ABSTRACT
A core-shell structured chitosan (CS)-based gene vector with a sustainable gene transfection effect was designed and successfully prepared in this study. The pEGFP was first combined with the thiolated and N-alkylated chitosan (TACS). Then, hydroxybutyl chitosan grafted with poly(ethylene glycol) (EG-HBC) was coated on the pEGFP-loaded TACS particles. The prepared pEGFP-loaded TACS@EG-HBC particles have a size of about 200 nm and little cytotoxicity. The in vitro and in vivo gene transfection experiments indicate that the pEGFP-loaded TACS@EG-HBC particles possess a better sustainable gene transfection capacity and a high transfection efficiency, which should be attributed to the biodegradation of the CS-based shell, the thiolation and N-alkylation modification on CS cores, and the grafted PEG chains with better biocompatibility. The in vivo gene expression of the loaded pEGFP can persist up to 60 days. This novel gene vector has a theoretical and practical significance for gene therapy with sustained transfection effect.
ABSTRACT
Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection.
Subject(s)
Chitosan/chemistry , DNA/chemistry , Delayed-Action Preparations/chemistry , Green Fluorescent Proteins/genetics , Nanoparticles/chemistry , Transfection/methods , Animals , Cell Survival/drug effects , DNA/genetics , DNA/metabolism , Delayed-Action Preparations/toxicity , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Male , Mice , Nanoparticles/toxicity , Plasmids/geneticsABSTRACT
Disulfide-exchange was found to cross-link the polyplex of disulfide-containing poly(amido amine) and pDNA with heating of the polyplex solution over a short time. The cross-linked polyplexes based on disulfide-containing poly(amido amine) have excellent stability under physiological salt conditions, and have significantly enhanced transfection activity in the serum media compared to non-cross-linked polyplexes. In vivo, ICR mice were injected with the polyplex through the tail vein, the results show that the transfection efficiency of the cross-linked polyplex is higher than that of the non-cross-linked variety. Furthermore, the polyplex containing Cy5 labelled DNA was also injected into the mice to illustrate the stability and distribution of the polyplex, cross-linked polyplexes show a much brighter luminescence than the non-cross-linked ones. This method does not need a cross-linker or catalyst, and there are no impurities produced, it may be an elegant approach to resolve the dilemma of in vivo application of a DNA polyplex, with excellent stability whilst in circulation and a rapid unpacking of the polyplex inside the cells.
ABSTRACT
A submicrometer-scaled polystyrene/melamine-formaldehyde hollow microsphere composite was prepared by self-assembling of sulfonated polystyrene (SPS) latex particles at the interface of emulsion droplets and then being fixed in place using a hard melamine-formaldehyde (MF) composite layer. For control-released purposes, the influential factors that control the size and uniformity of the packed-droplets and the permeability of the composite shell, including the initial particle location, the hydrophilicity and the size of colloidal templates, the oil phase solvent and reserving time of emulsions after the addition of MF prepolymer, were further studied. Relatively uniform sized particle packed-droplets with an average diameter of 10 microm were obtained. The assembled SPS particles kept ordering and minimal conglutination after the preparation of composite microspheres, which allows of controlling the permeability from the interstices between the particles. Porous-mesh-structured MF composite layer was formed to further control the permeability. The morphology of emulsions and composite microspheres were characterized by optical microscopy, scanning and transmission electron microscopy.
