The Research Of Dendritic Phthalocyanins Nano Particles Photodynamic Therapy On Choroidal Neovascularization In Brown Norway Rats

Objective: (1)To prepare the third generation photosensitizer (phthalocyanine nanoparticles) by incorporation of nano carrier. (2) To investigate the pharmacokinetics and pharmacodynamics of phthalocyanine nanoparticles in a rat model of CNV and in in vitro cultured human RPE and HREC, and to assess structure activity relationship of the incorporated nano carrier.Methods: ( 1 ) First and second generation of Poly(aryl ether) dendritic phthalocyanines zine namely G₁-ZnPc(COOH)₈ and G₂-ZnPc(COOH)₁₆ were synthesized and incorporated with PLL-PEG-PLL to prepare polymer nanoparticles. The morphology and photophysical properties of polymer nanoparticles loaded with dendritic phthaloeyanines were studied by AFM,TEM,UV/Vis and fluorescent spectrometer. (2) The cellular uptake of G₁-ZnPc(COOH)₈ and G₁-ZnPc(COOH)₈/m in RPE and HREC cultured in vitro was quantified by UV-spectra after exposure to both nanoparticles for 1～6 h. RPE cells exposed to 1.0×10-5mol/L G₁-ZnPc(COOH)₈ and G₁-ZnPc(COOH)₈/m were irradiated with laser at a dose of 2J/cm2. Cell viability was assayed via CCK-8 test and mitochondrial membrane potential ( m) were assessed by LSCM. (3) A total of 42 normal BN rats were enrolled, Six for control group and the rest for experiment groups.Rats were photocoagulated by 532nm laser irradiation on the retinas. Two ways of laser import, three surface contact lens and LIO, were used in two groups, respectively. Fundus examination,OCT, FFA,light microscope,and electron microscope were performed on various time point after photocoagulation to comfirm to development of CNV. The feasibility of both approaches for CNV model was assessed. (4) The efficacy of G₁-ZnPc(COOH)₈ -PDT and G₁-ZnPc(COOH)₈/m-PDT against CNV were validated through fundus examination,OCT, FFA,and electron microscope.Results: (1)AFM and TEM showed that two polymer nanoparticles formed core-shell structure, the shape of G₁-ZnPc(COOH)₈ /m is spherical with a diameter 50 nm and G₂-ZnPc(COOH)₁₆/m is spheroidicity with a diameter of 120nm. Both showed fine dispersibility. The peak absorption of G₁-ZnPc(COOH)₈ /m is 638nm, with a redshift 11nm in contrast to G₁-ZnPc(COOH)₈. For the G₂-ZnPc(COOH)₁₆/m, the peak absorption is 623nm which is redshift 13nm in contrast to G₂-ZnPc(COOH)₁₆. The duration of fluorescence of four photosensitizer are G₁-ZnPc(COOH)₈ (5.37ns),G₁-ZnPc(COOH)₈/m(0.49ns),G₂-ZnPc(COOH)₁₆ (5.93ns) and G₂-ZnPc(COOH)₁₆ /m(0.9ns) respectively. (2) There is a significant difference in the the uptake of the two photosensitizers between RPE and HREC without difference between the two drugs. Both photosensitizers mediated PDT significantly damaged RPE cells, the survival rate of RPE cells in nano photosensitizer group was significantly lower than the free phthalocyanine content complex group. Cells in the two groups are strong single green fluorescent under LSCM after PDT. (3) Three weeks after photocoagulation, the damage of fundus detected by OCT suggested the development of CNV. FFA also showed obvious fluorescence leakage at majority photocoagulation spot. Capillary-like structure were seen invade the retina. TEM showed that neovasculars formed in retina with erythrocyte surrounded by endothelial cells. No significant difference in the efficiency of CNV induction was observed in the two approaches of laser import. (4) One day after PDT, no significant improvement of in the leakage was found in the two experimental groups. Less leakage of CNV was observed in two experimental groups 1 week and 2 weeks later without significance differences between PDT1 group and PDT2 group. After 1 week, No leakage of CNV was found in the two experimental groups, but no significance differences was observed between PDT1 group and PDT2 group. But 2 weeks after PDT less leakage and closure of CNV were demonstrated by fundus examination,OCT,FFA and electron microscope with a significant difference between groups. No improvement of leakage was found in four control groups on three time point.Conclusion: (1)Polymer nanoparticles have lower fluorescence iniensity but higher singlet excited life by incorporated with polymeric micelles. (2) The photodynamic therapy efficiency of dendritic phthalocyanines was greatly enhanced in contrast to free dendritic phthalocyanines.(3) Frequency multiplication 532nm laser imported by LIO can induce CNV model effectively and is worth prevalence.(4) Nano dendritic phthalocyanines have higher CNV occlusion compared with free dendritic phthalocyanines. Improved Nano dendritic phthalocyanines have higher photodynamic effects by incorporated with polymeric micelles.