plane approximating the ultrasound and CT imaging planes. Sections were prepared for light microscopy with H & E, CD31 and factor VIII immunostain to evaluate overall morphology and vessel distribution. Ultrasound measurements of tumor volume, the spatial extent of contrast enhancement, and the time required for contrast replenishment within control tumors were significantly different from those of treated tumors. The time-integrated ultrasound contrast enhancement decreases and the time required for replenishment of the contrast agent within the tumor volume increases over the course of anti-angiogenic therapy. Parametric maps of integrated intensity are shown to correlate with the regions of viable tumor demonstrated on H & E and regions of elevated contrast intensity on CT. Contrast-enhanced ultrasound imaging of implanted tumors provides a tool to assess differences in the microcirculation of treated and control tumors in studies of anti-angiogenic agents.

  View details for PubMedID 12625773

• A new high resolution color flow system using an eigendecomposition-based adaptive filter for clutter rejection. IEEE transactions on ultrasonics, ferroelectrics, and frequency control Kruse, D. E., Ferrara, K. W. 2002; 49 (12): 1739-1754

  Abstract

  We present a new signal processing strategy for high frequency color flow mapping in moving tissue environments. A new application of an eigendecomposition-based clutter rejection filter is presented with modifications to deal with high blood-to-clutter ratios (BCR). Additionally, a new method for correcting blood velocity estimates with an estimated tissue motion profile is detailed. The performance of the clutter filter and velocity estimation strategies is quantified using a new swept-scan signal model. In vivo color flow images are presented to illustrate the potential of the system for mapping blood flow in the microcirculation with external tissue motion.

  View details for PubMedID 12546154

• Optical and acoustical interrogation of submicron contrast agents IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Patel, D., Dayton, P., Gut, J., Wisner, E., Ferrara, K. W. 2002; 49 (12): 1641-1651

  Abstract

  Unlike conventional ultrasound contrast agents with a diameter of several microns, in this paper we explore the use of submicron contrast agents for the detection and localization of lymph nodes. The submicron agents are gas-filled, double-walled microspheres that rupture when exposed to ultrasound energy at megahertz frequencies. In this study, three experimental systems are combined with model predictions to assist in understanding the response of these unique agents to a range of signal transmission parameters. Optical experimental results for each agent delineate the relative expansion as a function of acoustical peak negative pressure, pulse length, and center frequency. The optical images demonstrate an order of magnitude expansion in radius during the pulse rarefaction, in which the expansion magnitude is dependent on the transmitted pressure and frequency. Simulations using a modified Rayleigh-Plesset model predict an increasing relative expansion for the microbubbles (initial bubble radius ranging from 0.3-1.3 microm) with increasing pressure and decreasing initial radius. Acoustically recorded frequency spectra reveal the presence of harmonics for a range of transmitted pulses. In addition, in-vivo results from a normal canine model demonstrate marked contrast enhancement of first order lymph nodes. We hope to offer an alternative to present intra-operative procedures for sentinel node detection.

  View details for PubMedID 12546146

• Ultrasound measurement of the effect of temperature on microperfusion in the eye ULTRASOUND IN MEDICINE AND BIOLOGY Silverman, R. H., Ursea, R., Kruse, D., Ferrara, K. W., Rondeau, M. J., Coleman, D. J. 2002; 28 (11-12): 1413-1419

  Abstract

  Recent developments in ultrasound (US) technology have allowed the study of microperfusion in the anterior segment of the eye. Our aim was to determine the effect of the thermal environment on blood flow in the anterior segment. We measured blood flow in the major arterial circle of five rabbits. A 38-MHz US transducer was coupled to the eye with a normal saline water-bath with temperature controlled from 1 degrees C to 38 degrees C. The major arterial circle was localized and imaged using the swept-scan technique and M-mode data were then acquired for measurement of pulsatile flow. Peak systolic and mean velocity averaged 4.51 and 1.32 mm/s, respectively. Positive correlations were found between peak systolic (1.69%/ degrees C) and mean (1.76%/ degrees C) velocities and temperature. Vessel diameter (mean = 178 microm) did not show any significant change with temperature. High-resolution US flowmetry demonstrated decreasing flow rates in the iris with decreasing temperature.

  View details for Web of Science ID 000180041500006

  View details for PubMedID 12498936

• The magnitude of radiation force on ultrasound contrast agents JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Dayton, P. A., Allen, J. S., Ferrara, K. W. 2002; 112 (5): 2183-2192

  Abstract

  High-speed photography of insonified bubbles with a time resolution of 10 ns allows observations of translation due to radiation force, in addition to the visualization of radial oscillations. A modified version of the Rayleigh-Plesset equation is used to estimate the radius-time behavior of insonified microbubbles, and the accuracy of this model is verified experimentally. The translation of insonified microbubbles is calculated using a differential equation relating the acceleration of the bubble to the forces due to acoustic radiation and the drag imposed by the fluid. Simulations and experiments indicate that microbubbles translate significant distances with clinically relevant parameters. A 1.5 micron radius contrast agent can translate over 5 microns during a single 20-cycle, 2.25 MHz, 380 kPa acoustic pulse, achieving velocities over 0.5 m/s. Therefore, radiation force should be considered during an ultrasonic examination because of the possibility of influencing the position and flow velocity of the contrast agents with the interrogating acoustic beam.

  View details for DOI 10.1121/1.1509428

  View details for PubMedID 12430830

• Dynamics and fragmentation of thick-shelled microbubbles IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL May, D. J., Allen, J. S., Ferrara, K. W. 2002; 49 (10): 1400-1410

  Abstract

  Localized delivery could decrease the systemic side effects of toxic chemotherapy drugs. The unique delivery agents we examine consist of microbubbles with an outer lipid coating, an oil layer, and a perfluorobutane gas core. These structures are 0.5-12 microm in radius at rest. Oil layers of these acoustically active lipospheres (AALs) range from 0.3-1.5 microm in thickness and thus the agents can carry a large payload compared to nano-scale drug delivery systems. We show that triacetin-based drug-delivery vehicles can be fragmented using ultrasound. Compared with a lipid-shelled contrast agent, the expansion of the drug-delivery vehicle within the first cycle is similar, and a subharmonic component is demonstrated at an equivalent radius, frequency, and driving pressure. For the experimental conditions explored here, the pulse length required for destruction of the drug-delivery vehicle is significantly greater, with at least five cycles required, compared with one cycle for the contrast agent. For the drug-delivery vehicle, the observed destruction mechanism varies with the initial radius, with microbubbles smaller than resonance size undergoing a symmetric collapse and producing a set of small, equal-sized fragments. Between resonance size and twice resonance size, surface waves become visible, and the oscillations become asymmetrical. For agents larger than twice the resonance radius, the destruction mechanism changes to a pinch-off, with one fragment containing a large fraction of the original volume.

