The Preparation And Gene Delivery Efficiency Of Chondroitin Sulfate Modified Calcium Phosphate Nanoparticles

Efficient delivery and expression of exogenous genes is one of the keys of gene therapy.Common gene delivery vectors can be divided into viral carriers and non-viral carriers.Although viral gene carriers show high transfection efficiency,they have the risk of genome integration and their costs are much expensive.Non-viral delivery systems show decent biocompatibility and are easy to be modified,while their transfection efficiency is disappointing.Therefore,the development of biocompatible and efficient gene delivery system for gene therapy becomes imperative.In this research,a kind of anionic polysaccharide calcium phosphate gene delivery nanoparticles(CP-ALN-CS)was designed whose surface was modified by chondroitin sulfate(CS).This kind of delivery system possessed superb gene loading capability and biocompatibility.Unlike other non-viral nanocarriers which enter into cells mainly through the clathrin-mediated endocytosis pathway,CP-ALN-CS could be internalized by cells mainly via caveolin-dependent pathway,which can bypass lysosomes to avoid the degradation and destruction of genes,and ultimately improve the gene transfection efficiency.The specific research content is as follows:Calcium phosphate nanoparticles(CP NPs)were prepared by precipitation method with minor modification.Alendronate sodium(ALN)and chondroitin sulfate(CS)were adsorbed onto the surface of CP NPs to obtain CP-ALN-CS nanoparticles via electrostatic interaction.The physical and chemical properties of CP-ALN-CS nanoparticles such as particle size,surface potential,morphology,and stability were fully characterized and the results showed that CP-ALN-CS nanoparticles were spherical with a particle diameter of 130 nm and a surface potential of-5 m V and could stably exist in cell culture medium.Gene loading and release experiments showed that the maximum loading capacity of CP-ALN-CS for p EGFP was 230 μg/mg(p EGFP/NPs),and the loading efficiency of about 78%,which exhibiting a superb gene loading capability.Cellular uptake experiments demonstrated that CP-ALN-CS can be internalized by cells and showed higher uptake efficiency than CP,CP-ALN,and PEI did.Cytotoxicity experiments on HEK 293 T,NIH 3T3,and Raw264.7 cells indicated that ALN was toxic to cells,and the modification of CS could relieve cytoxicity.Organelle colocalization and cellular uptake inhibition experiments indicated that CP-ALN-CS could be internalized by cells through caveolin-mediated endocytosis pathway and transported to the endoplasmic reticulum and Golgi apparatus which can protect genes from lysosomal enzyme degradation.Finally,the p EGFP and m RNA which express green fluorescence were loaded in CP-ALN-CS to evaluate the in vitro transfection efficiency.The results showed that the transfection efficiency of CP-ALN-CS to p EGFP was higher than that of PEI with a positive cell rate of 72%.Likewise,the m RNA transfection efficiency of CP-ALN-CS was similar to that of the commercial transfection reagent lipofectamine 3000 which was significantly higher than that of PEI control.These results indicate that the caveolin-mediated endocytosis pathway is an effective way for nanoparticles to improve the gene transfection efficiency.In summary,this study constructed an anion chondroitin sulfate modified calcium phosphate gene delivery system to achieve efficient gene transfection through caveolin-mediated endocytosis pathway.This kind of strategies provides a new idea for the construction of next-generation gene delivery carriers and the development of in vitro transfection reagents.