Prostate-specific membrane antigen (PSMA) is the hallmark enzyme-biomarker for prostate cancer because it is expressed in the epithelium of nearly all prostate cancers. In addition to this unique expression in prostate cancer, which has led to targeted delivery of imaging and therapeutic agents, the endothelial expression of PSMA on the neovasculature of both prostatic and non-prostatic tumors offers a broader opportunity to modulate the vascularization and improve drug delivery. We recently demonstrated that the addition of an albumin-binding motif to small-molecule PSMA-targeted agents dramatically slows clearance and allows for tumor uptake of nearly 50% of an injected dose. Our phosphoramidate targeting molecules selectively target the active-site of PSMA, and rapidly penetrate prostate tumor cells, internalizing to endosomes/lysosomes. In addition, our Phos-Am linker system can rapidly release cargo at endosomal pH but is stable at physiologically pH. Based on these key achievements, we are currently positioned to develop the first effective PSMA-targeted SMDC. However, a key question remains for the successful development and optimization of PSMA-targeted drug-conjugates, ?is uptake of PSMA-targeted conjugates in tumor and non-target tissues directly correlated with their cargo release?? Our hypothesis is that a PSMA-targeted near-IR ratiometric fluorescence probe (PSMA-NIR-RFP) can report on both uptake and cargo-release in PSMA-positive cells and tumors using dual- channel in vivo imaging. We will test our hypothesis by pursuing the following specific aims. The in vivo performance of the probe will be evaluated using a novel multi-cell type, 3D bioprinted prostatic tumor model accurately represents PSMA(+) primary tumor vasculature and can be employed in xenograft mouse models.
Aim #1 : Determine the in vitro spatiotemporal subcellular accumulation and release of cargo from a PSMA-NIR-RFP in PSMA(+) prostate tumor cells and tumor-associated vasculature. It is our assertion that a PSMA-NIR-RFP can report on the uptake, trafficking, and cargo release in endosomes and lysosomes of PSMA(+) cells, through their pH-triggered dye release.
Aim #2 : Determine the extent of cargo-release from PSMA-targeted conjugates in target and non- target tissues in PSMA(+) tumor-xenograft mouse model. It is our expectation that quantification of tumor- specific uptake and controlled cargo-release can be assessed using a PSMA-NIR-RFP in combination with dual-channel in vivo imaging. The most important outcome of the proposed work is that a PSMA-NIR-RFP will be developed and employed to model and report on the in vivo uptake and cargo-release of PSMA-targeted conjugates, which can be subsequently applied to the optimization of PSMA and other biomarker-targeted drug-conjugates.
The objective of this application is to employ a small-molecule PSMA-targeted near-IR ratiometric probe (PSMA-NIR-RFP) to report on uptake and cargo release in PSMA-positive cells, tumors, and vasculature using dual-channel in vivo imaging. The in vitro and in vivo performance of the probe will be evaluated using a novel multi-cell type, 3D bioprinted prostatic tumor model accurately represents PSMA(+) primary tumor vasculature and can be employed in xenograft mouse models. The most important outcome of the proposed work is that a PSMA-targeted near-IR ratiometric probe will be developed and employed to report on the in vivo uptake and cargo-release of PSMA-targeted conjugates, which can be subsequently applied to the optimization of PSMA and other biomarker-targeted drug-conjugates.
