Experimental study on preparation of hydroxybutyl chitosan-thiolated N-alkylated chitosan/plasmid core-shell structure and its sustained-behavior in vitro and in vivo

Abstract

Objective To prepare and evaluate the core-shell nanoparticles hydroxybutyl chitosanthiolated N-alkylated chitosan encapsulated with enhanced green fluorescent protein encoded plasmids (pEGFP) as a sustained-release gene carrier.Methods The pEGFP-loaded thiolated N-alkylated chitosan (TACS/pEGFP) was prepared by complex coacervation method as the core,and the core was coated with the thermosensitive HBC through mixed wrap and freeze drying,then the core-shell nanoparticles (HBC-TACS/pEGFP) were prepared.The nanoparticles were characterized by dynamic light scattering (DLS)and transmission electron microscope (TEM).Gel agarose electrophoresis was done to test the protective effects of the nanoparticles on the pEGFP.The nanoparticles were transfect into HEK293 cells in vitro and mouse skeletal muscle in vivo.Results HBC-TACS/pEGFP core-shell nanoparticles were mainly spherical,with an average size of (317.6 ±55.9) nm,and zeta potential of (2.3 ± 1.1) mV.The gel agarose electrophoresis confirmed that the nanoparticles could effectively protect the pEGFP from degradation against DNase I.In vitro gene transfection efficiency of HBC-TACS/pEGFP was (8.00 ± 5.69) ％,significantly lower than that of TACS/pEGFP [(37.66 ± 5.18)％],P ＜ 0.01.However,the expression of fluorescent protein was increased in the animal muscle in a time-dependent manner.On the day 45,the fluorescence intensity of HBC-TACS/pEGFP was higher than TACS/pEGFP.Conclusion HBC-TACS/pEGFP can be used as a gene vector which can be continuously expressed in vivo. Key words: Core-shell structure; Chitosan ; Sustained-release ; Gene vector; Transfection