Aptamer And PCB Formulated Liposomes For Targeting Delivery Of STAT3 SiRNA To Lung Tumor

Objectives:Preparation of siRNA-loaded PCB and aptamers co-modified liposomes for pulmonary targeting,the ability of delivery siRNA was investigated,evaluation of its efficacy combined with anti-cancer drug cisplatin(DDP)in vitro and vivo level.Methods:PCB was synthesized by the polymerization of CB monomer and bromine initiator,EAAD(Epoxy Alkyl Amine Derivative),a cationic lipid material,which was synthesized by ring cleavage reaction.Their structures were characterized by ~1H-NMR,FTIR and LC-MS.AS1411-PCB-EAAD liposomes(Apt-CELs)were prepared by ethanol injection and EDC/NHS method.The particle size distribution,Zeta potential,siRNA encapsulation efficiency and stability were measured.Apt-CELs-siRNA was spray-dried to prepare dry powder inhalant,and its aerodynamic particle size and siRNA loading capacity were evaluated for in vivo experiments subsequent.MTT assay,flow cytometry(FCM)and confocal microscopy(CLSM)were used to evaluate the safety of the carrier,its ability of the cell uptake,the localization in the cells and the effect on the apoptosis and migration of the cells.The expression of STAT3 gene and protein was detected by RT-PCR and Western Blot.The effect of Apt-CELs-siRNA on the sensitivity of A549 cells to DDP was investigated by the combination treatment of DDP and siRNA.In situ lung cancer model of nude mice with Luc-A549 was established.The difference of the distribution about the two modes of administration by tail vein and lung inhalation was examined by Lumina II.During the DDP and siRNA administration period,the tumor growth was monitored and the antitumor effect was evaluated.The changes of H&E staining were observed in the main organs of nude mice.Results:The cationic liposomes(CELs)were successfully prepared by EAAD and PCB.After loading siRNA,the particle size was less than 200 nm and positive charged with 30mV,which has the high rate of encapsulation,in vitro anti-RNase experiments also show that liposomes can well protect against RNase degradation from the serum.The aerosol dry powder inhalant was also round,the wettability was low,the physical particle size was about 10μm,the aerodynamic particle size was about 3μm,the emptying rate was more than 90%,and the agarose gel electrophoresis showed no significant effect on siRNA stability after freeze drying.Safety experiments showed that the concentration of CELs in the 0~250μg/mL has no effect on cell viability,which has a lower cytotoxicity compared with lipofectamine 2000.The siRNA uptake rate in the Apt-CELs group was as high as 90%in A549 cells,and the uptake of CELs in H1299 cells was the best at 8 h in H1299 cells,and the uptake of macrophages RAW264.7 was lower than 40%.The confocal localization experiments showed that our vector could deliver siRNA to cytoplasm and avoid macrophages in A549/RAW264.7 co-culture system.The results of in vitro pharmacodynamics showed that the expression of STAT3 gene at mRNA and protein level was significantly down-regulated following the Apt-CELs-STAT3-siRNA transfected cells,which significantly increased the apoptosis efficiency and decreased the migration efficiency.The sensitivity of cells to cisplatin was significantly increased after transfection,and STAT3-siRNA and PD-L1-siRNA had some synergistic effect in H1299 cells.Fluorescence imaging of nude mice and in vitro tissue showed that inhalation of Apt-CEL in the lungs could deliver Cy5-siRNA to lung tissue.In vivo pharmacodynamics experiments showed that DDP was injected intravenously and Apt-CELs-STAT3-siRNA with lung inhalation combined can significantly enhance the anti-tumor effect,the pathological section also shows that after combined administration lung tissue tends to normal,and the liver and kidney have no significant damage.Conclusion:Aptamer and PCB formulated liposomes for targeting delivery system can effectively load siRNA and has excellent gene delivery capacity,which could significantly down-regulated the expression of related proteins,Apt-CELs-siRNA also significantly enhance the anti-tumor effect of DDP by pulmonary inhalation.