Experimental Study On Preparation Of Hydroxybutyl Chitosan-thiolated N-alkylated Chitosan/Plasmid Shell-core Structure And Its Sustained-behavior In Vitro And Vivo

Background&Objective: Chitosan is a cationic polysaccharide widely found in nature,and formed a complex with gene through the strong electrostatic interaction. SinceMumper reported using chitosan as gene vector firstly, chitosan and its derivatives as agene carrier has become a hotspot by researchers. The hydrophobic alkyl chain wasintroduced into the chitosan chain partially, and then thiol was introduced to improvethe the water solubility and its transfection efficiency in the previous test. The genecarried by these vectors was always expressed in short time (72h), in some conditions,gene is expected to be released slowly and achieve long-term expression. This study isaim to prepare and evaluate the shell-core nanoparticles hydroxybutyl chitosan-thiolated N-alkylated chitosan encapsulated with enhanced green fluorescent proteinencoded plasmids (pEGFP) as a sustained-release gene carrier.Methods: The pEGFP-loaded thiolated N-alkylated chitosan (TACS/pEGFP) wasprepared by complex coacervation method as the core, and the core was coated with thetemperature HBC through mixed wrap and freeze drying, then the core-shellnanoparticles (HBC-TACS/pEGFP) were prepared. The nanoparticles werecharacterized by Dynamic Light Scattering (DLS) and Transmission ElectronMicroscope (TEM).The capacity of protect the pEGFP from nuclease degradation wasshown by gel agarose electrophoresis. And it was tested to wonder the nanoparticlescould be used to transfect HEK293in vitro and mice skeletal muscle in vivo.Results: HBC-TACS/pEGFP shell-core nanoparticles were mainly spherical, with anaverage size of (317.6±55.9)nm, and zeta potential of (2.3±1.1) mV. The gel agaroseelectrophoresis analysis confirmed that they could effectively protect pEGFP fromdegradation against DNase I. In vitro gene transfection effect of HBC-TACS/pEGFPwas (8.00±5.69)%evaluated by flowcytometry, less than the transfection effect of TACS/pEGFP (37.66±5.18)%, P＜0.01. However, more expression of fluorescentprotein appeared in the animal muscle with a time-dependent manner. On Forty-fifthday，the fluorescence intensity of HBC-TACS/pEGFP was higher than TACS/pEGFP.Conclusion: This work indicates that the submicron shell/core-structuredchitosan-based composite particles could be potentially used as a promising gene vectorfor in vivo controlled gene transfection.