  View details for PubMedID 12403141

• A new high resolution color flow system using an eigendecomposition-based adaptive filter for clutter rejection IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Kruse, D. E., Ferrara, K. W. 2002; 49 (10): 1384-1399

  Abstract

  We present a new signal processing strategy for high frequency color flow mapping in moving tissue environments. A new application of an eigendecomposition-based clutter rejection filter is presented with modifications to deal with high blood-to-clutter ratios (BCR). Additionally, a new method for correcting blood velocity estimates with an estimated tissue motion profile is detailed. The performance of the clutter filter and velocity estimation strategies is quantified using a new swept-scan signal model. In vivo color flow images are presented to illustrate the potential of the system for mapping blood flow in the microcirculation with external tissue motion.

  View details for PubMedID 12403140

• Targeted imaging using ultrasound JOURNAL OF MAGNETIC RESONANCE IMAGING Dayton, P. A., Ferrara, K. W. 2002; 16 (4): 362-377

  Abstract

  The discipline of medical imaging is expanding to include both traditional anatomic modalities and new techniques for the functional assessment of the presence and extent of disease. Current FDA-approved ultrasound contrast agents are micron-sized bubbles with a stabilizing shell. Microbubble contrast agents can be used to estimate microvascular flow rate in a manner similar to dynamic contrast-enhanced magnetic resonance imaging (MRI). The concentration of these agents within the vasculature, reticulo-endothelial, or lymphatic systems produces an effective passive targeting of these areas. Liquid-filled nanoparticles and liposomes have also demonstrated echogenicity and are under evaluation as ultrasound contrast agents. Actively targeted ultrasound relies on specially designed contrast agents to localize the targeted molecular signature or physiologic system. These agents typically remain within the vascular space, and therefore possible targets include molecular markers on thrombus, endothelial cells, and leukocytes. The purpose of this review is to summarize the requirements, challenges, current progress, and future directions of targeted imaging with ultrasound.

  View details for DOI 10.1002/jmri.10173

  View details for PubMedID 12353252

• Contrast enhanced intermittent Power Doppler ultrasound with sub-micron bubbles for sentinel node detection ACADEMIC RADIOLOGY Wisner, E. R., Ferrara, K., Gabe, J. D., Patel, D., Nyland, T. G., Short, R. E., Ottoboni, T. B. 2002; 9: S389-S391

  View details for PubMedID 12188286

• Nondestructive subharmonic imaging IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Chomas, J., Dayton, P., May, D., Ferrara, K. 2002; 49 (7): 883-892

  Abstract

  Ultrasound contrast agent microbubbles are intravascular agents that can be used to estimate blood perfusion. Blood perfusion may be estimated by destroying the bubbles in a vascular bed and observing the refresh of contrast agents back into the vascular bed. Contrast agents can be readily destroyed by traditional imaging techniques. The design of a nondestructive imaging technique is necessary for the accurate quantification of contrast agent refresh. In this work, subharmonic imaging is investigated as a method for nondestructive imaging with the contrast agent microbubble MP1950 (Mallinckrodt, Inc., St. Louis, MO). Optical observation during insonation, in conjunction with a modified Rayleigh-Plesset (R-P) analysis, provides insight into the mechanisms of and parameters required for subharmonic frequency generation. Subharmonic imaging with a transmission frequency that is the same as the resonant frequency of the bubble is shown to require a minimum pressure of insonation that is greater than the experimentally-observed bubble destruction threshold. Subharmonic imaging with a transmission frequency that is twice the resonant frequency of the bubble produces a subharmonic frequency response while minimizing bubble instability. Optimization is performed using optical experimental analysis and R-P analysis.

  View details for PubMedID 12152942

• Dynamics of therapeutic ultrasound contrast agents ULTRASOUND IN MEDICINE AND BIOLOGY Allen, J. S., May, D. J., Ferrara, K. W. 2002; 28 (6): 805-816

  Abstract

  Novel therapeutic contrast agents offer great potential for localized drug delivery. Localized delivery should significantly improve the efficacy of drug delivery and reduce any toxic exposure to the healthy tissue. This work describes a preliminary theoretical description of agents, such as those developed by the ImaRx Corporation, enclosed by a relatively thick fluid shell. A theoretical extension is made to a generalized Rayleigh-Plesset formulation that allows it to be solved for an encapsulating liquid shell of arbitrary thickness and density. The equation is used to investigate the role of shell thickness, density and viscosity on the radial dynamics and velocity of the inner and outer radii. Comparisons are made with experimental measurements of the maximum radial expansions for agents with triacetin shells. For a seven-cycle driving acoustic pulse with a center frequency of 1.5 MHz and peak amplitude of 1.6 MPa, the equation predicts maximum expansions from 5.5 to 1.3 times the initial radius for agents 1 to 10 microm, respectively, in initial radius with a 500-nm (28.0 cP) encapsulating shell. These predictions have reasonable agreement with the maximum radial expansions obtained from optical experimental data of fragmenting and intact agents. Approximate agreement between theory and experiment for a similar range of agent sizes is also demonstrated for a pulse with the same pressure amplitude at 2.5 MHz. At 2.5 MHz, smaller radial expansion amplitudes from 1.1 to 4.1 times the initial radius were found for agents 1 to 10 microm in initial radius, respectively. Discrepancies are attributed to shape instabilities and their associated fragmentation effects not incorporated in the equation. A significant difference in the inner and outer wall velocities is predicted for agents with a 500-nm triacetin shell. A 2.5 microm initial radius agent driven with a seven-cycle pulse at 2.5 MHz and 1.6 MPa achieves a maximum negative inner wall velocity of 364 m/s and outer wall velocity of 63 m/s. For parameters that correspond to large differences between the inner and outer wall velocities, fragmentation is typically observed experimentally.

  View details for PubMedID 12113793

• Mean flow rate and integrated perfusion estimates obtained with contrast-assisted ultrasound Sadlowski, A., Chomas, J., Pollard, R., Bloch, S., Griffey, S., Wisner, E., Ferrara, K., Yuhas, D. E., Schneider, S. C. IEEE. 2002: 1977–80

  View details for Web of Science ID 000182111700446

• High frequency ultrasound with an eigen-decomposition filter to assess the effect of laser cyclophotocoagulation treatment on blood flow Kruse, D. E., Lim, M. C., Redline, D. E., Eisele, P. H., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2002: 1485–89

  View details for Web of Science ID 000182111700337

• Threshold of fragmentation for ultrasonic contrast agents JOURNAL OF BIOMEDICAL OPTICS Chomas, J. E., Dayton, P., May, D., Ferrara, K. 2001; 6 (2): 141-150

  Abstract

  Ultrasound contrast agents are small microbubbles that can be readily destroyed with sufficient acoustic pressure, typically, at a frequency in the low megaHertz range. Microvascular flow rate may be estimated by destroying the contrast agent in a vascular bed, and estimating the rate of flow of contrast agents back into the vascular bed. Characterization of contrast agent destruction provides important information for the design of this technique. In this paper, high-speed optical observation of an ultrasound contrast agent during acoustic insonation is performed. The resting diameter is shown to be a significant parameter in the prediction of microbubble destruction, with smaller diameters typically correlated with destruction. Pressure, center frequency, and transmission phase are each shown to have a significant effect on the fragmentation threshold. A linear prediction for the fragmentation threshold as a function of pressure, when normalized by the resting diameter, has a rate of change of 300 kPa/microm for the range of pressures from 310 to 1200 kPa, and a two-cycle excitation pulse with a center frequency of 2.25 MHz. A linear prediction for the fragmentation threshold as a function of frequency, when normalized by the resting diameter, has a rate of change of -1.2 MHz/microm for a transmission pressure of 800 kPa, and a two-cycle excitation pulse with a range of frequencies from 1 to 5 MHz.

