Development of theranostic agents for tumor imaging and photodynamic therapy (PDT)
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The idea of a single theranostic agent that can integrate real-time evaluation of tumor burden with improved treatment is highly desirable. These agents can help circumvent the current problems associated with combining multiple agents for tumor detection and subsequent therapy. Certain tumor-avid porphyrin-based photosensitizers (PS) used as drugs in PDT, with multi-functional groups, provide an opportunity to introduce desired key moieties in designing improved theranostic agents. Research carried out in our laboratory has led to the development of HPPH [3-(1'-hexyloxyethyl)-3-devinyl pyropheophorbide- a ] an effective PDT agent. A related analog, in which the hexyl ether chain is replaced with an iodo-benzyloxyethyl group, is also under preclinical investigation for tumor imaging by positron emission tomography (PET)/fluorescence and PDT. For improved tumor imaging and therapy, the high uptake of the theranostic agent and their tumor specificity are of utmost importance. Therefore, I hypothesize that use of targeted PS with optical/ PET imaging capabilities in combination with hyperthermia should improve tumor-imaging capability and long-term tumor cure by PDT . Published studies in our lab have shown the efficacy of 124 I- labeled methyl 3-(m-iodobenzyloxyethyl)pyropheophorbide- a as an effective agent for tumor imaging as well as PDT. Various isomers of the agent were synthesized in a bid to improve its tumor imaging as well PDT efficacy but the parent compound was found to be the most efficient. Imaging studies were able to highlight the tumor avid nature of the photosensitizer. The theranostic agent was able to image orthotopic tumors as well metastasis in a variety of tumor models both by PET as well as fluorescence imaging. Toxicology studies showed that the radioactive as well as the non-radioactive photosensitizer were nontoxic at extremely high doses. To further improve the tumor specific nature of the photosensitizer, cRGD peptides which target the integrin receptor were conjugated to the photosensitizer identified in Aim 1. The α v β 3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. cRGD (Arg-Gly-Asp) peptide represents a selective α v β 3 integrin ligand that has been extensively used for research, therapy, and diagnosis of neoangiogenesis. The 3-(1'-hexyloxyethyl)-3-devinylpyropheophorbide-a (HPPH), a chlorophyll-based photosensitizer was conjugated to cRGD and the related analogs. The cell uptake, in vitro/in vivo PDT efficacy and fluorescence-imaging potential of the conjugates were compared with the corresponding non-conjugated photosensitizer HPPH in α v β 3 integrin over-expressed U87 and 4T1 tumors. Compared to HPPH, the HPPH-cRGD conjugate in which the arginine and aspartic acid moieties were available for binding to two subunits of α v β 3 integrin showed faster clearance, enhanced tumor-imaging and PDT efficacy at 3-4 h post-injection. Molecular modeling studies also confirmed that the presence of HPPH moiety in HPPH-cRGD conjugate does not interfere with specific recognition of cRGD by α v β 3 integrin. Compared to U87 and 4T1 cells the HPPH-cRGD showed significantly low photosensitizing efficacy in A431 (α v β 3 negative) tumor cells, suggesting possible target-specificity of the conjugate. Conjugation of cRGD to methyl 3-(m-iodobenzyloxyethyl)pyropheophorbide- a showed similar results allowing us to replace the iodine with a fluorine for developing PET imaging agents. To further boost the PDT efficacy, mild hyperthermia was used to decrease tumor interstitial pressure and increase oxygen concentration within the tumor. Fever range hyperthermia helped increase the PDT efficacy of HPPH. Two modes of heating, whole animal as well localized heating were able to increase PDT efficiency. The uptake of the PS was unaffected by the heat treatment, as demonstrated through 14 C-labled PS based biodistribution studies. But there was a significant enhancement in the PDT efficacy in mice with colon 26 tumors following heat treatment as compared to tumored mice, which were kept at room temperature. Localized hyperthermia showed a better PDT response relative to whole body hyperthermia. Immunocompromised SCID mice with colon 26 tumors showed longer survival following PDT if they received either whole body or localized hyperthermia as compared to mice that did not receive the heat treatment. In conclusion, my thesis work allows for development of multifunctional agents that show promise as theranostic agents for both tumor imaging as well as therapeutic applications. The newly developed PS is highly tumor specific and shows enhanced PDT response as compared to the first generation compounds synthesized earlier.