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


    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


    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

  • 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


    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

  • 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


    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

  • Erratum to "Highly Integrated Multiplexing and Buffering Electronics for Large Aperture Ultrasonic Arrays". BME frontiers Wodnicki, R., Kang, H., Li, D., Stephens, D. N., Jung, H., Sun, Y., Chen, R., Jiang, L. M., Cabrera-Munoz, N. E., Foiret, J., Zhou, Q., Ferrara, K. W. 2022; 2022: 9818934


    [This corrects the article DOI: 10.34133/2022/9870386.].

    View details for DOI 10.34133/2022/9818934

    View details for PubMedID 37850159

    View details for PubMedCentralID PMC10521645

  • 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


    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

  • 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


    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

  • 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

  • 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
  • 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)


    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


    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
  • 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)


    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

  • 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
  • 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


    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

  • 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


    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

  • 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
  • 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


    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

  • Localized nanodelivery combined with immunotherapy promotes curative anti-tumor responses in a murine breast cancer model 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., Murphy, W. J., Ferrara, K. W. AMER ASSOC CANCER RESEARCH. 2019
  • Activatable nanodelivery of high payload gemcitabine augments therapeutic efficacy in murine breast and pancreatic cancer models 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., Ferrara, K. W. AMER ASSOC CANCER RESEARCH. 2019
  • 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
  • Combining activatable nanodelivery with immunotherapy in a murine breast cancer model. Journal of controlled release : official journal of the Controlled Release Society 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


    A successful chemotherapy-immunotherapy solid-tumor protocol should accomplish the following goals: debulk large tumors, release tumor antigen for cross-presentation and cross-priming, release cancer-suppressive cytokines and enhance anti-tumor immune cell populations. Thermally-activated drug delivery particles have the potential to synergize with immunotherapeutics to accomplish these goals; activation can release chemotherapy within bulky solid tumors and can enhance response when combined with immunotherapy. We set out to determine whether a single protocol, combining locally-activated chemotherapy and agonist immunotherapy, could accomplish these goals and yield a potentially translational therapy. For effective delivery of free doxorubicin to tumors with minimal toxicity, we stabilized doxorubicin with copper in temperature-sensitive liposomes that rapidly release free drug in the vasculature of cancer lesions upon exposure to ultrasound-mediated hyperthermia. We found that in vitro exposure of tumor cells to hyperthermia and doxorubicin resulted in immunogenic cell death and the local release of type I interferons across murine cancer cell lines. Following intravenous injection, local activation of the liposomes within a single tumor released doxorubicin and enhanced cross-presentation of a model antigen at distant tumor sites. While a variety of protocols achieved a complete response in >50% of treated mice, the complete response rate was greatest (90%) when 1 week of immunotherapy priming preceded a single activatable chemotherapeutic administration. While repeated chemotherapeutic delivery reduced local viable tumor, the complete response rate and a subset of tumor immune cells were also reduced. Taken together, the results suggest that activatable chemotherapy can enhance adjuvant immunotherapy; however, in a murine model the systemic adaptive immune response was greatest with a single administration of chemotherapy.

    View details for PubMedID 30978432

  • 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


    Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g., electroporation). We have previously used sonoporation to overexpress transgenes in different skeletal tissues in order to induce tissue regeneration. Here, we provide a protocol that could easily be adapted to address various other targets of tissue regeneration or additional applications, such as cancer and neurodegenerative diseases. This protocol describes how to prepare, conduct and optimize ultrasound-mediated gene delivery in both a murine and a porcine animal model. The protocol includes the preparation of a microbubble-DNA mix and in vivo sonoporation under ultrasound imaging. Ultrasound-mediated gene delivery can be accomplished within 10 min. After DNA delivery, animals can be followed to monitor gene expression, protein secretion and other transgene-specific outcomes, including tissue regeneration. This procedure can be accomplished by a competent graduate student or technician with prior experience in ultrasound imaging or in performing in vivo procedures.

    View details for PubMedID 30804568

  • 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


    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

  • 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


    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

  • 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
  • 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
  • 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
  • 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


    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

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


    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


    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

  • 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
  • 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


    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

  • 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


    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


    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

  • 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


    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

  • 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


    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

  • 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
  • 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