  View details for PubMedID 11375723

• Temperature-dependence of blood-flow in the major arterial circle. Ursea, R., Silverman, R. H., Kruse, D. E., Ferrara, K. W., Raevski, T., Lloyd, H. O., Coleman, D. J. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2001: S525

  View details for Web of Science ID 000168392102787

• Shell waves and acoustic scattering from ultrasound contrast agents IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Allen, J. S., Kruse, D. E., Ferrara, K. W. 2001; 48 (2): 409-418

  Abstract

  Ultrasound contrast agents are encapsulated microbubbles, filled either with air or a higher weight molecular gas, ranging in size from 1 to 10 microns in diameter. The agents are modeled as air-filled spherical elastic shells of variable thickness and material properties. The scattered acoustic field is computed from a modal series solution, and reflectivity and angular scattering are then determined from the computed fields for agents of various properties. We show that contrast agents also support shell resonance responses in addition to the monopole response, which has been the focus of previous contrast agent studies. Lamb waves appear to be the source of these additional responses. A shell or curvature Lamb wave generates dipole peaks in the 1- to 40-MHz range for 2.5 to 3.5 microns radius agents with elastic properties approximating those of albumin protein. The inclusion of damping affects the lower frequency dipole peaks but is less important for responses occurring above approximately 30 MHz. Moreover, these responses hold untapped potential for clinical ultrasound applications such as tissue perfusion studies and high frequency contrast agent imaging.

  View details for PubMedID 11370354

• Optical and acoustical dynamics of microbubble contrast agents inside neutrophils BIOPHYSICAL JOURNAL Dayton, P. A., Chomas, J. E., Lum, A. F., Allen, J. S., Lindner, J. R., Simon, S. I., Ferrara, K. W. 2001; 80 (3): 1547-1556

  Abstract

  Acoustically active microbubbles are used for contrast-enhanced ultrasound assessment of organ perfusion. In regions of inflammation, contrast agents are captured and phagocytosed by activated neutrophils adherent to the venular wall. Using direct optical observation with a high-speed camera and acoustical interrogation of individual bubbles and cells, we assessed the physical and acoustical responses of both phagocytosed and free microbubbles. Optical analysis of bubble radial oscillations during insonation demonstrated that phagocytosed microbubbles experience viscous damping within the cytoplasm and yet remain acoustically active and capable of large volumetric oscillations during an acoustic pulse. Fitting a modified version of the Rayleigh-Plesset equation that describes mechanical properties of thin shells to optical radius-time data of oscillating bubbles provided estimates of the apparent viscosity of the intracellular medium. Phagocytosed microbubbles experienced a viscous damping approximately sevenfold greater than free microbubbles. Acoustical comparison between free and phagocytosed microbubbles indicated that phagocytosed microbubbles produce an echo with a higher mean frequency than free microbubbles in response to a rarefaction-first single-cycle pulse. Moreover, this frequency increase is predicted using the modified Rayleigh-Plesset equation. We conclude that contrast-enhanced ultrasound can detect distinct acoustic signals from microbubbles inside of neutrophils and may provide a unique tool to identify activated neutrophils at sites of inflammation.

  View details for PubMedID 11222315

• Mechanisms of contrast agent destruction IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Chomas, J. E., Dayton, P., Allen, J., Morgan, K., Ferrara, K. W. 2001; 48 (1): 232-248

  Abstract

  Various applications of contrast-assisted ultrasound, including blood vessel detection, perfusion estimation, and drug delivery, require controlled destruction of contrast agent microbubbles. The lifetime of a bubble depends on properties of the bubble shell, the gas core, and the acoustic waveform impinging on the bubble. Three mechanisms of microbubble destruction are considered: fragmentation, acoustically driven diffusion, and static diffusion. Fragmentation is responsible for rapid destruction of contrast agents on a time scale of microseconds. The primary characteristics of fragmentation are a very large expansion and subsequent contraction, resulting in instability of the bubble. Optical studies using a novel pulsed-laser optical system show the expansion and contraction of ultrasound contrast agent microbubbles with the ratio of maximum diameter to minimum diameter greater than 10. Fragmentation is dependent on the transmission pressure, occurring in over 55% of bubbles insonified with a peak negative transmission pressure of 2.4 MPa and in less than 10% of bubbles insonified with a peak negative transmission pressure of 0.8 MPa. The echo received from a bubble decorrelates significantly within two pulses when the bubble is fragmented, creating an opportunity for rapid detection of bubbles via a decorrelation-based analysis. Preliminary findings with a mouse tumor model verify the occurrence of fragmentation in vivo. A much slower mechanism of bubble destruction is diffusion, which is driven by both a concentration gradient between the concentration of gas in the bubble compared with the concentration of gas in the liquid, as well as convective effects of motion of the gas-liquid interface. The rate of diffusion increases during insonation, because of acoustically driven diffusion, producing changes in diameter on the time scale of the acoustic pulse length, thus, on the order of microseconds. Gas bubbles diffuse while they are not being insonified, termed static diffusion. An air bubble with initial diameter of 2 microns in water at 37 degrees C is predicted to fully dissolve within 25 ms. Clinical ultrasound contrast agents are often designed with a high molecular weight core in an attempt to decrease the diffusion rate. C3F8 and C4F10 gas bubbles of the same size are predicted to fully dissolve within 400 ms and 4000 ms, respectively. Optical experiments involving gas diffusion of a contrast agent support the theoretical predictions; however, shelled agents diffuse at a much slower rate without insonation, on the order of minutes to hours. Shell properties play a significant role in the rate of static diffusion by blocking the gas-liquid interface and decreasing the transport of gas into the surrounding liquid. Static diffusion decreases the diameter of albumin-shelled agents to a greater extent than lipid-shelled agents after insonation.

