Bio

Dr. Spitler is the Deputy Director of the Precision Health and Integrated Diagnostics Center at Stanford University. He completed his Post Doctorial Research Fellowship (SCIT) at Stanford University School of Medicine, conducting research in the developing field of Magnetogenetics for remote controlled cellular reprogramming and developed smart MRI cell tracking tools for oncology cell tracking studies. He has designed numerous biological models, synthetic biology approaches and worked on the development of new technologies in a number of scientific areas ranging from medical devices to gene therapy. Prior to his position at Stanford, Dr. Spitler received his Ph.D. in Cellular and Developmental Biology at the Beckman Laser Institute at the University of California, Irvine. His research at the Beckman Laser Institute included developing and characterizing new nitric oxide-based drugs, laser, and LED-based multimodal wound healing therapies some of which are currently being used in the clinic as a result of his work.
Dr. Spitler received his Bachelor’s of Science degree in Molecular Cell and Developmental Biology from the University of California, Santa Cruz, where he worked in the area of structural biology. Over the past two decades he has held a number of academic and industrial positions and has served as an advisor or advisory board member for a number of Bay Area companies. Dr. Spitler is the recipient of the Stanford Cancer Imaging Fellowship Training Award, RSL Innovation Challenge Award, the Biophotas Research Fellowship, and the Stanford Center for Biomedical Imaging Achievement Award.  

Honors & Awards

  • RSL Innovation Challenge Grant, Radiological Sciences Laboratory - Stanford School of Medicine (2015-2016)
  • Stanford Cancer Imaging Training Fellowship, National Cancer Institute (2014-2016)
  • Biophotas Research Fellowship, Biophotas Inc., Beckman Laser Institute, University of California Irvine (2013-2014)
  • Military Photomedicine Program, Beckman Laser Institute, University of California Irvine (2010-2014)
  • Achievement Award in Biomedical Imaging, Stanford University (2010)

Contact

  • Academic
    rspitler@stanford.edu
    University - Academic staff Department: Rad/Precision Health and Integrated Diagnostics Position: Deputy Director, Precision Health and Integrated Diagnostics and Canary Centers
    • 3155 Porter Dr Rm 2248
    • Palo Alto,  California  94304-1213 

Additional Info

  • Mail Code: 5483

Links

All Publications

  • The Project Baseline Health Study: a step towards a broader mission to map human health NPJ DIGITAL MEDICINE Arges, K., Assimes, T., Bajaj, V., Balu, S., Bashir, M. R., Beskow, L., Blanco, R., Califf, R., Campbell, P., Carin, L., Christian, V., Cousins, S., Das, M., Dockery, M., Douglas, P. S., Dunham, A., Eckstrand, J., Fleischmann, D., Ford, E., Fraulo, E., French, J., Gambhir, S. S., Ginsburg, G. S., Green, R. C., Haddad, F., Hernandez, A., Hernandez, J., Huang, E. S., Jaffe, G., King, D., Koweek, L. H., Langlotz, C., Liao, Y. J., Mahaffey, K. W., Marcom, K., Marks, W. J., Maron, D., McCabe, R., McCall, S., McCue, R., Mega, J., Miller, D., Muhlbaier, L. H., Munshi, R., Newby, L., Pak-Harvey, E., Patrick-Lake, B., Pencina, M., Peterson, E. D., Rodriguez, F., Shore, S., Shah, S., Shipes, S., Sledge, G., Spielman, S., Spitler, R., Schaack, T., Swamy, G., Willemink, M. J., Wong, C. A. 2020; 3 (1): 84

    Abstract

    The Project Baseline Health Study (PBHS) was launched to map human health through a comprehensive understanding of both the health of an individual and how it relates to the broader population. The study will contribute to the creation of a biomedical information system that accounts for the highly complex interplay of biological, behavioral, environmental, and social systems. The PBHS is a prospective, multicenter, longitudinal cohort study that aims to enroll thousands of participants with diverse backgrounds who are representative of the entire health spectrum. Enrolled participants will be evaluated serially using clinical, molecular, imaging, sensor, self-reported, behavioral, psychological, environmental, and other health-related measurements. An initial deeply phenotyped cohort will inform the development of a large, expanded virtual cohort. The PBHS will contribute to precision health and medicine by integrating state of the art testing, longitudinal monitoring and participant engagement, and by contributing to the development of an improved platform for data sharing and analysis.

