ABSTRACT
BACKGROUND: Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. METHODS: pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at -30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. RESULTS: Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. CONCLUSIONS: The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration.
Subject(s)
Chitosan/therapeutic use , Genetic Therapy/methods , Hyaluronic Acid/therapeutic use , Animals , DNA/genetics , Electrophoretic Mobility Shift Assay , Freeze Drying , Gene Transfer Techniques , Genes, Reporter , Genetic Vectors , Humans , Luciferases/genetics , Mice , Nanoparticles , Particle Size , Plasmids , Thymidine Kinase/genetics , TransfectionABSTRACT
pDNA/chitosan complexes have been investigated as promising non-viral vectors for gene delivery. However, an increase in transfection efficiency and enhancement of physicochemical stability are required for their practical use. In this study, chondroitin sulfate (CS) was employed as a coating agent to increase the stability and transfection efficiency of a pDNA/chitosan complex. The pDNA/chitosan/CS ternary complexes formed with six kinds of CSs having different limiting viscosities (0.2-1.6) and sulfation degrees (5.0-7.0%) showed considerable differences in particle size, surface charge, and morphology. Among them, CS having a medium limiting viscosity (0.5-0.6) and a high sulfation degree (6.9%) showed significant enhancements in cell transfection efficiency. Analyses of cellular uptake and intracellular trafficking revealed that increased cellular uptake via macropinocytosis, together with reduced entry into lysosomes, may explain the promotion of transfection efficiency of ternary complexes.
Subject(s)
Chitosan/chemistry , Chondroitin Sulfates/chemistry , DNA/chemistry , Gene Transfer Techniques , Animals , COS Cells , Cattle , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microscopy, Atomic Force/methods , Nanoparticles/chemistry , Pinocytosis , Polysaccharides/chemistry , Sepharose/chemistry , Transfection , ViscosityABSTRACT
Successful gene therapy depends on the development of effective gene carriers. Naturally occurring chitosan has been employed widely as a non-viral gene carrier because of its low toxicity, low immunogenicity, biocompatibility, and biodegradability. In this review, we summarize the utilization of chitosan, modified chitosan, and chitosan-containing ternary complexes as gene carriers. In particular, we discuss the influence of the physicochemical features of pDNA/chitosan complexes on their functions, such as stability and gene transfer into cells.
