The Preparation Of SiBcl-2-loaded Calcium Phosphate Lipid Hybrid Nanoparticles And Its Antitumor Activity

Objective:siRNA can knock down the expression of target genes effectively and specifically. Free siRNA injected into the systemic circulation will meet enzymatic degradation. It seems that free siRNA is difficult to accumulate in the tumor tissue and enter the tumor cell to play its therapeutic function. Developing a safe and efficient nanocarrier for systemic administration of siRNA is a major challenge in RNAi-based therapy currently. The pH-sensitive and charge-conversional conjugate, derivative of cholesterol modified by the citraconic anhydride, was synthesized in order to enhance the antitumor activity of si RNA in vivo. In order to construct a really efficient siRNA delivery system and improve the stability of siRNA in the blood circulation, the derivative of cholesterol was used to decorate the calcium phosphate lipid hybrid nanoparticles. It’s helpful to disassemble the calcium phosphate lipid hybrid nanoparticles in the endolysosomes because of the degradation of pH-sensitive conjuate. The siRNA could be released to the cytosol to improve antitumor activity of siRNA in vivo.Method:(1) The pH-sensitive and charge-conversional conjugate(CHOL-AA-Cit) was synthesized. The structure of conjugate was identified by spectrum analysis.(2) The siRNA targeted to Bcl-2 was chosen as a model gene. The water-in-oil microemulsion method was applied to prepare si Bcl-2-loaded calcium phosphate nanoparticles and the film dispersion was used to prepare lipid hybrid nanoparticles(LNPS@siBcl-2). The formulation was optimizied to gain the best prescription by adjusting the amount of outer lipids for lipid hybrid nanoparticles. The morphology of lipid hybrid nanoparticle was observed by transmission electron microscopy. The zeta potential and particle size were detected by Laser Particle Size Analyzer.(3) The toxicity of lipid hybrid nanoparticles was tested by MTT method. And the antitumor activity of LNPS@siBcl-2 was conducted on A549 cells by MTT method in vitro. The Caspase-3 Activity Assay Kit was applied to evaluate the effect of LNPS@siBcl-2 on the activity of Caspase-3 in A549 cell. The Western Blot was used to detect the expression of the target protein Bcl-2, and the expression of apoptosis-related protein BAX and Caspase-3 was detected to study the mechanism of inducing apoptosis of A549 cells by LNPS@siBcl-2.(4) Flow cytometry and laser confocal scan microscopy were applied to detect the cellular uptake and observe the sub-cellular distribution of LNPS@Cy5-siBcl-2 in A549 cells. The cellular uptake mechanism of LNPS@Cy5-siBcl-2 was detected in A549 cells. Laser confocal scan microscopy was used to observe the tumor spheroid uptake of LNPS@Cy5-siBcl-2 in A459 three-dimensional tumor spheroids to evaluate the penetrability of LNPS@Cy5-siBcl-2.(5) The xenograft tumor model was created by subcutaneous injection of A549 cells in nude mice to evaluate the antitumor activity of LNPS@siBcl-2. The body weight and tumor volume were detected after intravenous injection LNPS@siBcl-2 into nude mice every three days. The bio-distribution of Cy5-siBcl-2 in tumor tissue and in normal organs was detected by the Caliper IVIS Lumina II in vivo image system after LNPS@Cy5-siBcl-2 and free Cy5-siBcl-2 were injected via the tail vein into the nude mice. After living imaging, mice were sacrificed and the tissues were collected for further experiment. The tumor tissues and organs were cut into 4 μm slices, and the slices were stained with DAPI(100 μg/m L) and fixed by 4% paraformaldehyde solution. After that, the slices were subjected to laser confocal scan microscopy to observe Cy5-siBcl-2 fluorescence in the slices of tumor tissues and organs. The morphological changes of major organs and tumor tissue were observed in H&E staining sections by fluorescence microscope. The protein expression in tumor tissue was detected by Western Blot. Results:(1) The result of conjugate identification by spectrum analysis showed that the synthesized compound was objective production.The average diameter and zeta potential of the siBcl-2-loaded lipid hybrid nanoparticles(LNPS@siBcl-2) were 80 nm and-13 mV at pH 7.4, respectively. The average diameter and zeta potential changed to 1506 nm and +9 mV at pH 5.0, respectively.(2) LNPS@siBcl-2 exhibited excellent antitumor activity in concentration-dependent manner in vitro. The Kit assay showed that LNPS@siBcl-2 can significantly increase the activity of Caspase-3 in concentration-dependent manner.The results of Western Blot showed that the expression of Bcl-2 was siginificantly down-regulated and the expression of BAX and Caspase-3 was up-regulated in concentration-dependent manner, which lead to the death of A549 cells in vitro.(3) The results of flow cytometry and laser confocal scan microscopy showed that the cellular uptake would augment with the increase of incubation time and decrease of the medium pH. This indicated that the cellular uptake of LNPS@Cy5-siBcl-2 was time- and pH-dependent in A549 cells. The cellular uptake of LNPS@Cy5-siBcl-2 could be inhibited by low temperature(4℃) and ATP depletion. The results indicated that the cellular uptake process of LNPS@Cy5-siBcl-2 was energy-consuming in A549 cells. The cellular uptake inhibitors such as sucrose(the clathrin-associated pathway inhibitor), methyl-β-cyclodextrin(the caveolae-associated pathway inhibitor) and colchicine(macropinocytosis pathway inhibitor) can inhibit the cellular uptake of LNPS@Cy5-siBcl-2 in A549 cells. A distinct reduction of nanoparticles cellular uptake was observed after treated with colchicine indicated that the major path way involved in the internalization of LNPS@Cy5-siBcl-2 was macropinocytosis. The results of laser confocal scan microscopy indicated that the LNPS@Cy5-siBcl-2 could escape from the lysosome and release the Cy5-siBcl-2 into cytosol.(4) LNPS@Cy5-si Bcl-2 could deliver Cy5-si Bcl-2 more deeper area in A549 tumor spheroids.(5) The proliferation of A549 cell was inhibited by LNPS@siBcl-2 on the xenograft tumor model in the nude mice. After the injection of LNPS@siBcl-2 into the tumor-bearing nude mice, the body weight didn’t reduce markedly among all the nude mice. Compared with free Cy5-siBcl-2 treated mice, most of the Cy5-si Bcl-2 accumulated in tumor tissues specifically after LNPS@siBcl-2 was administered to tumor-bearing mice. The H&E staining results indicated that the degree of malignancy of A549 cells were decreased in tumor-bearing mice treated with LNPS@siBcl-2. According to the results of Western Blot, the expression of Bcl-2 was down-regulated, and the expression of BAX and Caspase-3 was up-regulated in tumor tissues of tumor-bearing mice treated with LNPS@siBcl-2. Conclusions:LNPS@siBcl-2 was pH-sensitive which could improve the transfection efficacy of siBcl-2. After the injection of LNPS@siBcl-2 into the tumor-bearing nude mice, Cy5-siBcl-2 could accumulate in the tumor site. LNPS@siBcl-2 could significantly delay the tumor growth in tumor-bearing nude mice, which resulted from the down-regulation of the expression of Bcl-2 and up-regulation of the expression of the BAX and Caspase-3. The lipid hybrid nanoparticle modified by pH-sensitive conjugate was an ideal siRNA delivery system.