    View details for DOI 10.1038/s41746-020-0290-y

    View details for Web of Science ID 000538242900001

    View details for PubMedID 32550652

    View details for PubMedCentralID PMC7275087

  • Publisher Correction: A mountable toilet system for personalized health monitoring via the analysis of excreta. Nature biomedical engineering Park, S. M., Won, D. D., Lee, B. J., Escobedo, D. n., Esteva, A. n., Aalipour, A. n., Ge, T. J., Kim, J. H., Suh, S. n., Choi, E. H., Lozano, A. X., Yao, C. n., Bodapati, S. n., Achterberg, F. B., Kim, J. n., Park, H. n., Choi, Y. n., Kim, W. J., Yu, J. H., Bhatt, A. M., Lee, J. K., Spitler, R. n., Wang, S. X., Gambhir, S. S. 2020

    Abstract

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    View details for DOI 10.1038/s41551-020-0562-5

    View details for PubMedID 32382068

  • A mountable toilet system for personalized health monitoring via the analysis of excreta. Nature biomedical engineering Park, S. M., Won, D. D., Lee, B. J., Escobedo, D. n., Esteva, A. n., Aalipour, A. n., Ge, T. J., Kim, J. H., Suh, S. n., Choi, E. H., Lozano, A. X., Yao, C. n., Bodapati, S. n., Achterberg, F. B., Kim, J. n., Park, H. n., Choi, Y. n., Kim, W. J., Yu, J. H., Bhatt, A. M., Lee, J. K., Spitler, R. n., Wang, S. X., Gambhir, S. S. 2020

    Abstract

    Technologies for the longitudinal monitoring of a person's health are poorly integrated with clinical workflows, and have rarely produced actionable biometric data for healthcare providers. Here, we describe easily deployable hardware and software for the long-term analysis of a user's excreta through data collection and models of human health. The 'smart' toilet, which is self-contained and operates autonomously by leveraging pressure and motion sensors, analyses the user's urine using a standard-of-care colorimetric assay that traces red-green-blue values from images of urinalysis strips, calculates the flow rate and volume of urine using computer vision as a uroflowmeter, and classifies stool according to the Bristol stool form scale using deep learning, with performance that is comparable to the performance of trained medical personnel. Each user of the toilet is identified through their fingerprint and the distinctive features of their anoderm, and the data are securely stored and analysed in an encrypted cloud server. The toilet may find uses in the screening, diagnosis and longitudinal monitoring of specific patient populations.

    View details for DOI 10.1038/s41551-020-0534-9

    View details for PubMedID 32251391

  • Toward achieving precision health SCIENCE TRANSLATIONAL MEDICINE Gambhir, S., Ge, T., Vermesh, O., Spitler, R. 2018; 10 (430)

    View details for DOI 10.1126/scitranslmed.aao3612

    View details for Web of Science ID 000426291800005

  • Toward achieving precision health. Science translational medicine Gambhir, S. S., Ge, T. J., Vermesh, O., Spitler, R. 2018; 10 (430)

    Abstract

    Health care systems primarily focus on patients after they present with disease, not before. The emerging field of precision health encourages disease prevention and earlier detection by monitoring health and disease based on an individual's risk. Active participation in health care can be encouraged with continuous health-monitoring devices, providing a higher-resolution picture of human health and disease. However, the development of monitoring technologies must prioritize the collection of actionable data and long-term user engagement.

    View details for DOI 10.1126/scitranslmed.aao3612

    View details for PubMedID 29491186

    View details for PubMedCentralID PMC5985668

  • Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents MOLECULAR IMAGING AND BIOLOGY Brewer, K. D., Spitler, R., Lee, K. R., Chan, A. C., Barrozo, J. C., Wakeel, A., Foote, C. S., Machtaler, S., Rioux, J., Willmann, J. K., Chakraborty, P., Rice, B. W., Contag, C. H., Bell, C. B., Rutt, B. K. 2018; 20 (1): 65–73

