Construction And Application Of Redox-Responsive Organic-Inorganic Hybrid Calcium Phosphate Gene Delivery System Through Caveolae-Mediated Endocytosis

ObjectiveThe calcium phosphate（CaP）transfection is a routine method of gene transfection.However,traditional CaP nanoparticles are unstable,easy to gather into large particles,and hard to be uptaken by cells.Moreover,most of the CaP nanoparticles that enter the cells will be transported via the endo-lysosome pathway,resulting in the degradation of the loaded nucleic acid by the acidic environment and nuclease of the endolysosomal system.To improve the stability,increase the transfection efficiency,and reduce the toxicity,this thesis designed a functional organic small molecule Man-SS-Aln that was composed of a mannitol group,a disulfide bond and a diphosphate group based on the previous study.By the coordination interaction between diphosphate groups of the Man-SS-Aln and Ca²⁺of calcium phosphate to form organic-inorganic hybrid calcium phosphate gene delivery system,it achieved that the Man-SS-Aln was modified onto the surface of the CaP core and thus increased the stability of the CaP nanoparticles.The mannitol groups distributed at the outermost end would specifically stimulate and enhance the caveolae-mediated endocytosis,making the intracellular transport of nanoparticles bypass the endolysosome system so as to avoid the damage of nucleic acid drug by the endo-lysosome system and improve the transfection efficiency.The redox-responsive disulfide bonds introduced in the modification materials Man-SS-Aln would be broken down under the high level of glutathione in the cytoplasm,which would enable the controlled release of loaded nucleic acid and high gene transfection efficiency.Methods1.The organic small molecule Man-SS-Aln was synthesized by using alendronate sodium,L-cysteine,D-mannose and SPDP.The structure was characterized by HRMS,¹H NMR and ¹³C NMR.2.A set of organic-inorganic hybrid CaP-MSSA nanoparticles with different Man-SS-Aln modifications at 1%,2%,4%,and 15%（w/w）was prepared by the coprecipitation method,which was named as CaP-MSSA-1,CaP-MSSA-2,CaP-MSSA-4,and CaP-MSSA-15 respectively.The particle size,PDI,zeta potential and storage stability of all kinds of nanoparticles were explored by dynamic light scattering laser particle sizer.The morphology was observed by transmission electron microscopy and scanning electron microscopy.The surface elements mapping analysis was performed by field emission transmission electron microscopy.The gene loading and protective ability of CaP-MSSA nanoparticles was investigated by agarose gel electrophoresis.Agarose gel electrophoresis was also used to detect the gene release under the different levels of glutathione（GSH）in order to evaluate the redox-responsive ability of CaP-MSSA nanoparticles.3.Cytotoxicity of Man-SS-Aln,CaP and CaP-MSSA nanoparticles was studied by MTT assay.The safety of CaP and CaP-MSSA nanoparticles was further investigated by hemolysis test.Plasmid DNA encoding green fluorescent protein（GFP）and si RNA 443was adopted as the model gene.Flow cytometry and fluorescence microscopy were used to evaluate the gene transfection efficiency of CaP and CaP-MSSA nanoparticles in HEK293T and He La cells in vitro.4.The endocytic mechanism was investigated through evaluating gene transfection efficiency with or without specific inhibitors of different endocytic pathways.The cellular uptake pathway and intracellular transport behavior were investigated by labeling control plasmid and organelles with specific fluorescent probes.The effects of Man-SS-Aln,CaP and CaP-MSSA nanoparticles on the phosphorylation of caveolin-1were detected by western blot.5.The transfection efficiency of CaP,CaP-MSSA-1 and CaP-MA-1 nanoparticles in vivo was investigated in nude mice after single administration via tail vein injection.Results1.Man-SS-Aln composed of a mannitol group,a disulfide bond and a diphosphate group was obtained.The molecular structure of the product was identified as expected by¹H NMR,¹³C NMR and HRMS.2.Four kinds of organic-inorganic hybrid CaP-MSSA nanoparticles modified by the different mass ratio of Man-SS-Aln（CaP-MSSA-1,CaP-MSSA-2,CaP-MSSA-4,and CaP-MSSA-15 nanoparticles）were prepared by the coprecipitation method.The data of particle size,PDI and zeta potential showed that all the nanoparticles were negatively charged（-12 m V to-10 m V）,the average particle size was about 200～300 nm and they were evenly dispersed.SEM and TEM showed that the nanoparticles were spherical.According to the surface element mapping