| Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer. The development of efficient and safe gene transfer systems could be one of the most important directions for gene therapy. Low transfection efficiency and instability in vivo are the main barriers for siRNA delivery. To overcome these barriers, the novel core-membrane siRNA delivery system will be designed, which combined the advantage of cationic polymers and calcium phosphate nanoparticles. The calcium phosphate-siRNA nanoparticles are encapsulated in liposomes, and the surface is modified with cationic polymers. The preparation, character, cytotoxicity, cellular uptake, transfection efficiency and gene silence efficiency in vitro of the vector were studied. Additionally, VEGF-siRNA mediated by this vector would be studied for tumor angiogenesis inhibition effect. This project would provide new methods for reasonable design of non-viral siRNA vectors.Objective:By analyzing the main barriers for siRNA delivery and according to the intracellular mechanisms and targets, the novel core-membrane siRNA delivery system will be designed, which combined the advantage of cationic polymers and cationic lipids. This system possesses considerable endosomal escape capability and high efficiency in target tumor inhibition. This project would provide a reference for reasonable design of non-viral siRNA vectors.Methods:①Synthesis and characterization of PEI-Chol. PEI-Chol (PEI 800-Chol) was synthesized by PEI (800Da) and cholesterol, which was covalently conjugated to the backbone of PEL The basic properties of the compound were examined.②Preparation and characterization of calcium phosphate nanoparticles. Calcium phosphate nanoparticles were prepared by microemulsion and co-precipitation method. The influence factors of the preparation of nanoparticles and the basic properties of nanoparticles were studied, then one of the most suitable one was selected. The ability of carrying siRNA and the ratio of incubation were determined by agarose gel electrophoresis.③Preparation of polycation liposomes/calcium phosphate nanoparticles complex (PLCP). We used liposome-encapsulated technology to control the particle size of calcium phosphate nanoparticles, and modified the polymersurface by cationic liposome. Also we investigated the shape, size and zeta potential of PCL and PLCP, determinated the buffer capacity, cytotoxicity of PLCP, filtered the lyoprotectants, and made a preliminary assessment of its ability in vitro transfection.④Evaluation of the PLCP’s Cellular uptake capacity. We studied the cellular uptake in MCF-7 cells, which mediated by CaP NP, PCL, LipofectamineTM 2000 and PLCP. Observations of cellular uptake mediated by PLCP at various N/P ratios. We detected the impact of DOPE on cellular uptake through constructing carrier by different lipids.⑤In vitro gene silencing activity of siRNA mediated by PLCP. One hand, we designed the siRNA sequences against GFP, using breast cancer MCF-7 cells as cell model, verified the transfection effect, and detected the inhibition of GFP-mRNA expression on tumor cells mediated by PLCP. On the other hand, we designed the siRNA sequences against VEGF, using breast cancer MCF-7 cells as cell model, and detected the inhibition of VEGF mRNA and VEGF protein expression mediated by PLCP on tumor cells by real-time quantitative PCR and Western blot assay.⑥Tumor growth inhibition study mediated by PLCP. The nude mice model was established by injected MCF-7 cells, and the effect of anti-VEGF siRNA mediated by PLCP in the inhibition of tumor growth and anti-angiogenesis was studied.Results:①PEI 800-Chol was synthesized by PEI (800Da) and cholesterol. The 1H-NMR and IR results indicated that cholesterol was covalently conjugated to the backbone of PEI. The chloride group of Cholesterol and the amide of PEI were reacted to form an amide group. The product has neither package monomer cholesterol benzoyl chloride nor its hydrolysis product and carboxyl.②Calcium phosphate nanoparticles were prepared by microemulsion and co-precipitation methods. During the preparation of microemulsion, the optimal ratio of Triton X-100/n-hexyl=3:2 (w/w) and (Triton X-100+n-hexyl)/cyclohexane=6:4 (w/w) was selected, the particle sizes of CaP NPs were 15.2±1.7 nm, PDI were 0.206±0.029, and the negative zeta potentials were-5.4±1.6 mV. During the preparation of co-precipitation, the system was stable when the pH of the solution was maintained at 6.95±0.65, and the different proportions of A and B solution had obvious effect on the particle size of the nanoparticles. The optimal particle size was obtained when B:A was 1. The particle sizes of CaP NPs were 148.9±21.8 nm, PDI were 0.248±0.017, and the negative zeta potentials were-13.6±1.0 mV. We finally selected the co-precipitation method as the more suitable preparation of calcium phosphate nanoparticles for the study. Agarose gel electrophoresis indicated that siRNA swimming can completely blocked when the mass ratio of CaP NP and siRNA was 15:1.