    Abstract

    Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo.By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking. This work presents a biomedical imaging characterization of MEs including: MRI transverse relaxivity (r 2) for MEs and ME-labeled cells (compared to a commercially available iron oxide nanoparticle); microscopic validation of labeling efficiency and subcellular locations; and in vivo imaging of a MDA-MB-231BR (231BR) human breast cancer cells in a mouse brain.At 7T, r 2 relaxivity of bare MEs was higher (250 s-1 mM-1) than that of conventional SPIO (178 s-1 mM-1). Optimized in vitro loading of MEs into 231BR cells yielded 1-4 pg iron/cell (compared to 5-10 pg iron/cell for conventional SPIO). r 2 relaxivity dropped by a factor of ~3 upon loading into cells, and was on the same order of magnitude for ME-loaded cells compared to SPIO-loaded cells. In vivo, ME-labeled cells exhibited strong MR contrast, allowing as few as 100 cells to be detected in mice using an optimized 3D SPGR gradient-echo sequence.Our results demonstrate the potential of magneto-endosymbionts as living MR contrast agents. They have r 2 relaxivity values comparable to traditional iron oxide nanoparticle contrast agents, and provide strong MR contrast when loaded into cells and implanted in tissue.

    View details for PubMedID 28616842

    View details for PubMedCentralID PMC5730509

  • Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions MOLECULAR IMAGING AND BIOLOGY Lee, K. R., Wakeel, A., Chakraborty, P., Foote, C. S., Kajiura, L., Barrozo, J. C., Chan, A. C., Bazarov, A. V., Spitler, R., Kutny, P. M., Denegre, J. M., Taft, R. A., Seemann, J., Rice, B. W., Contag, C. H., Rutt, B. K., Bell, C. B. 2018; 20 (1): 55–64

    Abstract

    The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing.The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking.MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

    View details for PubMedID 28631141

    View details for PubMedCentralID PMC5736464

  • Tumor-associated macrophages, nanomedicine and imaging: the axis of success in the future of cancer immunotherapy. Immunotherapy Zanganeh, S., Spitler, R., Hutter, G., Ho, J. Q., Pauliah, M., Mahmoudi, M. 2017; 9 (10): 819-835

    Abstract

    The success of any given cancer immunotherapy relies on several key factors. In particular, success hinges on the ability to stimulate the immune system in a controlled and precise fashion, select the best treatment options and appropriate therapeutic agents, and use highly effective tools to accurately and efficiently assess the outcome of the immunotherapeutic intervention. Furthermore, a deep understanding and effective utilization of tumor-associated macrophages (TAMs), nanomedicine and biomedical imaging must be harmonized to improve treatment efficacy. Additionally, a keen appreciation of the dynamic interplay that occurs between immune cells and the tumor microenvironment (TME) is also essential. New advances toward the modulation of the immune TME have led to many novel translational research approaches focusing on the targeting of TAMs, enhanced drug and nucleic acid delivery, and the development of theranostic probes and nanoparticles for clinical trials. In this review, we discuss the key cogitations that influence TME, TAM modulations and immunotherapy in solid tumors as well as the methods and resources of tracking the tumor response. The vast array of current nanomedicine technologies can be readily modified to modulate immune function, target specific cell types, deliver therapeutic payloads and be monitored using several different imaging modalities. This allows for the development of more effective treatments, which can be specifically designed for particular types of cancer or on an individual basis. Our current capacities have allowed for greater use of theranostic probes and multimodal imaging strategies that have led to better image contrast, real-time imaging capabilities leveraging targeting moieties, tracer kinetics and enabling more detailed response profiles at the cellular and molecular levels. These novel capabilities along with new discoveries in cancer biology should drive innovation for improved biomarkers for efficient and individualized cancer therapy.

