The Pharmaceutical Research On Lyophilized Her2-specific Pegylated Immunoliposomes For Sirna Delivery

It can be envisaged that every human disease is caused by activity from one or a few genes and some of these genes should be amenable for RNAi intervention. The use of RNAi for cancer therapeutics has the potentiality of revolutionizing the treatment of this devastating disease[1]. For a successful siRNA delivery, the final destination is the cytoplasm of the target cells. This all-through progress remains a great challenge. The first concern is the stability of siRNA itself. Their relatively small size also leads to rapid clearance by kidney filtration after systemic injection. Together with the negatively charged nature of siRNA that prevent it from having association with cell membrane, it needs a carrier to load siRNA. When formulate into a delivery system, siRNA also needs to escape from the non-specific uptake by the reticuloendothelial system (RES). Then the siRNA formulation has to extravasate and gain access to the target tissue. At last they need to get across the cell membrane, followed by escaping from the endosome successfully to reach the cellular cytoplasm[2-5]. Up to the present day, the development of PEGylated immunoliposome represents a practical way in cancer gene therapy which may have the potential possibility to resolve these issues above.In this study, the HER2 overexpressing SK-BR-3 breast cancer cells were adopted. Through many methods such as PEGylation, anti-HER2 Fab'modification and lyophilization, we obtained excellent preparation which provides a better protection for siRNA, a better target for tumor, a better endosome release and a better gene silencing ability.We have gained some valuable results via consulting the basic work before[6], which demonstrsted that the most efficient DC-Chol/DOPE liposomes for siRNA delivery was at 1:1 molar ratio of DC-Chol to DOPE, PEGylation decreased siRNA transfection efficiency, HER2-Flip have a high specific expression in SK-BR-3 of breast cancer cell and endocytosis. On these basises, firstly we developed the PEGylated DC-Chol/DOPE liposomes (PL), then we attached the Fab of recombinant humanized anti-HER2 monoclonal antibody to the terminus of PEG on PL (called PEGylated immunoliposomes, PIL). Subsequently PIL was lyophilized (LPIL).In the first study, we evaluated the characterization of liposomes, such as particle size, zata potential, Fab conjugation, encapsulation efficiency of siRNA and the siRNA serum stability. Particle size and zeta potential results showed all prepared PIL and PL had a particle size between 130 and 150 nm. After lyophilization and rehydration with water, LPIL and LPL had a size increase of about 10²0 nm. The weight ratio of DC-Chol and siRNA of the LPIL entrapping siRNA which had the largest size was 5. Accompanying with increased weight ratio of DC-Chol and siRNA, the zeta potential of LPIL entrapping siRNA gradually increased, reaching a plateau when the ratio was over 10. The presentation and integrity of anti-HER2 Fab after conjugated to the liposomes surface were detected by SDS-PAGE. The conjugation efficiency was up to 12μg/ml or so. The siRNA encapsulation efficiency (EE) was determined by a Quant-ItTM RiboGreen^? RNA assay. At the ratio of 3 or 5, the EE of 2.5% PEG LPIL was significantly higher than that of 2.5% PEG PIL (P<0.01). Serum stability of siRNA in aqueous solution versus in liposomes preparations was characterized using agarose gel electrophoresis. The siRNA in 2.5% PEG LPIL and 2.5% PEG LPL showed the lowest degrading rate, indicating that 2.5% PEG LPIL and 2.5% PEG LPL provide a better protection for siRNA than 2.5% PEG PIL and 2.5% PEG PL.Recognition property, effect of PEGylation degree on gene silencing of liposomes and effect of RhoA silencing on cell migration were studied in vitro experiment. The binding ability and nternalization of LPIL to SK-BR-3 cells were analyzed by laser scanning confocal microscopy (LSCM). These results confirmed that 2.5% PEG LPIL specifically bind to HER2 and internalize into the cancer cells via receptor-mediated endocytosis. The transfection efficiency and gene knockdown efficiency were demonstrated by flow cytometry (FCM). The FCM results confirmed that the lyophilization/rehydration method, anti-HER2 Fab and optimal PEGylation degree are indispensable for superior gene silencing of 2.5%PEG LPIL. Moreover, we used RhoA as a cancer therapeutic target to demonstrate the potential of 2.5% PEG LPIL in cancer gene therapy. Compared with the untreated cells, transfection of SK-BR-3 cells with 2.5% PEG LPIL entrapping anti-RhoA siRNA down-regulated RhoA mRNA expression by 85% and inhibited cell invasion by 79.5%.In conclusion, our data showed the promise of 2.5%PEG LPIL in specifically binding to and silencing siRNA gene expression in HER2-overexpressing cancer cells and have the potential possibility to cure breast cancer.