  View details for PubMedID 11367791

• Acoustic fragmentation of therapeutic contrast agents designed for localized drug delivery May, D., Allen, J., Gut, J., Ferrara, K., Yuhas, D. E., Schneider, S. C. IEEE. 2001: 1385–88

  View details for Web of Science ID 000176890800304

• A new high resolution color flow system using an eigendecomposition-based adaptive filter for clutter rejection Kruse, D. E., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2001: 1439–43

  View details for Web of Science ID 000176890800316

• A hand-held, high frequency ultrasound scanner Erickson, S., Kruse, D., Ferrara, K., Yuhas, D. E., Schneider, S. C. IEEE. 2001: 1465–68

  View details for Web of Science ID 000176890800321

• Experimental validation of a theoretical framework to predict radiation force displacement of contrast agents Dayton, P. A., Allen, J. S., Kruse, D. E., Ferrara, K. W., Yuhas, D. E., Schneider, S. C. IEEE. 2001: 1687–90

  View details for Web of Science ID 000176890800371

• Submicron contrast agents for the detection and localization of at risk lymph nodes Patel, D., Dayton, P., Gut, J., Wisner, E., Ferrara, K., Yuhas, D. E., Schneider, S. C. IEEE. 2001: 1717–20

  View details for Web of Science ID 000176890800377

• Subharmonic phase-inversion for tumor perfusion estimation Chomas, J. E., Pollard, R., Wisner, E., Ferrara, K., Yuhas, D. E., Schneider, S. C. IEEE. 2001: 1713–16

  View details for Web of Science ID 000176890800376

• Optimization of real-time high frequency ultrasound for blood flow imaging in the microcirculation Kruse, D. E., Mai, J. J., Silverman, R. H., Insana, M. F., Coleman, D. J., Ferrara, K. W., Insana, M. F., Shung, K. K. SPIE-INT SOC OPTICAL ENGINEERING. 2001: 284–92

  View details for DOI 10.1117/12.428203

  View details for Web of Science ID 000171334900028

• Experimental and theoretical evaluation of microbubble behavior: Effect of transmitted phase and bubble size IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Morgan, K. E., Allen, J. S., Dayton, P. A., Chomas, J. E., Klibanov, A. L., Ferrara, K. W. 2000; 47 (6): 1494–1509

  Abstract

  Ultrasound contrast agents provide new opportunities to image vascular volume and flow rate directly. To accomplish this goal, new pulse sequences can be developed to detect specifically the presence of a microbubble or group of microbubbles. We consider a new scheme to detect the presence of contrast agents in the body by examining the effect of transmitted phase on the received echoes from single bubbles. In this study, three tools are uniquely combined to aid in the understanding of the effects of transmission parameters and bubble radius on the received echo. These tools allow for optical measurement of radial oscillations of single bubbles during insonation, acoustical study of echoes from single contrast agent bubbles, and the comparison of these experimental observations with theoretical predictions. A modified Herring equation with shell terms is solved for the time-dependent bubble radius and wall velocity, and these outputs are used to formulate the predicted echo from a single encapsulated bubble. The model is validated by direct comparison of the predicted radial oscillations with those measured optically. The transient bubble response is evaluated with a transducer excitation consisting of one-cycle pulses with a center frequency of 2.4-MHz. The experimental and theoretical results are in good agreement and predict that the transmission of two pulses with opposite polarity will yield similar time domain echoes with the first significant portion of the echo generated when the rarefactional half-cycle reaches the bubble.

  View details for PubMedID 18238696

• Evaluation of tumor angiogenesis with US: Imaging, Doppler, and contrast agents ACADEMIC RADIOLOGY Ferrara, K. W., Merritt, C. R., Burns, P. N., Foster, F. S., Mattrey, R. F., Wickline, S. A. 2000; 7 (10): 824-839

  View details for PubMedID 11048880

• Optical observation of contrast agent destruction APPLIED PHYSICS LETTERS Chomas, J. E., Dayton, P. A., May, D., Allen, J., Klibanov, A., Ferrara, K. 2000; 77 (7): 1056–58

  View details for DOI 10.1063/1.1287519

  View details for Web of Science ID 000088788000046

• Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles CIRCULATION Lindner, J. R., Dayton, P. A., Coggins, M. P., Ley, K., Song, J., Ferrara, K., Kaul, S. 2000; 102 (5): 531-538

  Abstract

  We have previously shown that microbubbles adhere to leukocytes in regions of inflammation. We hypothesized that these microbubbles are phagocytosed by neutrophils and monocytes and remain acoustically active, permitting their detection in inflamed tissue.In vitro studies were performed in which activated leukocytes were incubated with albumin or lipid microbubbles and observed under microscopy. Microbubbles attached to the surface of activated neutrophils and monocytes, were phagocytosed, and remained intact for up to 30 minutes. The rate of destruction of the phagocytosed microbubbles on exposure to ultrasound was less (P

  View details for PubMedID 10920065

• 3aBB4. Ultrasound contrast agents retain acoustic activity post phagocytosis by leukocytes (vol 106, pg 2192(A), 1999) JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Dayton, P., Chomas, J., Ferrara, K., Lindner, J., Coggins, M. 2000; 107 (4): 2298

  View details for DOI 10.1121/1.428559

  View details for Web of Science ID 000086367000053

• Imaging of the eyelid with very-high frequency ultrasound. Ursea, R., Ursea, B. G., Silverman, R. H., Reinstein, D. Z., Kruse, D., Ferrara, K. W., Coleman, D. J. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2000: S124

  View details for Web of Science ID 000086246700632

• Slow Intensive Home Hemodialysis (SIHD): the University Hospitals of Cleveland experience. Nephrology news & issues Cacho, C., Ferrara, K., Guthrie, B., Priester, A., Murray, E., Newman, L., Blankschaen, S., Weiss, M. 2000; 14 (4): 36-41

  View details for PubMedID 11075101

• Color flow mapping ULTRASOUND IN MEDICINE AND BIOLOGY Kruse, D., Ferrara, K. 2000; 26: S16-S18

  View details for PubMedID 10794865

• Ultrasound contrast agents used for localized drug delivery May, D., Dayton, P., Chomas, J., Allen, J., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 2000: 1429–32

  View details for Web of Science ID 000171881300313

• Acoustical and physical dynamics of phagocytosed microbubble contrast agents Dayton, P., Chomas, J., Lum, A., Simon, S., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 2000: 1877–80

  View details for Web of Science ID 000171881300412

• Wideband sub-forcing harmonic phase inversion imaging Morgan, K. E., Allen, J. S., Ferrara, K. W., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 2000: 1889–92

  View details for Web of Science ID 000171881300415

• Non-destructive subharmonic imaging Chomas, J., Dayton, P., May, D., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 2000: 1939–42

  View details for Web of Science ID 000171881300425

• Surface wave phenomena on ultrasound contrast agents Allen, J., Kruse, D., May, D., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 2000: 1881–84

  View details for Web of Science ID 000171881300413

• Optimization of real-time high frequency ultrasound for blood flow imaging in the microcirculation Kruse, D., Silverman, R., Erickson, S., Coleman, D. J., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 2000: 1461–64

  View details for Web of Science ID 000171881300321

• Effect of ultrasound on microbubbles after leukoycte phagocytosis Lindner, Dayton, P. A., Coggins, M. P., Kaul, S., Ferrara, K. LIPPINCOTT WILLIAMS & WILKINS. 1999: 203

  View details for Web of Science ID 000083417101050

• Acoustic radiation force in vivo: A mechanism to assist targeting of microbubbles ULTRASOUND IN MEDICINE AND BIOLOGY Dayton, P., Klibanov, A., Brandenburger, G., Ferrara, K. 1999; 25 (8): 1195-1201

  Abstract

  The goal of targeted imaging is to produce an enhanced view of physiological processes or pathological tissue components. Contrast agents may improve the specificity of imaging modalities through selective targeting, and this may be particularly significant when using ultrasound (US) to image inflammatory processes or thrombi. One means of selective targeting involves the attachment of contrast agents to the desired site with the use of a specific binding mechanism. Because molecular binding mechanisms are effective over distances on the order of nanometers, targeting effectiveness would be greatly increased if the agent is initially concentrated in a particular region, and if the velocity of the agent is decreased as it passes the potential binding site. Ultrasonic transmission produces a primary radiation force that can manipulate microbubbles with each acoustic pulse. Observations demonstrate that primary radiation force can displace US contrast agents from the center of the streamline to the wall of a 200-microm cellulose vessel in vitro. Here, the effects of radiation force on contrast agents in vivo are presented for the first time. Experimental results demonstrate that radiation force can displace a contrast agent to the wall of a 50-microm blood vessel in the mouse cremaster muscle, can significantly reduce the velocity of flowing contrast agents, and can produce a reversible aggregation. Acoustic radiation force presents a means to localize and concentrate contrast agents near a vessel wall, which may assist the delivery of targeted agents.