    View details for DOI 10.2217/imt-2017-0041

    View details for PubMedID 28877626

  • Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues. Nature nanotechnology Zanganeh, S., Hutter, G., Spitler, R., Lenkov, O., Mahmoudi, M., Shaw, A., Pajarinen, J. S., Nejadnik, H., Goodman, S., Moseley, M., Coussens, L. M., Daldrup-Link, H. E. 2016; 11 (11): 986-994

    Abstract

    Until now, the Food and Drug Administration (FDA)-approved iron supplement ferumoxytol and other iron oxide nanoparticles have been used for treating iron deficiency, as contrast agents for magnetic resonance imaging and as drug carriers. Here, we show an intrinsic therapeutic effect of ferumoxytol on the growth of early mammary cancers, and lung cancer metastases in liver and lungs. In vitro, adenocarcinoma cells co-incubated with ferumoxytol and macrophages showed increased caspase-3 activity. Macrophages exposed to ferumoxytol displayed increased mRNA associated with pro-inflammatory Th1-type responses. In vivo, ferumoxytol significantly inhibited growth of subcutaneous adenocarcinomas in mice. In addition, intravenous ferumoxytol treatment before intravenous tumour cell challenge prevented development of liver metastasis. Fluorescence-activated cell sorting (FACS) and histopathology studies showed that the observed tumour growth inhibition was accompanied by increased presence of pro-inflammatory M1 macrophages in the tumour tissues. Our results suggest that ferumoxytol could be applied 'off label' to protect the liver from metastatic seeds and potentiate macrophage-modulating cancer immunotherapies.

    View details for DOI 10.1038/nnano.2016.168

    View details for PubMedID 27668795

  • Protein corona: Opportunities and challenges INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY Zanganeh, S., Spitler, R., Erfanzadeh, M., Alkilany, A. M., Mahmoudi, M. 2016; 75: 143-147

    Abstract

    In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field.

    View details for DOI 10.1016/j.biocel.2016.01.005

    View details for Web of Science ID 000376832600018

    View details for PubMedID 26783938

  • Combination of low level light therapy and nitrosyl-cobinamide accelerates wound healing JOURNAL OF BIOMEDICAL OPTICS Spitler, R., Ho, H., Norpetlian, F., Kong, X., Jiang, J., Yokomori, K., Andersen, B., Boss, G. R., Berns, M. W. 2015; 20 (5)

    Abstract

    Low level light therapy (LLLT) has numerous therapeutic benefits, including improving wound healing, but the precise mechanisms involved are not well established; in particular, the underlying role of cytochrome C oxidase (C-ox) as the primary photoacceptor and the associated biochemical mechanisms still require further investigation. We previously showed the nitric oxide (NO) donating drug nitrosyl-cobinamide (NO-Cbi) enhances wound healing through a cGMP/cGMP-dependent protein kinase/ERK1/2 mechanism. Here, we show that the combination of LLLT and NO-Cbi markedly improves wound healing compared to either treatment alone. LLLT-enhanced wound healing proceeded through an electron transport chain-C-ox-dependent mechanism with a reduction of reactive oxygen species and increased adenosine triphosphate production. C-ox was validated as the primary photoacceptor by three observations: increased oxygen consumption, reduced wound healing in the presence of sodium azide, and disassociation of cyanide, a known C-ox ligand, following LLLT. We conclude that LLLT and NO-Cbi accelerate wound healing through two independent mechanisms, the electron transport chain-C-ox pathway and cGMP signaling, respectively, with both resulting in ERK1/2 activation.

    View details for DOI 10.1117/1.JBO.20.5.051022

    View details for Web of Science ID 000356241900027

    View details for PubMedID 25562608

  • Comparison of laser and diode sources for acceleration of in vitro wound healing by low-level light therapy JOURNAL OF BIOMEDICAL OPTICS Spitler, R., Berns, M. W. 2014; 19 (3)

    View details for DOI 10.1117/1.JBO.19.3.038001

    View details for Web of Science ID 000338333400042

  • Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor, improves wound healing through cGMP/cGMP-dependent protein kinase. Cellular signalling Spitler, R., Schwappacher, R., Wu, T., Kong, X., Yokomori, K., Pilz, R. B., Boss, G. R., Berns, M. W. 2013; 25 (12): 2374-2382

    Abstract

    Nitric oxide (NO) donors have been shown to improve wound healing, but the mechanism is not well defined. Here we show that the novel NO donor nitrosyl-cobinamide (NO-Cbi) improved in vitro wound healing in several cell types, including an established line of lung epithelial cells and primary human lung fibroblasts. On a molar basis, NO-Cbi was more effective than two other NO donors, with the effective NO-Cbi concentration ranging from 3 to 10μM, depending on the cell type. Improved wound healing was secondary to increased cell migration and not cell proliferation. The wound healing effect of NO-Cbi was mediated by cGMP, mainly through cGMP-dependent protein kinase type I (PKGI), as determined using pharmacological inhibitors and activators, and siRNAs targeting PKG type I and II. Moreover, we found that Src and ERK were two downstream mediators of NO-Cbi's effect. We conclude that NO-Cbi is a potent inducer of cell migration and wound closure, acting via cGMP, PKG, Src, and extracellular signal regulated kinase (ERK).

