PROJECT SUMMARY Prostate cancer (PC) is a leading cause of cancer death among male population. While treatments for early stage PC often utilize anti-androgen therapy, most patients eventually develop androgen-independent castration-resistant prostate cancer (CRPC) that requires surgical resection followed by radiation and chemotherapy. There are increasing interests in employing mitochondria-targeting therapeutics to overcome chemo-resistance in treating CRPC. Amphipathic tail-anchoring peptide (ATAP) targets mitochondria to induce apoptosis. The cytotoxic effects of ATAP do not require pro-apoptotic proteins, nor they are influenced by anti- apoptotic factors. By linking ATAP to an internalizing RGD peptide (iRGD), ATAP can be used to selectively target cancer cells to suppress prostate tumor growth. Meanwhile, it is highly desired that a chemotherapeutic drug can be co-delivered, so that synergistic dual drug effects can be achieved. Unfortunately, no delivery vehicles are available in the market for dual drug delivery in PC treatments. Self-assembled peptide nanoparticles hold great promise for targeted drug delivery and imaging, as they are inherently biocompatible and can be easily modified to interface with biomolecules. Studies from the investigators' groups showed that cyclic peptides consisting of di- or octa-amino acids can self-assemble into nanoparticles exhibiting stable fluorescence in the range of visible and near infrared ranges, which are suitable for cell and deep tissue imaging. The cyclic peptide nanoparticles (cPNP) can be tethered with tumor homing moieties and serve as a vehicle for targeted co-delivery of therapeutic reagents. This project is aimed to develop a cPNP-based platform to co-deliver ATAP-iRGD and paclitaxel for PC treatments, so that synergetic effects of mitochondria- dependent apoptosis and chemotherapy can be achieved. The platform also provides fluorescent tracking capability for drug release, thus allows us to uncover the targeting mechanism and to optimize therapeutic strategies in vivo. ATAP-iRGD is linked to cPNPs via carboxyl-to-amine crosslinking, and paclitaxel is conjugated onto cPNPs through ?-? stacking and electrostatic interactions. Here, an iterative approach is proposed to design and optimize biophysical and biochemical properties of various amino acid combinations in achieving a cPNP tumor-targeting vehicle that is biocompatible and suitable for dual drug loading and release. The pathways of iRGD-guided cPNP-ATAP/paclitaxel penetration and release will be studies. Its efficacy in inducing death of PC cells will be obtained by taking advantage of the nanoparticle's intrinsic fluorescence. The in vivo studies for treatments of mouse models of CRPC will be conducted using the cPNP-ATAP/paclitaxel. In addition, pharmacokinetic and bio-distribution analyses will be conducted to evaluate safety and toxicity of the dual drug delivery platform. The technology developed in this application will lay down the foundation of building a highly effective and selective method for CRPC treatment.