  View details for PubMedID 10576262

• High-resolution ultrasonic imaging of blood flow in the anterior segment of the eye INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Silverman, R. H., Kruse, D. E., Coleman, D. J., Ferrara, K. W. 1999; 40 (7): 1373-1381

  Abstract

  To develop a noninvasive technique to visualize and measure blood flow in the iris and ciliary body.Echo data from 50-MHz ultrasound scans of the iris and ciliary body of rabbits were digitized using a new "swept scan" modality. The method makes use of spatial oversampling to identify regions with scatterers whose range changes with time. The data allowed construction of high-resolution B-mode images with embedded flow information. Pulsatility over the cardiac cycle was evaluated by sending a series of pulses along a single line of sight containing a vessel of interest. Local blood flow and changes over the cardiac cycle before and after application of atropine were quantified.Flow was identified in the radial vessels and major arterial circle of the iris. Vessels with lumens as small as 40 microm in diameter and flow velocities as low as 0.6 mm/sec were measured. Change in blood velocity over the cardiac cycle was determined to be approximately 27%. Peak systolic velocity after administration of topical atropine increased by 72%.This technique allowed visualization of flow using the same type of very-high-frequency transducer now widely used for imaging the anterior segment. The technique can also be used at lower frequencies for more posterior tissues with similar improvement of resolution over Doppler. The ability to examine flow in the anterior segment of the eye offers a new tool for study of glaucoma, hypotony, tumors, and other disorders.

  View details for Web of Science ID 000080531900009

  View details for PubMedID 10359319

• Ultrasonic blood-flow imaging and measurement in the iris and cilliary body Silverman, R. H., Kruse, D., Coleman, D. J., Ferrara, K. W. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 1999: S275

  View details for Web of Science ID 000079269201453

• Optical and acoustical observations of the effects of ultrasound on contrast agents IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Dayton, P. A., Morgan, K. E., Klibanov, A. L., Brandenburger, G. H., Ferrara, K. W. 1999; 46 (1): 220–32

  Abstract

  Optimal use of encapsulated microbubbles for ultrasound contrast agents and drug delivery requires an understanding of the complex set of phenomena that affect the contrast agent echo and persistence. With the use of a video microscopy system coupled to either an ultrasound flow phantom or a chamber for insonifying stationary bubbles, we show that ultrasound has significant effects on encapsulated microbubbles. In vitro studies show that a train of ultrasound pulses can alter the structure of an albumin-shelled bubble, initiate various mechanisms of bubble destruction or produce aggregation that changes the echo spectrum. In this analysis, changes observed optically are compared with those observed acoustically for both albumin and lipid-shelled agents. We show that, when insonified with a narrowband pulse at an acoustic pressure of several hundred kPa, a phospholipid-shelled bubble can undergo net radius fluctuations of at least 15%; and an albumin-shelled bubble initially demonstrates constrained expansion and contraction. If the albumin shell contains air, the shell may not initially experience surface tension; therefore, the echo changes more significantly with repeated pulsing. A set of observations of contrast agent destruction is presented, which includes the slow diffusion of gas through the shell and formation of a shell defect followed by rapid diffusion of gas into the surrounding liquid. These observations demonstrate that the low-solubility gas used in these agents can persist for several hundred milliseconds in solution. With the transmission of a high-pulse repetition rate and a low pressure, the echoes from, contrast agents can be affected by secondary radiation force. Secondary radiation force is an attractive force for these experimental conditions, creating aggregates with distinct echo characteristics and extended persistence. The scattered echo from an aggregate is several times stronger and more narrowband than echoes from individual bubbles.

  View details for PubMedID 18238417

• High resolution blood flow mapping in the anterior segment of the eye Kruse, D., Silverman, R., Coleman, D. J., Erickson, S., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1999: 1477–80

  View details for Web of Science ID 000088356000320

• Optimization of microbubble destruction Chomas, J., Dayton, P., Morgan, K., Allen, J., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1999: 1689–92

  View details for Web of Science ID 000088356000366

• Shell resonance responses in ultrasound contrast agents Allen, J., Kruse, D., Jones, J., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1999: 1677–79

  View details for Web of Science ID 000088356000363

• Experimental and theoretical analysis of individual contrast agent behavior Morgan, K. E., Allen, J. S., Chomas, J. E., Dayton, P. A., Ferrara, K. W., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1999: 1685–88

  View details for Web of Science ID 000088356000365

• Ultrasound contrast agents phagocytosed by neutrophils demonstrate acoustic activity Dayton, P. A., Lindner, Chomas, J. E., Morgan, K. E., Simon, S., Lum, A., May, D., Coggins, M. P., Ferrara, K. W., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1999: 1705–8

  View details for Web of Science ID 000088356000370

• Optical and acoustical studies of ultrasound contrast agents Allen, J., Dayton, P., Chomas, J., Ferrara, K., Nowak, M. D., Adrezin, R. S., Leone, D. J. IEEE. 1999: 81–82

  View details for DOI 10.1109/NEBC.1999.755774

  View details for Web of Science ID 000081429400040

• Direct video microscopic observation of the dynamic effects of medical ultrasound on ultrasound contrast microspheres INVESTIGATIVE RADIOLOGY Klibanov, A. L., Ferrara, K. W., Hughes, M. S., Wible, J. H., Wojdyla, J. K., Dayton, P. A., Morgan, K. E., Brandenburger, G. H. 1998; 33 (12): 863-870

  Abstract

  Ultrasound can cause destruction of microbubble contrast agents used to enhance medical ultrasound imaging. This study sought to characterize the dynamics of this interaction by direct visual observation of microbubbles during insonification in vitro by a medical ultrasound imaging system.Video microscopy was used to observe air-filled sonicated albumin microspheres adsorbed to a solid support during insonation.Deflation was not observed at lowest transmit power settings. At higher intensities, gas left the microparticle gradually, apparently dissolving into the surrounding medium. Deflation was slower for higher microsphere surface densities. Intermittent ultrasound imaging (0.5 Hz refresh rate) caused slower deflation than continuous imaging (33 Hz).Higher concentrations of microbubbles, lower ultrasound transmit power settings, and intermittent imaging each can reduce the rate of destruction of microspheres resulting from medical ultrasound insonation.

