Bladder cancer (estimated 83,730 new cases and 17,200 death in 2021) is unique in that most (70-80%) of patients are diagnosed with early-stage, non-muscle invasive bladder cancer (NMIBC). NMIBC is first treated by transurethral resection (TUR) and then adjuvant transurethral therapies, such as intravesical BCG and chemotherapy, for patients with high risk for advanced stages. However, recurrence remains a significant problem. There has been no breakthrough in NMIBC treatment since BCG was FDA-approved for bladder cancer treatment in 1990. Last year, the FDA approved pembrolizumab for treating BCG-unresponsive, high-risk NMIBCs, but it is very expensive and its complete response rate is not very satisfactory. These days, the shortage of BCG has become a problem in clinical practice. Our long-term goal is to develop a clinically translatable NMIBC-treatment strategy to effectively and selectively kill cancer cells without collateral damage to normal bladder tissue. Our hypothesis is that a mitochondria-localizing and singlet oxygen (SO)-activatable prodrug is effectively activated by HAL (hexyl-5-aminolevulinate)-PDT in the mitochondria of cancer cells, thus greatly improving therapeutic efficacy of HAL-PDT with minimal collateral damage. Recently, we proved and advanced this novel SO-activatable prodrug concept in both intramolecular and intermolecular activation manners. The combination of green light and intravesical administration is expected to avoid collateral damage to normal bladder wall unlike traditional PDT. The objectives of this proposed work are 1) to develop SO- activatable prodrugs of clinically used anticancer drugs for NMIBC and 2) to prove enhanced efficacy of this combination treatment in both in vitro and in vivo models. An orthotopic rat model of bladder cancer will be used to better mimic physiology of human NMIBC. Three specific aim are proposed: Aim 1) Develop mitochondria- targeting and SO-activatable prodrugs of clinically used drugs for NMIBC, Aim 2) Determine uptake and the combination effect using 3D cell culture and healthy bladders (rat and rabbit), and Aim 3) Determine the anticancer effects and local side effects of the combination treatment using the orthotopic rat bladder cancer model. This project is highly innovative in both conceptual and technical aspects. It is a novel approach to address an important clinical problem using a highly multidisciplinary and integrative strategy. If successful, the proposed research will provide new effective treatment strategy and prodrugs for treating NMIBC with high likelihood of rapid clinical translation, because HAL and anticancer drugs have historically been used for NMIBC. Due to the advances in optical and transurethral surgical technology and the unique accessibility of bladder, there is no major technical barrier for implementing such strategy to clinical practice. We have been consulting with practicing urological clinicians to gear our strategy towards clinical tra...