Study On The New Photosensitizer ZnPcS4-BSA For Photodynamic Therapy Of The CNV In Vivo And In Vitro

Part IEffects on retinal pigment epithelium after PDT with ZnPcS4-BSAObjective: To investigate the effects on retinal pigment epithelial (RPE) cells after Zinc phthalocyanine tetra-sulfonated bound bovine serum albumin (ZnPcS4-BSA) based photodynamic therapy (PDT). These studies provide a rational basis for new clinical perspectives in future CNV therapy.Methods: Firstly, the cellular uptake of ZnPcS4 and ZnPcS4-BSA was quantified by UV-spectra after confluent human retinal pigment epithelial cells were exposed to various doses of ZnPcS4 and ZnPcS4-BSA for 1-24 h. Secondly, RPE cells exposed to 10 mg/ml ZnPcS4-BSA were irradiated with 50 or 100J/cm² light. After ZnPcS4-BSA based PDT, cell viability was evaluated by MTT, and reactive oxygen species (ROS ) and mitochondrial membrane potential (â–³(?)m) were assessed by flow cytometry and confocal microscopy. Then, investigate the expression of proangiogenic and antiangiogenic factors, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in retinal pigment epithelial (RPE) cells after photodynamic therapy (PDT).Results: After four hours incubation, the ratio of ZnPcS4-BSA content in RPE cells reached its peak. Therefore, four hours after incubation was selected as the optimal time point to irradiate the cell. PDT results in a significant increase in reactive oxygen species (ROS) and loss of mitochondrial membrane potential (â–³(?)m). Together, these results suggest that PDT induces apoptosis by inducing ROS production and a loss ofâ–³(?)m, in which ROS plays a critical role in these events. VEGF was increased and PEDF reduced in cultured RPE cells shortly after PDT.Conclusion: The cellular uptake of ZnPcS4-BSA is much more than that of ZnPcS4. The ratio of ZnPcS4-BSA content in RPE cells reached its peak after four hours incubation. In this study, intracellular ROS formation and loss ofâ–³(?)m were observed to play important roles in mediating apoptotic processes of RPE cells induced by ZnPcS₄-BSA based PDT . Part IILocalization of a targeted- photosensitizer: ZnPcS4-BSA in the rat eyeObjective : To investigate the intraocular localization of Zinc phthalocyanine tetra-sulfonated photosensitizer by using fluorescence microscopy and digital fundus fluorescence angiography.The localization and site of action of photosensitizers in the eye is important for photodynamic therapy for fundus disorders.Methods: Normal BN rats were divided into two groups: the control group and the experiment group. ZnPCS₄-BSA (2.0mg ? kg^-1) was intravenously administered to rats. The eyes were enucleated and examined by means of fluorescence microscopy 5, 10, 15, 30 and 60 minutes after dye injection. In vivo digital fundus fluorescence angiography with use of ZnPcS₄-BSA was performed.Results: Fluorescence from the choriod, choriocapillaris, and retinal pigment epithelial cells was increased quickly. Crest-time is 10-20 minutes after dye intravenous injection. 30 minutes later, mild fluorescence detected from the retina, fluorescence almost fadeaway at 60 minutes. Angiographic studies with use of ZnPcS₄-BSA revealed differences in the pattern and rate of photosensitizer accumulation.Conclusion: ZnPcS₄-BSA accumulated rapidly in the choroid and retinal pigment epithelium and less rapidly in the outer retina. Differences in ocular localization of the photosensitizer were demonstrated. ZnPcS₄-BSA based photodynamic therapy CNV optimal timing of laser irradiation after dye administration is 10-20 minutes.Part IIIEvaluation of the New Photosensitizer ZnPcS4-BSA for Photodynamictherapy of the Choroidal neovascularization in Rats Objective: To investigated the ability of ZnPcS₄-BSA to produce photochemical closure of experimental choroidal neovascularization (CNV) upon irradiation with 670nm laser light.Methods: CNV was created in the fundi of Brown-Norway rats using the argon laser model and documented by fluorescein angiography (FFA) and optical coherence tomography(OCT). PDT was performed at the doses of 2.0 mg/m2 on the CNV and on normal retina and choroid. The rats were divided into groups of PDT1 and PDT2 by the laser of 50 and 100J/cm2. Treatment outcomes were assessed by FFA and OCT and confirmed by light and electron microscopy.Result: Seven and fourteen days after laser injury, the OCT demonstrated nervous layer of retina thinningz in the photocoagulated zone. Fluorescein angiography didn't find hyperfluorescence. Twenty-one days after laser injury, Fluorescein angiography revealed hyperfluorescence, with later frames of the angiogram demonstrating increased leakage of fluorescein in the photocoagulated zone. The CNV is represented on the OCT as a highly reflective band merging with RPE/choriocapillaris layer obliterating the double band of fibrosis observed; the reflective fibrotic CNV that had merged with the RPE/choriocapillaris band as an elevated reflective mound. FFA and OCT indicated that irradiation with 670nm laser light 20 minutes after ZnPCS₄-BSA injection produced complete closure of choroidal neovascularization with minimal damage to overlying retina. Histology of light and electron microscopy demonstrated necrosis of CNV endothelial cells with minimal damage to surrounding tissues.Conclusion: This study demonstrated that Optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) can be efficiently and objectively used to assess the CNV in the animal model. ZnPCS₄ -BSA selectively localizes to choriocapillaris and CNV in rats, resulting in occlusion of laser-induced CNV with minimal damage to retina tissues. The effect was depended on the laser power. ZnPcS₄ -BSA-PDT is a potential new strategy for the treatment of macular degeneration and other human diseases manifesting choroidal neovascularization.