analysis,the elemental composition of CaP nanoparticles included Ca,O and P,while there was an additional S element in CaP-MSSA-1 nanoparticles,indicating that Man-SS-Aln was successfully modified on the surface of the CaP core.The stability result showed that the size of the CaP nanoparticles increased dramatically to 1718±415 nm after storage at 4℃for 1 day,and became too large to be detected due to the aggregation and sedimentation on the third day.The CaP-MSSA nanoparticles had remained stable for 30 days,indicating that introducing Man-SS-Aln modification significantly improved the stability of CaP nanoparticles.By agarose gel electrophoresis for the supernatant of the nanoparticles,no clear p DNA band was found in the CaP-MSSA group,indicating the efficiently loading of the genes.After treated with endonuclease,the p DNA bands were detected clearly,indicating the effective protection ability of CaP-MSSA nanoparticles for the loaded gene.Under the condition that simulates a high concentration of GSH at 10 m M in cells,CaP-MSSA-1 nanoparticles with disulfide bonds showed clear p DNA bands,and the electron microscopy showed the fuzzy edge and incomplete sphere of the nanoparticles.When incubated with the different concentrations of GSH at 0.01,0.1,1 and 10 m M,CaP-MSSA-1 nanoparticles showed clear p DNA bands only within a high level of GSH（≥1 m M）,suggesting that CaP-MSSA nanoparticles had good redox-responsive characteristics.3.MTT assay showed non-significant cytotoxicity between the modification material Man-SS-Aln and the control group.Compared with the CaP nanoparticles,the biocompatibility of the Man-SS-Aln modified CaP-MSSA nanoparticles was remarkedly improved.The hemolysis test revealed that the hemolytic rate of CaP-MSSA nanoparticles was less than 5%,and it did not affect the normal morphology of red blood cells,suggesting the safety of CaP-MSSA nanoparticles met the requirement of gene delivery vector.By flow cytometry and fluorescence microscopy,it showed that the transfection efficiency of CaP-MSSA-1 nanoparticles was about 77.9±1.0%for HEK293T cells and53.7±1.2%for Hela cells.The CaP-MSSA-1 nanoparticles had the highest gene transfection activity than other CaP-MSSA nanoparticles and CaP nanoparticles in vitro.Compared with CaP and CaP-MA-1 nanoparticles that didn’t contain disulfide bonds,CaP-MSSA-1 nanoparticles had higher transfection efficiency,suggesting that the redox-responsive disulfide bond could effectively improve the transfection efficiency of nanoparticles.4.The endocytic mechanism study showed that genistein,an inhibitor of caveolae-mediated endocytosis pathway,inhibited the cellular uptake and transfection efficiency of CaP-MSSA-1 nanoparticles more than that of CaP nanoparticles.According to the intracellular behavior of nanoparticles,the co-localization rate of CaP-MSSA-1nanoparticles with lysosome was only 17.8±3.3%but reached 82.6±3.0%with caveosome.Western blot analysis showed that CaP-MSSA-1 nanoparticles obviously enhanced the level of phosphorylated caveolin-1.These data revealed that the Man-SS-Aln modified CaP-MSSA-1 nanoparticles could specifically stimulate and enhance the caveolae-mediated endocytosis,reduce the degradation of nucleic acid drugs by the endo-lysosomal system,and improve the gene transfection efficiency.5.Gene transfection in vivo showed that CaP-MSSA-1 nanoparticles had the highest fluorescence intensity than that of CaP and CaP-MA-1 nanoparticles that didn’t contain disulfide bonds.After a single administration of CaP-MSSA-1 nanoparticles,the transfection efficiency could last for at least 72 hours,showing the sustained gene transfection efficacy in vivo.ConclusionsIn this study,an organic small molecule Man-SS-Aln consisting of a mannitol group,a disulfide bond,and a diphosphate group was designed and synthesized.Four kinds of uniform and stable organic-inorganic hybrid CaP-MSSA nanoparticles were prepared by surface modification with Man-SS-Aln at the different mass ratios.CaP-MSSA nanoparticles could effectively improve the phosphorylation level of caveolin-1,promote the nanoparticles to enter the cell by caveolae-mediated endocytosis,and avoid the degradation of loaded genes via the clathrin-mediated endocytosis.Meanwhile,the redox-responsive property of disulfide bonds realized the rapid and controlled release of loaded genes in cells,so as to exert good gene transfection efficiency in vitro and in vivo.This novel gene delivery system can be further investigated,which is expected to provide a safe and efficient delivery vector for the clinical gene therapy.