    View details for DOI 10.1016/j.cellsig.2013.07.029

    View details for PubMedID 23920342

  • Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice MOLECULAR THERAPY-NUCLEIC ACIDS Dallas, A., Ilves, H., Shorenstein, J., Judge, A., Spitler, R., Contag, C., Wong, S. P., Harbottle, R. P., MacLachlan, I., Johnston, B. H. 2013; 2

    Abstract

    We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and (3)H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% 1 day after injection of 2.5 mg/kg sshRNA) with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.Molecular Therapy-Nucleic Acids (2013) 2, e123; doi:10.1038/mtna.2013.50; published online 17 September 2013.

    View details for DOI 10.1038/mtna.2013.50

    View details for Web of Science ID 000332467500006

    View details for PubMedID 24045712

  • cGMP-dependent protein kinase Iß regulates breast cancer cell migration and invasion via interaction with the actin/myosin-associated protein caldesmon. Journal of cell science Schwappacher, R., Rangaswami, H., Su-Yuo, J., Hassad, A., Spitler, R., Casteel, D. E. 2013; 126: 1626-1636

    Abstract

    The two isoforms of type I cGMP-dependent protein kinase (PKGIα and PKGIβ) differ in their first ∼100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The dimerization domains form coiled-coil structures and serve as platforms for isoform-specific protein-protein interactions. Using the PKGIβ dimerization domain as an affinity probe in a proteomic screen, we identified the actin/myosin-associated protein caldesmon (CaD) as a PKGIβ-specific binding protein. PKGIβ phosphorylated human CaD on serine 12 in vitro and in intact cells. Phosphorylation on serine 12 or mutation of serine 12 to glutamic acid (S12E) reduced the interaction between CaD and myosin IIA. Because CaD inhibits myosin ATPase activity and regulates cell motility, we examined the effects of PKGIβ and CaD on cell migration and invasion. Inhibition of the NO/cGMP/PKG pathway reduced migration and invasion of human breast cancer cells, whereas PKG activation enhanced their motility and invasion. siRNA-mediated knockdown of endogenous CaD had pro-migratory and pro-invasive effects in human breast cancer cells. Reconstituting cells with wild-type CaD slowed migration and invasion; however, CaD containing a phospho-mimetic S12E mutation failed to reverse the pro-migratory and pro-invasive activity of CaD depletion. Our data suggest that PKGIβ enhances breast cancer cell motility and invasive capacity, at least in part, by phosphorylating CaD. These findings identify a pro-migratory and pro-invasive function for PKGIβ in human breast cancer cells, suggesting that PKGIβ is a potential target for breast cancer treatment.

    View details for DOI 10.1242/jcs.118190

    View details for PubMedID 23418348

  • Visualization of plasmid delivery to keratinocytes in mouse and human epidermis SCIENTIFIC REPORTS Gonzalez-Gonzalez, E., Kim, Y., Speaker, T. J., Hickerson, R. P., Spitler, R., Birchall, J. C., Lara, M. F., Hu, R., Liang, Y., Kirkiles-Smith, N., Prausnitz, M. R., Milstone, L. M., Contag, C. H., Kaspar, R. L. 2011; 1

    Abstract

    The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

    View details for DOI 10.1038/srep00158

    View details for Web of Science ID 000300557200002

    View details for PubMedID 22355673

    View details for PubMedCentralID PMC3240989

  • In Vitro Evolution of Ligands to the Membrane Protein Caveolin JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Majumdar, S., Hajduczki, A., Vithayathil, R., Olsen, T. J., Spitler, R. M., Mendez, A. S., Thompson, T. D., Weiss, G. A. 2011; 133 (25): 9855-9862

    Abstract

    Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, although implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with >1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to caveolin by the T20 variants with K(d) values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.