③The novel core-membrane siRNA delivery system was designed based on the Calcium phosphate nanoparticles and polycation liposomes. The calcium phosphate-siRNA nanoparticles were encapsulated in liposomes, and the surface was modified with cationic polymers. PCL was prepared using film hydration method. The particle sizes of PCL/siRNA were 129.6±14.0 nm, PDI were 0.200±0.023, and the zeta potentials were 39.3±6.2 mV. The particle sizes of PLCP were approximately 260.4±23.4 nm, PDI were 0.188±0.038, and the zeta potentials were 0.3±0.2 mV. Determination of buffer capacity experiments showed that the buffer capacity of PLCP fell in between that of PCL and PEI 800, but still has a strong buffering capacity. Filter results of the lyoprotectants for PLCP showed that 5% system quality of mannitol was the best. The cytotoxicity of PLCP was less than that of PEI (molecular weight 25kDa)/siRNA mixture, showed a higher security, while the transfection efficiency was almost equal to PEI (molecular weight 25kDa)/siRNA mixture.④Through comparing the cell uptake ability of CaP NP, PCL, LipofectamineTM 2000 and PLCP in the MCF-7 cells. The results showed that the PLCP mediated siRNA exhibited the strongest fluorescence intensity. Different N/P ratios has a significant impact on the cellular uptake of PLCP, with the increase of N/P, the fluorescence intensity increases, when N/P was 20, the fluorescence intensity reached the maximum and then slowly decreased. The effect of DOPE on the cell uptake was studied through the construction of PLCPs and PLCP. The results showed that the siRNA expression mediated by PLCP was significantly better than that of PLCPs.⑤Through the design of siRNA sequences aimed at GFP, the MCF-7 cells stably GFP expressing were used as the cell model, and the PLCP was used to verify the results. The results showed that GFP-443 was the best sequence. In vitro gene silencing assay, anti-GFP siRNA mediated by vectors, the GFP mRNA expression was detected by the fluorescent image and RT-qPCR method. The results indicated that the CaP NP group did not show obvious effects of gene silencing, PCL group can effectively inhibit the expression of GFP, however, with the increase of transfection time, the inhibition efficiency decreased, PLCP and LipofectamineTM 2000 can significantly inhibit the expression of GFP in the transfection time (0-72h), PLCP silencing efficiency reached 80%, significantly higher than LipofectamineTM 2000 (-60%) during 48-72h. Through the design of siRNA sequences aimed at VEGF, the MCF-7 cells were used as the cell model. The inhibition effect of the expression of VEGF mRNA and VEGF protein in tumor cells was determined by RT-qPCR and Western blot. RT-qPCR results showed that the PLCP/VEGF siRNA2 group got the best silencing efficiency, VEGF siRNA group has more significant gene silencing efficiency than the random sequences of siRNA. Through the comparison of three sequences of VEGF siRNA and inter group comparison of LipofectamineTM 2000, the sequence of VEGF-1137 siRNA can get very low expression of VEGF. Western blot results showed that, the inhibition of protein expression of designed VEGF siRNA was stronger than the random siRNA. Overall analysis with 72h, the inhibition of VEGF-A expression of PLCP/VEGF siRNA group was sustained and stable, and has certain advantages compared with LipofectamineTM 2000/VEGF siRNA group.⑥ The nude mice model was established by injected MCF-7 cells, and the effect of siRNA-VEGF mediated by PLCP in the inhibition of tumor growth and anti-angiogenesis was studied. Nude mice subcutaneous injection of MCF-7 cells suspension in the 10 day smoothly into a tumor. When the tumor grew to 50-100mm3, the mice were randomly divided into seven groups for the subsequent experiments. The tumor volume of injection PLCP/siRNA-VEGF combined with Adriamycin group decreased obviously. Compared to the control group, the micro vascular density of injection PLCP/siRNA-VEGF combined with Adriamycin group and a single injection of PLCP/siRNA-VEGF group decreased significantly. Compared to the control group, the VEGF, AKT2 and HIF-laprotein expression of injection PLCP/siRNA-VEGF combined with Adriamycin group and a single injection of PLCP/siRNA-VEGF group decreased significantly.Conclusion:In this study, we demonstrated a new approach for RNAi gene silencing using a PLCP delivery system. CaP NPs core were prepared by a co-precipitation method and further coated with PCL to form PLCP. Since the considerable endosomal escape capability, low cytotoxicity, high efficiency in target gene silencing and tumor growth inhibition, the PLCP offers a potential application for delivering therapeutic siRNA. |
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