University of Pennsylvania · Department of Bioengineering

Targeted Magnetic Resonance Imaging Contrast Agents

The non-invasive imaging of cancer biomarkers in living subjects could provide a powerful technique for locating metastatic disease, staging tumors, evaluating the availability of therapeutic targets, and monitoring the efficacy of treatment. Magnetic resonance (MR) imaging is a particularly attractive platform for such molecular imaging applications due to its ability to acquire high-resolution anatomical images in conjunction with measures of biomarker expression. However, a major obstacle faced by MR is overcoming the relatively low sensitivity of targeted MR contrast agents. In general, the number of cell receptors at a disease site is too low to recruit enough MR contrast agents to generate sufficient contrast. Therefore, there remains a need to develop new imaging agents capable of generating higher contrast and/or novel amplification strategies that will result in improved targeting. Our lab is pursuing both of these avenues. Specifically, we are developing new formulations of both iron oxide- and gadolinium-based nanoparticles, to improve the contrast-enhancing capabilities per nanoparticle. In parallel, we are developing new amplification schemes that allow for an improvement in the accumulation of contrast agents at the target site. Below are several representative publications:

1) Cheng, Z., Thorek, D.L.J, Tsourkas, A. (2009) Porous polymersomes with encapsulated Gd-labeled dendrimers as highly efficient MRI contrast agents. Advanced Functional Materials, 19(23), 3753-3759. PMCID: PMC3536029
2) Cheng, Z., Thorek, D.L.J., Tsourkas, A. (2010) Gd-conjugated dendrimer nanoclusters as a tumor targeted T1 magnetic resonance imaging contrast agent.  Angewandte Chemie International Edition, 49(2), 346-350.  PMCID: PMC2862691
3) Huang, C-H., Nwe, K., Al Zaki, A., Brechbiel, M. Tsourkas, A. (2012) Biodegradable polydisulfide dendrimer nanoclusters as MRI contrast agents. ACS Nano, 6(11), 9416-9424. PMCID: PMC3508381.
4) Yan, L., Higbee, E., Liu, R., Tsourkas, A., Cheng, Z. (2016). A simple method for the synthesis of porous polymeric vesicles and their application as MR contrast agent. Journal of Materials Chemistry B, 3(48), 9277-9284. PMCID:PMC4675335.


Site-Specific Bioconjugation Chemistries

Personalized medicine promises advancements in healthcare by emphasizing early diagnosis of disease, more accurate disease classification and targeted, rather than systemic, treatments. These goals are increasingly within reach thanks to the use of immunoassays to detect and classify diseases and the emergence of targeted therapies, many of which rely on nanoplatforms. A central element of many immunoassays and targeted therapies is the use of antibodies or other protein-based targeting ligands. In order to utilize targeting ligands, they frequently need to be conjugated to solid supports, as in the case of immunoassays, nanoparticles, as is increasingly the case with therapeutic or diagnostic payloads, or other functional moieities (e.g. proteins, drugs, etc.). While ideal conjugation methods should possess several features, including biocompatibility, high efficiency, site-specificity, and broad applicability, most existing conjugation methods unfortunately do not meet these requirements. To overcome this challenge, we recently created a suite of bioconjugation techniques that allow for the site-specific and efficient (~100%) attachment of targeting ligands to solid supports, nanoparticles, drugs (e.g. antibody-drug conjugates), or additional targeting ligands (e.g. bispecific antibodies). Our approaches are simple, rapid, scalable and can be used with any targeting ligands ranging in size from a peptide to a full-length antibody. Below are several representative publications:

1) Warden-Rothman, R., Caturegli, I., Popik, V., Tsourkas, A. (2013) Sortase-Tag Expressed Protein Ligation (STEPL): combining protein purification and site-specific bioconjugation into a single step. Analytical Chemistry, 85(22), 11090-11097. PMCID: PMC3843242.
2) Hui, J.Z., Tamsen, S., Song, Y., Tsourkas, A. (2015) LASIC: Light activated site-specific conjugation of native IgGs. Bioconjugate Chemistry, 26(8), 1456-1460. PMCID in process.
3) Wang*, H.H., Altun*, B., Nwe, K., Tsourkas, A. (2017) Proximity-based sortase-mediated ligation. Angewandte Chemie, 56(19), 5349-5352. (*contributed equally). PMCID:PMC5537000.
4) Greineder, C.F., Villa, C.H., Walsh, L.R., Kiseleva, R., Hood, E.D., Khoshnejad, M., Warden-Rothman, R., Tsourkas, A., Muzykantov, V.R. (2018). Site-specific modification of single-chain antibody fragments for bioconjugation and vascular immunotargeting. Bioconjugate Chemistry, 29(1), 56-66.