  View details for Web of Science ID 000077187000004

  View details for PubMedID 9851820

• Changes in the echoes from ultrasonic contrast agents with imaging parameters IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Morgan, K. E., Dayton, P. A., Kruse, D. E., Klibanov, A. L., Brandenburger, G. H., Ferrara, K. W. 1998; 45 (6): 1537–48

  Abstract

  Current harmonic imaging scanners transmit a narrowband signal that limits spatial resolution in order to differentiate the echoes from tissue from the echoes from microbubbles. Because spatial resolution is particularly important in applications, including mapping vessel density in tumors, we explore the use of wideband signals in contrast imaging. It is first demonstrated that microspheres can be destroyed using one or two pulses of ultrasound. Thus, temporal signal processing strategies that use the change in the echo over time can be used to differentiate echoes from bubbles and echoes from tissue. Echo parameters, including intensity and spectral shape for narrowband and wideband transmission, are then evaluated. Through these experiments, the echo intensity received from bubbles after wideband transmission is shown to be at least as large as that for narrowband transmission, and can be larger. In each case, the echo intensity increases in a nonlinear fashion in comparison with the transmitted signal intensity. Although the echo intensity at harmonic multiples of the transmitted wave center frequency can be larger for narrowband insonation, echoes received after wideband insonation demonstrate a broadband spectrum with significant amplitude over a very wide range of frequencies.

  View details for PubMedID 18250001

• A swept-scanning mode for estimation of blood velocity in the microvasculature IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Kruse, D. E., Silverman, R. H., Fornaris, R. J., Coleman, D. J., Ferrara, K. W. 1998; 45 (6): 1437–40

  Abstract

  In contrast to previous systems in which an ultrasonic pulse was repeatedly directed to a discrete line of sight, a new method has been developed to continuously scan over a region in order to rapidly assess blood velocities in superficial small blood vessels. Using this technique, which we call swept-scan, a high frequency transducer can rapidly translate across a region of interest, and sensitive maps of blood velocity in small blood vessels can be constructed. This system has been applied to flow mapping in the anterior segment of the eye, which is clinically significant in cases of trauma and glaucoma. No previous imaging technique has been capable of estimating blood velocities within this region in a clinically useful manner. With this new technique, each 2-D scan of the eye can be obtained in an interval on the order of 1 second, and blood flow through the iris and ciliary body can be detected in vessels as small as 40 microns. A major implication of this new technique is that a wall filter can be applied continuously to the return from all regions, thus eliminating the transient response that occurs along each line of sight in traditional Doppler systems.

  View details for PubMedID 18249990

• Ultrasonic mapping of the microvasculature: Signal alignment ULTRASOUND IN MEDICINE AND BIOLOGY Zagar, B. G., Fornaris, R. J., Ferrara, K. W. 1998; 24 (6): 809-824

  Abstract

  The ultimate goal of this work was the development of a system capable of estimating the low flow velocities in the microvasculature. Estimation of low velocity flow within these vessels is challenging due to the small signal levels and the effect of cardiac and respiratory motion. Realignment of the signal from a single line-of-sight to remove physiological tissue motion is a critical part of the process of small-vessel flow mapping, and our methods for this alignment are considered in this paper. Each method involves the correlation of pulses acquired from the same line-of-sight. The first method involves the correlation of adjacent pulses (nearest-neighbor), the second involves a single reference line and the third involves averaging the correlation over a set of reference lines. We find that a nearest-neighbor strategy is suboptimal, and that strategies involving a global reference line are superior. A bound on the variance of estimates of the location of the correlation peak is presented. This bound allows us to consider our results in comparison with an absolute limit. Finally, a new algorithm allowing for alignment between lines-of-sight is described, and initial results are presented. Such an algorithm does, in fact, reduce jitter, correct for tissue motion and enables us to better visualize vessel continuity. We find that vessels as small as 40 microm can be mapped in two dimensions using a 50-MHz transducer.

  View details for Web of Science ID 000075026000004

  View details for PubMedID 9740383

• The effect of the phase of transmission on contrast agent echoes IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Morgan, K., Averkiou, M., Ferrara, K. 1998; 45 (4): 872–75

  Abstract

  Ultrasound contrast agents consist of a gas bubble, encapsulated by a shell for stabilization. The shell dampens the fluctuations in the bubble radius when insonified. The detection of contrast microbubbles during a medical examination can indicate whether a region is perfused with blood. Here, the authors consider the effect of the phase of sonification signal on the backscatter by the bubble echo. By transmitting two short pulses of ultrasound with opposite phases, the authors demonstrate that a unique pair of echoes can be generated by a single microbubble, and that the properties of these echoes may be useful in the discrimination of bubble and tissue echoes. Specifically, the significant echo amplitude begins coincident with each transmitted rarefactional half-cycle, and the mean frequency of this echo depends on the transmitted phase. When rarefaction is transmitted first for a 2.25 MHz signal, the mean frequency is 0.8 MHz higher for an albumin-shelled bubble and 0.9 MHz higher for a lipid-shelled bubble. The experimental results agree with the predictions of the Gilmore-Akulichev equation.

  View details for PubMedID 18244239

• In response to S.K. Alam and K.J. Parker ULTRASOUND IN MEDICINE AND BIOLOGY Ferrara, K. 1998; 24 (4): 611–12

  View details for Web of Science ID 000073747900017

• Correlation analysis of received echoes from contrast agents in-vitro and in-vivo Chomas, J. E., Sikes, R. A., Ferrara, K. W., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1998: 1803–6

  View details for Web of Science ID 000079968900401

• A preliminary evaluation of the effects of primary and secondary radiation forces on acoustic contrast agents IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Dayton, P. A., Morgan, K. E., Klibanov, A. L., Brandenburger, G., Nightingale, K. R., Ferrara, K. W. 1997; 44 (6): 1264–77

  View details for DOI 10.1109/58.656630

  View details for Web of Science ID A1997YH55700013

• Ultrasonic measurement of breast tissue motion and the implications for velocity estimation ULTRASOUND IN MEDICINE AND BIOLOGY ElFallah, A. I., Plantec, M. B., Ferrara, K. W. 1997; 23 (7): 1047-1057

  Abstract

  A high-resolution study of breast tissue motion during cardiac systole and respiration is presented. An experimental system was designed to achieve a velocity resolution on the order of 1 mm/s with high spatial resolution. The peak velocity of tissue motion estimated during cardiac systole ranged from 0.2 mm/s to 5.6 mm/s among the subjects studied. It is shown that motion due to the cardiac cycle is less significant when the subject is positioned on the side rather than supine. The mean tissue velocity among subjects in the supine position is 2.88 mm/s and drops to 0.81 mm/s for the side position, with a corresponding spatial displacement of 0.095 mm, dropping to 0.027 mm. The velocity profiles indicate that 100 ms is required for the entire ribcage contraction-relaxation process to occur. Experiments using a prone biopsy table show the almost complete elimination of tissue motion due to cardiac systole, suggesting that the use of the table eliminates this motion, thus allowing for high-resolution blood velocity estimates. Features resulting from respiratory motion are also presented. We found this motion to be of a much longer time duration, and of a much higher magnitude, with velocities as high as 29 mm/s. The implications of the study on the high-resolution estimation of blood velocity and high-resolution breast imaging are discussed.

