The Evaluation Of SiRNA Loaded Lipopolyamine Co-modified Liposomes Targetinganti-EGFR MAb

Objective: In order to structure Staramine co-modified cationic liposomes loadedwith Survivin-siRNA targeting Anti-EGFR mAb (EGFR-PEG-SCLs-siRNA). We evaluateits pharmaceutical properties, cytotoxicity, as well as the uptake rate, the mechanism andSurvivin-siRNA play a role in apoptosis in vitro. Some preliminary work we have doneincluding screening siRNA sequence in vitro, in order to parpare dry powder inhalation forpulmonary delivery in the future.Method: Staramine was synthesized by the substitution reaction and amidationreaction. On the other hand, synthesis of DSPE-PEG2000-Mal modified cationic liposomeswas parpared. All of them were characterized by TLC, FT-IR,1H-NMR. Then we screenedthe siRNA sequence of the best silence by western blot. Liposomes were prepared usingthe ethanol injection method. The characteristics of PEG-SCLs-siRNA on size, Zetapotential, encapsulation efficiency, RNase stability were investigated. As A549/DDP cellsare overpressed survivin proteins, we choose A549/DDP cells as the tumor cell models.Cell cytotoxity were investigated by MTT. Lipofectamine2000as the positive control,subsequently, the cellular uptake, mechanisms, and apoptosis in intro were analyzed byFlow Cytometry and Atomic force microscopy respectively.Results: Staramine and DSPE-PEG2000-Mal were successfully synthesized and wecalculated thier purity by1H-NMR were87.0%and86.2%respectively. Then wescreened the siRNA sequence of the highest silence of38.4%. The average size and zetapotential of EGFR-PEG-SCLs-siRNA were (172.7±6.6) nm and (+7.78±0.5) mV,respectively. The entrapment efficiency of siRNA was (90.8±0.8)%. The liposomes canprotect siRNA well without degrading by RNase. Atomic force microscopy showedendocytosis of cellular uptake. The Anti-EGFR mAb targeted (p＜0.05) increased cellularuptake as its mean fluorescence intensity was1.4-fold higher by Flow Cytometry. Theresults of uptake pathways of EGFR-PEG-SCLs indicated that clathrin-mediatedendocytosis and caveolin-mediated endocytosis were the major routes with energy consuming. We used Flow cytometry to detect the apoptosis of A549/DDP cells, theapoptotic Index by EGFR-PEG-SCLs-siRNA group was (41.19±1.83)%, significantlyhigher than that in Lipofectamine-2000transfection group (42.84±2.53).Conclusion: We parpared Staramine co-modified cationic liposomes loaded withSurvivin-siRNA targeting Anti-EGFR mAb (EGFR-PEG-SCLs-siRNA) with anappropriate particle size, zeta potential and a high encapsulation efficiency. TheAnti-EGFR mAb targeted increased cellular uptake dramatically. The primary uptakepathways of EGFR-PEG-SCLs were clathrin-mediated endocytosis and caveolin-mediatedendocytosis. EGFR-PEG-SCLs can delivery Survivin-siRNA to cells to play a role topromotes apoptosis in vitro..