Rational Optimization Of Hyaluronic Acid-based Gene Delivery Carrier And Its Mediated Gene Silencing For Breast Cancer

Cationic polysaccharides are currently used as a class of non-viral gene delivery carriers.This kind of carrier has the very high load capacity,but in vivo transport is easily associated with plasma proteins and clearance by the reticuloendothelial system.Therefore,how to improve the transfection efficiency in vivo is an urgent problem to be solved based on the cationic polysaccharide gene therapy.Study on the core-shell structure of the virus gene vector in the prophase laboratory research,hyaluronic acid(HA)and the cationic functional groups of polyethyleneimine(PEI)were introduced into the bridge chain,has formed “polysaccharide-bridge chain-cationic functional group” structure of polysaccharide.When the modified polysaccharide interacts with nucleic acid,the cationic functional group PEI can compress DNA by electrostatic interaction.Under the traction of the bridge chain,the spatial conformation of the polysaccharide skeleton is changed,and it is wrapped on the outer side of PEI-DNA to form self-assembled nano vesicle structure.The nano-gene carrier can significantly improve the efficiency of nucleic acid transfection,which has the advantages of good biocompatibility,long circulation,and tumor targeting.However,the introduction of the bridge chain how to affect the carrier's nucleic acid delivery has not been further studied.Based on this,we constructed cationic hyaluronic acid with different length of bridge chain,and investigated the changes of nucleic acid delivery ability and its application in gene therapy of breast cancer.The main research are as follows:1.Synthesis and characterization of the vehicles.PEI was grafted onto HA to synthesize HA-PEI by amide reaction.Using hexamethylenediamine and 1,12-dodecanediamine as bridging chains,PEI was grafted onto HA to synthesize HA-6-PEI and HA-12-PEI,respectively.The structure was characterized by proton nuclear magnetic resonance spectroscopy and fourier infrared spectroscopy.The particle size of the carrier was characterized by Laser nano-particle size analyzer and transmission electron microscope.The grafting rate of cationic functional groups in the carrier was measured by field emission scanning electron microscope.The experimental results showed that HA-PEI,HA-6-PEI and HA-12-PEI were successfully prepared.DNA gel retardation experiments showed that HA-PEI,HA-6-PEI,and HA-12-PEI could successfully load DNA.Based on the dynamic light scattering and circular dichroism spectra,the HA-6-PEI,HA-12-PEI loaded DNA can be self-assembled.2.In vitro study of mouse 4T1 breast cancer cells.(1)In order to investigate the gene delivery properties of HA-PEI,HA-6-PEI and HA-12-PEI in vitro,cell proliferation,cell uptake,intracellular distribution,cell transfection and cellular uptake mechanism were investigated.The results showed that HA-12-PEI had no significant effect on the proliferation,cycle and apoptosis of 4T1 cells.HA-PEI and HA-6-PEI had different effects on cell cycle,but there was no significant difference in proliferation and apoptosis.After cell uptake was 6 h,the uptake could be over 90 %.The complex of the carrier and its loaded nucleic acid are mainly taken into cells through clathrin-mediated endocytosis pathway and has a dose-dependent manner,but it has a certain energy dependence after the bridging chain modification.Carriers transport of green fluorescent protein expression vector into the cell can observe bright green fluorescence,which shows that carriers can load nucleic acid transfection to cells and successfully express target protein.(2)Three carriers were loaded targeting of programmed cell death 1 ligand(PD-L1)RNAi expression plasmid(shPD-L1)was constructed for gene therapy of nano drug delivery system HA-PEI/shPD-L1,HA-6-PEI/shPD-L1 and HA-12-PEI/shPD-L1.The RNAi effect of the nanoparticle drug delivery system was investigated at the 4T1 cell level.The expression of PD-L1 mRNA in HA-PEI/shPD-L1,HA-6-PEI/shPD-L1 and HA-12-PEI/shPD-L1 was significantly different from that of the blank control group(P<0.01),and decreased to 7.53 %,11.23 %,9.40 %,separately.The expression of PD-L1 protein in HA-PEI/shPD-L1,HA-6-PEI/shPD-L1 and HA-12-PEI/shPD-L1 was significantly different from that in the blank control group(P<0.01),and decreased to 9.49 %,11.07 % and 11.73 %,separately.It is suggested that HA-PEI/shPD-L1,HA-6-PEI/shPD-L1,and HA-12-PEI/shPD-L1 can successfully induce the RNAi effect at the cell level in vitro.3.In vivo study of BALB/c mice loaded with breast cancer cell 4T1.(1)The results of blood level detection showed that the erythrocytes agglutination effect of HA-6-PEI group and HA-12-PEI group were lower than that of HA-PEI group,the serum stability of DNA in the HA-6-PEI group and HA-12-PEI group increased.(2)In situ tumor inhibition assay showed that the inhibition rates of HA-PEI/shPD-L1 group,HA-6-PEI/shPD-L1 group and HA-12-PEI/shPD-L1 group were 59.72 %,66.67 % and 69.44 %,separately.The inhibition rates of PD-L1 mRNA in HA-PEI/shPD-L1,HA-6-PEI/shPD-L1 and HA-12-PEI/shPD-L1 drug delivery systems were 51.21 %,57.29 % and 63.02 %,separately,and the inhibition rates of PD-L1 protein expression were 39.20 %,50.23 % and 63.44 %,separately.In situ tumor suppressor activity results showed that compared with HA-PEI/shPD-L1,HA-12-PEI/shPD-L1 could significantly inhibit the proliferation of tumor in situ(P<0.01),while HA-6-PEI/shPD-L1 inhibited the proliferation activity of the tumor in situ between them.(3)Tumor metastasis inhibition activity showed that lung metastasis inhibition rates in HA-PEI/shPD-L1 group,HA-6-PEI/shPD-L1 group and HA-12-PEI/shPD-L1 group were 76.06 %,78.26 % and 86.95 %,separately.Compared with HA-PEI/shPD-L1 group,HA-12-PEI/shPD-L1 nanoparticle delivery system could significantly inhibit lung metastasis of 4T1 cells(P<0.05),but HA-6-PEI/shPD-L1 had no significant difference.(4)In vivo safety evaluation showed that the security of HA-12-PEI/shPD-L1 was significantly higher than that of HA-PEI/shPD-L1,but HA-6-PEI/shPD-L1 between them.