  View details for PubMedID 9330448

• High resolution 3D flow mapping in tumors Kruse, D., Chomas, J., Salerno, M., El-Fallah, A., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1997: 1211–14

  View details for Web of Science ID 000072927100258

• Correlation techniques for ultrasonic signal alignment Fornaris, R. J., Ferrara, K. W., Zagar, B. G., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1997: 1215–18

  View details for Web of Science ID 000072927100259

• Effect of imaging parameters on the echoes from ultrasonic contrast agents Morgan, K. E., Dayton, P. A., Kruse, D. E., Klibanov, A. L., Brandenburger, G. H., Ferrara, K. W., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1997: 1593–96

  View details for Web of Science ID 000072927100342

• Simultaneous optical and acoustical observations of contrast agents Dayton, P., Morgan, K., Allietta, M., Klibanov, A., Brandenburger, G., Ferrara, K., Schneider, S. C., Levy, M., McAvoy, B. R. IEEE. 1997: 1583–91

  View details for Web of Science ID 000072927100341

• Color flow mapping ULTRASOUND IN MEDICINE AND BIOLOGY Ferrara, K., DeAngelis, G. 1997; 23 (3): 321-345

  Abstract

  Color flow mapping systems have become widely used in the short time since their development. These systems overlay a pseudo-color velocity map upon the gray-scale two-dimensional image. Between 4 and 16 pulses are directed to each line-of-sight, and this requirement reduces the frame rate in comparison with the gray-scale image. Other limitations of color flow mapping include its ability to estimate only the velocity toward or away from the transducer and an increase in the variance in comparison with spectral Doppler. Potential artifacts include aliased velocities and the detection of flow in hypoechoic or hyperechoic nonvascular structures. Clinical applications include cardiology, studies of the abdominal and peripheral vasculature, evaluation of organ perfusion and the differentiation of tumors. Most current systems use narrowband estimators that examine a fixed sample volume and detect a change in phase between two pulses. Wideband estimators that can track red blood cells in two or three dimensions are under evaluation. Narrowband estimators, including the autocorrelator, the short Fourier transform and second order autoregressive filters, are compared with wideband estimators including cross-correlation, sum-absolute-difference and the wideband maximum likelihood estimator. Because the intensity of blood echoes is far smaller than echoes from surrounding tissue, high pass filters have been developed that can reject the larger signal from tissue using the return from a small number of pulses. Other areas of research include strategies for flow estimation with contrast agents, three-dimensional color flow mapping and power Doppler flow mapping.

  View details for PubMedID 9160902

• Mapping of the vasculature of the ciliary body Ferrara, K. W., Silverman, R. H., Zagar, B., Aslanides, I. M., Coleman, D. J. LIPPINCOTT-RAVEN PUBL. 1996: 3878

  View details for Web of Science ID A1996TX39703872

• High resolution 3D color flow mapping: Applied to the assessment of breast vasculature ULTRASOUND IN MEDICINE AND BIOLOGY Ferrara, K. W., Zagar, B., SOKILMELGAR, J., ALGAZI, V. R. 1996; 22 (3): 293-304

  Abstract

  In order to develop a three dimensional (3D) color flow map of low velocity flow through small vessels, we explore the use of a high resolution velocity estimation technique and a new technique for the differentiation of regions of flow and stationary tissue. Following the transmission of a wideband signal, a signal processing strategy that tracks the motion of small regions of blood is used to estimate velocity. We find that the use of wideband transmission, with coherent estimation over a long pulse train, provides the opportunity to map very low velocity flow, and to detect flow at beam-vessel angles near 90 degrees. The use of 3D continuity processing to differentiate flow and stationary tissue is shown to improve noise immunity. In-vitro and in-vivo velocity maps are presented. The 3D velocity profile is then constructed.

  View details for PubMedID 8783461

• Properties of contrast agents insonified at frequencies above 10 MHz Morgan, K., Dayton, P., Klibanov, S., Brandenburger, G., Kaul, S., Wei, K., Ferrara, K., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1996: 1127–30

  View details for Web of Science ID A1996BH56C00236

• Action of microbubbles when insonified: Experimental evidence Dayton, P., Goode, A., Morgan, K., Klibanov, S., Brandenburger, G., Ferrara, K., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1996: 1131–34

  View details for Web of Science ID A1996BH56C00237

• 3D ultrasonic mapping of the microvasculature Stith, A., Zagar, B. G., Fornaris, R., Silverman, R., Ferrara, K., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1996: 1473–76

  View details for Web of Science ID A1996BH56C00311

• Estimation of blood velocity with high frequency ultrasound IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Ferrara, K. W., Zagar, B. G., SokilMelgar, J. B., Silverman, R. H., Aslanidis, I. M. 1996; 43 (1): 149–57

  View details for DOI 10.1109/58.484474

  View details for Web of Science ID A1996TQ35100019

• EFFECT OF THE BEAM-VESSEL ANGLE ON THE RECEIVED ACOUSTIC-SIGNAL FROM BLOOD IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL FERRARA, K. W. 1995; 42 (3): 416–28

  View details for DOI 10.1109/58.384452

  View details for Web of Science ID A1995QV97300014

• PARAMETER MAPPING FOR THE DETECTION OF DISTURBED BLOOD-FLOW ULTRASOUND IN MEDICINE AND BIOLOGY Ferrara, K. W., OSTROMOGILSKY, M., Rosenberg, S., SOKILMELGAR, J. 1995; 21 (4): 517-525

  Abstract

  Particularly in color flow mapping, the detection of minor stenoses is a significant outstanding challenge. With the goal of spatially mapping changes in velocity and shear rate to locate the source of the flow disturbance, two indicators of the magnitude of the signal correlation are presented and evaluated for known experimental flow conditions. A normalized parameter is presented, which is proportional to the magnitude of the signal correlation evaluated over a set of received pulses. Its performance is compared to that of a single-lag correlation measure for the development of spatial maps that indicate shear rate and velocity.

