Specific Aim: We aim to design and fabricate multifunctional targeted Indocyanine green(ICG)-Mitomycin C (MMC) incorporated perfluorocarbon nano-double emulsions (TIMPNDEs), and exploretheir potential for uses in 1) photodynamic-, 2) photothermal-, and 3) chemo-therapies.Background: Multifunctional therapeutic agent has been demonstrated as an emerging and promisingstrategy for cancer resistance. MMC is one of the most commonly used anticancer drugs. However, seriousside effects caused from high dose hamper its application. In terms of the alternative anticancer treatment,phototherapy has been widely explored in the clinic of cancer therapy due to its robust efficacy, highaccessibility, and easy-to-use capability, whereas ICG, a water-soluble tricarbocyanine dye, is one of fewclinically proved photosensitizers. Instead of serving as contrast agent in near infrared (NIR) tomography,ICG is further used as the agent of phototherapy for cancer treatment since ICG can produce singlet oxygenand hyperthermia effect upon NIR irradiation. However, many drawbacks of ICG such as 1)concentration-dependent aggregation which is detrimental to its photosensitivity, 2) irreversible andaccelerated degradation in aqueous solution, and 3) short half-life (3 – 4 min) in vivo tremendously hamperits applicability. Taken all together, development of a targeted ICG-MMC incorporated agent with highstability and effectiveness is urgently needed for bladder cancer resistance.Features: To enhance the applicability of the developed therapeutical agent, TIMPNDEs were assembledby using perfluorocarbon liquid, ICG, and MMC molecules that enable to provide following advantages:[1] Multifunctional TIMPNDEs can provide both physical and chemical treatments for bladder cancer cellswhereby the efficacy of cancer eradication can be dramatically enhanced.[2] ICG and MMC can be protected by the emulsion vehicles and thus the stabilities of both drug moleculesespecially ICG can be enhanced.[3] Since perfluorocarbon possesses high O2-dissolving capability, the developed TIMPNDEs enable toprovide abundance of oxygen molecules for use in photodynamic therapy.[4] Based on the binding specificity of the TIMPNDEs, the amount of both ICG and MMC as wells asefficiency of drug delivery to the tumor cells can be enhanced and thus raise the effectiveness of cancereradication. Furthermore, since the TIMPNDEs can provide both photo- and chemo-therapies, the doseof MMC while treated by TIMPNDEs may be lower than that used in normal chemotherapy and thatmay reduce the chemotherapy-induced side effect accordingly.Research Schedule: The following tasks will be performed orderly in the next two years: Stage 1: Optimization and characterization of TIMPNDEs (The 1st Year)[1] To evaluate the loading efficiency, content percentage, degradation rate and drug release efficiency forencapsulated ICG and/or MMC under static setting without laser irradiation[2] Cytotoxicity of the TIMPNDEs[3] Capability of oxygen carry of the TIMPNDEs[4] Targetability of the TIPNDEs to the specific bladder cancer cells Stage 2: Applicability of the TIMPNDEs for both photo- and chemotherapy (The 1st Year)[1] To investigate the effect of TIMPNDEs concentration on singlet oxygen generation[2] To investigate the effect of TIMPNDEs on temperature increase[3] To investigate the release profile of encapsulated MMC under static and NIR-irradiated conditions[4] Repeat the above three studies by using different NIR exposure conditions (intensity and irradiation time)[5] To optimize the TIMPNDEs fabrication procedures and operation parameters in cancer eradication Stage 3: In vitro and in vivo examinations for the developed TIMPNDEs (The 1st - 2nd Year)[1] Cell assay using human bladder cancer cell line (in vitro assay)[2] Animal study. Modification of the TIMPNDEs and reproducibility validation (in vivo assay)