| Summary: | 碩士 === 臺北醫學大學 === 藥學研究所 === 98 === The aim of this study is to delineate the entry mechanism of polymeric micelle-formulated plasmid DNA into Raw 264.7 macrophage cells. We used seven inhibitors to block different endocytosis pathways : (1) acetic acid (2) chlropromazine (3) PMA (phorbol 12-myristate 13-acetate) (4) amiloride (5) ammonium chloride (6) chlroquine (7) cytochalasin B for calthrin-mediated endocytosis (CME), caveolae-mediated endocytosis, macropinocytosis, clathrin- and caveolin-independent endocytosis, respectively.
When analyzing the toxicity of polymeric micelles by MTT assay and apoptotic array, there were no differences between control and the polymeric micelles treated cell within 2-24 hours in MTT assay. In apoptotic array, we found that seven pro-apoptotic genes and two anti-apoptotic genes of mRNA levels were up-regulated after treatment of polymeric micelles for 48 hours. In microscopic examination, the results suggested that cellular morphology were similar between control and treated with polymeric micelles cells. Therefore, the polymeric micelles may not have toxicity to cells.
To analyzing the amount of plasmid DNA, we extract total genome from cells and proceeding PCR with Lac Z gene, the entry of plasmid DNA was evaluated by electrophoresis. After continuously incubate P/ PM with RAW264.7 cells, the highest plasmid DNA amount in cell was estimated in 2 - 4 hr, and then decreased at 8, 12, 24 hr. The elimination rate of plasmid DNA was 0.0014 ?b 0.00017 min-1. Incubating cell with P/ PM for 2 hr and then washout, the plasmid DNA amount in cell was decreased as time increased, and the elimination rate was found in 0.0012 min-1.
In additions, delivering FITC-labeled nanoparticle and plasmid DNA mixed with superfect in to cells. As a result, both of them entered into cell were increased within time. And in the DNase stability test, the protected plasmid DNA by polymeric micelle were stable from 1 hour (P only) to 3 hours (P/ PM) in DNase-containing solution.
After pre-treat inhibitors with P/ PM, the amount of plasmid DNA in cells were analyzed at indicated time. The results revealed that the addition of acetic acid which inhibit the clathrin-mediated endocytosis and cytochalasin B which non-selectively inhibit the endocytosis could decrease the entry of plasmid DNA into cells. This result indicated that the entry mechanism of the plasmid DNA was related to clathrin-mediated endocytosis. Other inhibitors didn’t affect the entry of plasmid DNA. In additions, amiloride significantly increased the DNA entering into cell at 24 hr.
In conclusion, the internalization of P/ PM into cell was through clathrin-mediated endocytosis.
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