 

Therapeutic and Imaging Agents for Intra-Operative Procedures

Incomplete tumor resection is a common cause of local tumor recurrence following surgery. Patient outcomes strongly correlate with the extent of surgical resection. To improve removal of residual cancer cells at the time of surgery, we have developed nanoparticle-based agents for both image-guided surgery and photodynamic therapy (PDT). We have shown that in a preclinical surgical resection model in mice, following introduction or our nanoparticles, animals undergoing image-guided surgery demonstrate increased progression-free survival compared to animals undergoing microscopic surgery. Moreover, we have shown that animals that receive PDT demonstrate prolonged survival. These agents can be molecularly targeted and were designed to enable seamless integration into the current standard of care. Below are several representative publications:

1) Yan, L., Amirshaghaghi, A., Huang, D. Miller, J., Stein, J.M., Busch, T.M., Cheng, Z.*, Tsourkas, A.* (2018) Protoporphyrin IX (PpIX)-coated superparamagnetic iron oxide nanoparticle (SPION) nanoclusers for magnetic resonance imaging and photodynamic therapy. Advanced Functional Materials. In press. PMCID in progress.
2) Thawani, J.P., Amirshaghaghi, A., Yan, L., Stein, J., Liu, J., Tsourkas, A. (2017) Photoacoustic-guided surgery with indocyanine green-coated superparamagnetic iron oxide nanoparticle clusters extends progression-free survival in a pre-clinical mouse tumor model. Small. 13(37). In press. PMCID in progress.
3) Yan, L., Miller, J., Yuan, M, Liu, J., Higbee, E., Busch, T., Tsourkas, A., Cheng, Z. (2017) Improved photodynamic therapy efficacy of protoporphyrin IX loaded polymeric micelles using erlotinib pretreatment. Biomacromolecules. In press.

 

Theranostic Agents for Radiation Therapy and Imaging

Despite substantial advances in the treatment of many solid malignancies, poor outcomes is often due to the relative radiation dose limitations of the surrounding normal tissue constraining dose escalation to tumor, difficult of accurately identifiying the tumor margin, and the radioresistance of some cancer cells. To resolve some of these challenges, we have developed a theranostic nanoplatform, consisting of gold- and superparamagnetic iron oxide (SPIO) nanoparticles, with well-aligned radiotherapeutic and diagnostic properties. When GSMs were intravenously administered into tumor-bearing mice selective tumoral accumulation enabled MR imaging of tumor margins. Subsequent irradiation led to improved survival, due to gold-mediated radiosensitization, compared to mice receiving radiation alone. Moreover, contrast-enhanced MR was predictive or tumor response, providing a promising mechanism to guide follow-up treatment. Below are several representative publications:

1) Al Zaki, A., Joh, D., Cheng, Z., de Barros, A.L., Kao, G.D., Dorsey, J.F., Tsourkas, A. (2014) Gold-loaded polymeric micelles for computed tomography–guided radiation therapy treatment and radiosensitization. ACS Nano, 8(1), 104-112. PMCID: PMC3906892.
2) Joh, D.Y., Sun, L., Stangl, M., Al Zaki, A., Murty, S., Davis, J.J., Racharla, L., Bhang, D.H., Baumann, B.C., Alonso-Basanta, M., Ryeom, S.W., Kao, G.D., Tsourkas, A., Dorsey, J.F. (2013) Selective targeting of brain tumors with nanoparticle-induced radiosensitization. PLoS One, 8(4), e62425. PMCID: PMC3640092.
3) McQuade*, C., Al Zaki*, A., Desai, Y., Vido, M., Sakhuja, T., Cheng, Z., Hickey, R., Joh, D., Park, S-J, Kao, G.D., Dorsey, J.F., Tsourkas, A. (2015).  A multi-functional nanoplatform for imaging, radiotherapy, and the prediction of therapeutic response. Small, 11(7), 834-43 (*contributed equally). PMCID: PMC4329028
4) Al Zaki, A., Hui, J.Z., Higbee, E., Tsourkas, A. (2015) Biodistribution and clearance of gold-loaded polymeric micelles using 0.9 and 5 nm gold nanoparticles. Journal of Biomedical Nanotechnology, 11(10), 1836-1846. PMCID:PMC4942304.

 

Absolute Quantification of RNA Expression

Variations in gene expression are commonly considered the major determinants for dictating cell behavior. Accordingly, methods to measure gene expression, such as reverse-transcriptase (RT) PCR and DNA microarrays, have proven to be invaluable in regards to understanding cell regulatory processes and disease mechanisms. However, these methods generally provide only the relative change in gene expression for a population of cells and not an absolute measure of RNA copies at the single cell level. Under many circumstances it is the aberrant behavior of only a few cells or the stochasticity of RNA expression within a population that leads to disease evolution. Currently, we are developing optical imaging probes that are capable of the real-time visualization and absolute quantification of gene expression in single living cells. We envision that the development of an optical probe that can provide a more complete profile of gene expression, with spatial and temporal resolution in single living cells, will lead to significant advancements in molecular medicine, clinical diagnosis and biotechnology, and will also facilitate our understanding of human health and disease. Below are several representative publications:

1) Zhang, X., Zajac, A.L., Huang, L., Behlke, M.A., Tsourkas, A. (2014) Imaging the directed transport of single engineered RNA transcripts in real-time using ratiometric bimolecular beacons. PLoS One. 9(1), e85813. PMCID:PMC3893274.
2) Zhang, X., Song, Y., Shah, A.Y., Lekova, V., Raj, A., Huang, L., Behlke, M.A., Tsourkas, A. (2013) Quantitative Assessment of Ratiometric BiMolecular Beacons as a Tool for Imaging Single Engineered RNA Transcripts and Measuring Gene Expression in Living Cells. Nucleic Acids Research, 41(15), e152. PMCID: PMC3753654.
3) Chen, A., Davydenko, O., Behlke, M.A., Tsourkas, A. (2010) Ratiometric BiMolecular Beacons for the sensitive detection of RNA in single living cells.  Nucleic Acids Research, 38(14), e148.  PMCID: PMC2919734
4) Chen, A.K., Behlke, M.A., Tsourkas, A. (2008) Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA.  Nucleic Acids Research, 36(12), e69.  PMCID: PMC2475621