  View details for Web of Science ID A1995RA23600009

  View details for PubMedID 7571144

• High resolution 3D color flow mapping of breast vasculature Ferrara, K. W., Zagar, B., Plantec, M., SokilMelgar, J., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1995: 1467–70

  View details for Web of Science ID A1995BF57F00300

• 50 MHz color flow mapping Ferrara, K. W., Zagar, B., Silverman, R., Aggarwal, K., Dayton, P., SokilMelgar, J., Aslanidis, Y., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1995: 1497–1500

  View details for Web of Science ID A1995BF57F00307

• A STATISTICAL-ANALYSIS OF THE RECEIVED SIGNAL FROM BLOOD DURING LAMINAR-FLOW IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL FERRARA, K. W., ALGAZI, V. R. 1994; 41 (2): 185–98

  View details for DOI 10.1109/58.279131

  View details for Web of Science ID A1994NH37300003

• A THEORETICAL AND EXPERIMENTAL-ANALYSIS OF THE RECEIVED SIGNAL FROM DISTURBED BLOOD-FLOW IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL FERRARA, K. W., ALGAZI, V. R. 1994; 41 (2): 172–84

  View details for DOI 10.1109/58.279130

  View details for Web of Science ID A1994NH37300002

• AN EXPERIMENTAL EVALUATION OF THE EFFECT OF THE BEAM-VESSEL ANGLE ON THE RECEIVED ACOUSTIC SIGNAL FROM BLOOD FERRARA, K. W., KALISZ, A., ZAGAR, B., ALGAZI, V. R., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1994: 1691–95

  View details for Web of Science ID A1994BC66H00345

• AN EXPERIMENTAL EVALUATION OF THE RECEIVED SIGNAL FROM BLOOD AT 50-MHZ FERRARA, K. W., ZAGAR, B., ALGAZI, R., SILVERMAN, R., SOKILMELGAR, J., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1994: 1603–7

  View details for Web of Science ID A1994BC66H00327

• SIGNAL AND IMAGE PROCESSING STRATEGIES FOR RECONSTRUCTING 3-D COLOR FLOW MAPS FROM ULTRASONIC DATA ZAGAR, B. G., ALGAZI, V. R., FERRARA, K. W., Levy, M., Schneider, S. C., McAvoy, B. R. I E E E. 1994: 1649–52

  View details for Web of Science ID A1994BC66H00335

• EVALUATION OF THE PERFORMANCE OF STRATEGIES FOR THE ESTIMATION OF LOW-VELOCITY BLOOD-FLOW FERRARA, K., ALGAZI, V. R., DARROCH, T., RAGHAVENDRA, P., Levy, M., McAvoy, B. R. I E E E. 1993: 1049–53

  View details for Web of Science ID A1993BA31N00208

• AN EXPERIMENTAL COMPARISON OF THE SIGNAL FROM LAMINAR AND DISTURBED BLOOD-FLOW FERRARA, K. W., ALGAZI, V. R., Levy, M., McAvoy, B. R. I E E E. 1993: 1043–47

  View details for Web of Science ID A1993BA31N00207

• THE EFFECT OF FREQUENCY-DEPENDENT SCATTERING AND ATTENUATION ON THE ESTIMATION OF BLOOD VELOCITY USING ULTRASOUND IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Ferrara, K. W., ALGAZI, V. R., Liu, J. X. 1992; 39 (6): 754-767

  Abstract

  A comprehensive theoretical performance comparison of the wideband maximum-likelihood (WMLE) and cross-correlation strategies, previously proposed and evaluated for the estimation of blood velocity using ultrasound is presented. It is based on evaluation of the bias, local and global accuracy, and signal-to-noise ratio (SNR) performance. The results show that the intervening medium does not bias either wideband estimation, due to the effect of tracking the scattering target. The presence of intervening tissue actually improves the global accuracy of both wideband estimators, without a significant change in the local accuracy of either wideband estimator. After the transmission of P pulses, a comparison of the performance of the two strategies shows that the cross-correlation estimator requires P(2 ) correlations to achieve performance similar to that of the WMLE with P operations. In addition, the WMLE can increase the effective SNR in comparison with cross correlation.

  View details for PubMedID 18267692

• COMPARISON OF NARROW-BAND AND WIDE-BAND COLOR-FLOW ESTIMATORS IN THE PRESENCE OF INTERVENING TISSUE FERRARA, K. W., ALGAZI, V. R., LIU, J., Mcavoy, B. R. I E E E. 1992: 1169–73

  View details for DOI 10.1109/ULTSYM.1992.275892

  View details for Web of Science ID A1992BY55S00231

• A NEW WIDE-BAND SPREAD TARGET MAXIMUM-LIKELIHOOD ESTIMATOR FOR BLOOD VELOCITY ESTIMATION .2. EVALUATION OF ESTIMATORS WITH EXPERIMENTAL-DATA IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Ferrara, K. W., ALGAZI, V. R. 1991; 38 (1): 17-26

  Abstract

  For pt.I see ibid., vol.38, p.1-16, Jan. 1991. The signal models and performance of the estimation strategies described in pt.I are tested with experimental ultrasonic data. The ultrasonic data analyzed verify the theoretical model and predicted performance. The averaged correlation, verified experimentally, confirms that the correlation envelope can be used to estimate the velocity of scatterers and that the shape of the correlation function conveys information regarding the velocity profile within the sample volume. For both the wideband point and range spread estimators, the predicted improvement in velocity resolution and the reduction in height of subsidiary velocity peaks are demonstrated. Through the use of these estimation strategies, information regarding the mean velocity and velocity variation are available for each spatial location within the vessel. This information is presented using a three-dimensional spatial velocity profile display, which appears to offer a number of advantages in the rapid identification of pathology.

  View details for PubMedID 18267552

• A NEW WIDE-BAND SPREAD TARGET MAXIMUM-LIKELIHOOD ESTIMATOR FOR BLOOD VELOCITY ESTIMATION .1. THEORY IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Ferrara, K. W., ALGAZI, V. R. 1991; 38 (1): 1-16

  Abstract

  The derivation and theoretical evaluation of new wideband maximum-likelihood strategies for the estimation of blood velocity using acoustic signals are presented. A model for the received signal from blood scatterers, using a train of short wideband pulses, is described. Evaluation of the autocorrelation of the signal based on this model shows that the magnitude, periodicity, and phase of the autocorrelation are affected by the mean scatterer velocity and the presence of a velocity spread target. New velocity estimators are then derived that exploit the effect of the scatterer velocity on both the signal delay and the shift in frequency. The wideband range spread estimator is derived using a statistical model of the target. Based on the point target assumption, a simpler wideband maximum-likelihood estimator is also obtained. These new estimation strategies are analyzed for their local and global performance. Evaluation of the Cramer-Rao bound shows that the bound on the estimator variance is reduced using these estimators, in comparison with narrowband strategies. In order to study global accuracy, the expected estimator output is evaluated, and it is determined that the width of the mainlobe is reduced. In addition, it is shown that the height of subsidiary velocity peaks is reduced through the use of these new estimators.

  View details for PubMedID 18267551

• IMPROVED COLOR FLOW MAPPING USING THE WIDE-BAND MAXIMUM-LIKELIHOOD ESTIMATOR FERRARA, K. W., ALGAZI, V. R., MCAVOY, B. R. I E E E. 1990: 1517–21

  View details for DOI 10.1109/ULTSYM.1990.171620

  View details for Web of Science ID A1990BT34L00301

• ESTIMATION OF BLOOD VELOCITY USING THE WIDE-BAND MAXIMUM-LIKELIHOOD ESTIMATOR FERRARA, K. W., ALGAZI, V. R., MCAVOY, B. R. I E E E. 1989: 897–901

  View details for Web of Science ID A1989BQ92K00171