| MicroRNA(miRNA)is a non-coding endogenous RNA in nearly all eukaryotes,which regulate almost all cellular pathways and play an important role in many diseases.However,naked miRNAs are extremely easily degraded by nucleases in both external and internal environments,making it difficult to enter cells directly.Therefore,it is urgent to prepare a gene vector with low cytotoxicity,high cytocompatibility,high transfection efficiency and low cost.Chitosan(CS),a natural cationic polysaccharide,is abundant and biodegradable in nature,and it is demonstrated that mi RNA was protected from nuclease degradation,so it is considered as a promising nucleic acid delivery carrier.Alveolar echinococcosis caused by Echinococcus multilocularis(Emu.)is an important zoonotic disease,a great threat to human health due to the limited interventions.Emu-mi R-4989-3p is one of the "virulence" factors in echinococcus multilocularis extracellular vesicles,and play a key role in the pathogenesis of AE.Moreover,emu-mi R-4989-3p may also be involved in host-parasite interactions.In this study,chitosan nanoparticles(CSNPs)for miRNA delivery were synthesized,their physicochemical properties were studied,and the feasibility of CSNPs as transfection reagent was evaluated and applied in the study of Echinococcus multilocularis.This study mainly consists of the following four parts:In chapter 1,the status quo of chitosan,miRNA and AE were introduced,and the research progress of chitosan delivery of mi RNA was summarized,and the application potential of chitosan in AE was emphasized.In chapter 2,using Sodium Tripolyphosphate(TPP)as crosslinking agent,chitosan nanoparticles(CSNPs)with different proportions were prepared by ion crosslinking method.And the influence of the rito of CS and TPP for its morphology,particle size,binding and loading capacity,cytotoxicity,encapsulation rate and release rate were studied,and the influence of p H and temperature on CS stability and binding time on CS-mi RNA binding ability was investigated.It was found that the size of CSNPs increased from 100 nm to 150 nm with the decrease of TPP content.In the study of cytotoxicity,the nanoparticles of 10:1ratio of CS and TPP showed lower cytotoxicity and the release rate reached 90% at 24 h and showed good binding and loading capacity(69.62%)with mi RNA.Based on this,the stability of CSNPs was tested,the results showed that CSNPs were relatively stable and could be stored for a long time in-80 ℃ and PBS buffer,and the size and morphology have not significant change was found after several freeze-thaw cycles,furthermore,CSNPs structures are almost undamaged in the p H = 4-10.In the analysis of binding time,it was found that the particle size of CS-mi RNA decreased with the increase of binding time,and 15 minutes of binding was the best condition.In chapter 3,MTT assay and CLSM characterization were used to study the feasibility of CSNPs as transfection reagent,and the cytotoxicity and transfection efficiency of CSNPs were compared with liposomes.The results showed that CS-mi RNA entered cells via endocytosis,and the cell uptake rate reached the highest at 24 h after transfection.CS-mi RNA had a broad spectrum and was internally digested by the three cell lines(RAW,AML12,293T),with almost no difference in cell uptake efficiency.The results showed that CSNPs performed better than Liposome(LPS)in groups of hepatic cells(HC)and Kupffer cells(KC)groups.24 h after transfection,the uptake rate of cells in the LPS group and the CS group,namely,the internalization rate,was about 88.7% in the CS group and 90.9% in the LPS group.In chapter 4,CS-miR-4989 complex was synthesized through electrostatic interaction to study its role in vitro and vivo study of Emu.infection respectively.In addition,FITC-CS was synthesized and injected into the tail vein to study the distribution of CS in mice.The results showed that the expression level of ube2 n,the target gene of mi R-4989,was significantly down-regulated,about 40 % down-regulated 24 h after transfection in vitro,60 % downregulated 40 h after transfection,and 40 % down-regulated in vivo.Western Blot(WB)results showed that UBE2 N protein expression was down-regulated by 40 % and 60 % in vitro,while UBE2 N expression was down-regulated by about 30 % in vivo.The fluorescence of mouse organs showed that most CSNPs were concentrated in liver,followed by kidney,while there was almost no CSNPs in heart,spleen and blood.In summary,we conclude that CSNPs were successful as a superior vehicle for efficient liver-targeted delivery of mi RNAs and were developed as a potential mi RNA-bases therapeutic which was used to against E.multilocularis infection. |