    View details for DOI 10.1021/ja201792q

    View details for Web of Science ID 000292439700041

    View details for PubMedID 21615158

  • Non-damaging Retinal Phototherapy: Dynamic Range of Heat Shock Protein Expression INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Sramek, C., Mackanos, M., Spitler, R., Leung, L., Nomoto, H., Contag, C. H., Palanker, D. 2011; 52 (3): 1780-1787

    Abstract

    Subthreshold retinal phototherapy demonstrated clinical efficacy for the treatment of diabetic macular edema without visible signs of retinal damage. To assess the range of cellular responses to sublethal hyperthermia, expression of the gene encoding a 70 kDa heat shock protein (HSP70) was evaluated after laser irradiation using a transgenic reporter mouse.One hundred millisecond, 532 nm laser exposures with 400 μm beam diameter were applied to the retina surrounding the optic nerve in 32 mice. Transcription from the HSP70 promoter was assessed relative to the control eye using a bioluminescence assay at 7 hours after laser application. The retinal pigmented epithelium (RPE) viability threshold was determined with a fluorescence assay. A computational model was developed to estimate temperature and the extent of cell damage.A significant increase in HSP70 transcription was found at exposures over 20 mW, half the threshold power for RPE cell death. Computational modeling estimated peak temperature T = 49°C at HSP70 expression threshold. At RPE viability threshold, T = 57°C. Similar temperatures and damage indices were calculated for clinical subvisible retinal treatment parameters.Beneficial effects of laser therapy have been previously shown to extend beyond those resulting from destruction of tissue. One hundred millisecond laser exposures at approximately half the threshold power of RPE damage induced transcription of HSP70, an indication of cellular response to sublethal thermal stress. A computational model of retinal hyperthermia can guide further optimization of laser parameters for nondamaging phototherapy.

    View details for DOI 10.1167/iovs.10-5917

    View details for Web of Science ID 000288965300070

    View details for PubMedID 21087969

  • Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters GENE THERAPY Gonzalez-Gonzalez, E., Ra, H., Spitler, R., Hickerson, R. P., Contag, C. H., Kaspar, R. L. 2010; 17 (10): 1270-1278

    Abstract

    Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.

    View details for DOI 10.1038/gt.2010.74

    View details for Web of Science ID 000282948600011

    View details for PubMedID 20463756

  • Biodegradable Nanoparticles With Sustained Release of Functional siRNA in Skin JOURNAL OF PHARMACEUTICAL SCIENCES Jacobson, G. B., Gonzalez-Gonzalez, E., Spitler, R., Shinde, R., Leake, D., Kaspar, R. L., Contag, C. H., Zare, R. N. 2010; 99 (10): 4261-4266

    Abstract

    A key challenge in developing RNAi-based therapeutics is efficient delivery of functional short interfering RNA (siRNA) to target cells. To address this need, we have used a supercritical CO(2) process to incorporate siRNA in biodegradable polymer nanoparticles (NPs) for in vivo sustained release. By this means we have obtained complete encapsulation of the siRNA with minimal initial burst effect from the surface of the NPs. The slow release of a fluorescently labeled siRNA mimic (siGLO Red) was observed for up to 80 days in vivo after intradermal injection into mouse footpads. In vivo gene silencing experiments were also performed, showing reduction of GFP signal in the epidermis of a reporter transgenic mouse model, which demonstrates that the siRNA retained activity following release from the polymer NPs.

    View details for DOI 10.1002/jps.22147

    View details for Web of Science ID 000282473400012

    View details for PubMedID 20737633

  • Silencing of Reporter Gene Expression in Skin Using siRNAs and Expression of Plasmid DNA Delivered by a Soluble Protrusion Array Device (PAD) MOLECULAR THERAPY Gonzalez-Gonzalez, E., Speaker, T. J., Hickerson, R. P., Spitler, R., Flores, M. A., Leake, D., Contag, C. H., Kaspar, R. L. 2010; 18 (9): 1667-1674

    Abstract

    Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation.

    View details for DOI 10.1038/mt.2010.126

    View details for Web of Science ID 000281502300013

    View details for PubMedID 20571543

    View details for PubMedCentralID PMC2956931