DNA Nanocomposites Based On Chitosan Quaternary Ammonium Salt - Arginine Polymer Carrier To Improve Gene Transfection In Vitro And In

Efficient gene delivery vector is the prerequisite for successful gene therapy. Chitosan can condense plasmid DNA (pDNA) into nanocomplex and has been widely used as gene carrier due to its biocompatibility and biodegradability. However, chitosan/pDNA nanocomplex has suffered from instability under neutral condition and difficulty in timely dissociation in the cytoplasm, thus precluding efficient gene transfer. Modification of chitosan is supposed to improve such limitations and increase transfection efficiency. To mediate highly effective transfection, cationic polymeric vectors have to traverse multiple extra-and intracellular obstructions. Therefore, gaining insight into the key parameters and rate-limiting steps involved in the cellular processing of polymer-based delivery systems is of vital importance for rational design of gene delivery carrier and the realization of expression of exogenous genes both in vitro and in vivo.Cationic chitosan derivative-trimethyl chitosan-arginine conjugate (TMC-Arg), which was synthesized by the trimethylation of chitosan and arginine conjugation, could condense plasmid encoding Enhanced Green Fluorescence Protein (pEGFP) into nanocomplexes (TANC). The transfection efficiency of TANC and the mechanisms for improved gene transfer ability were studied. Different proportions of sodium tripolyphosphate (TPP) and poly(y-glutamic acid)(y-PGA) were added into TANC to investigate the influence of the composition of crosslinking agents on transfection efficiency both in vitro and in vivo.Four kinds of TANC were obtained by modulating the proportion of TPP and y-PGA, termed TANC1,TANC2, TANC3and TANC4. TMC/pEGFP namocomplex (TNC) and TANC possessed similar particle sizes, ranging from100nm to150nm, and Zeta potentials, ranging from20mV to30mV. The Zeta potentials of TANC decreased with increasing amount of TPP. TNC and TANC could effectively condense pEGFP and protect it from the degradation of DNase. TANC showed good stability against ionic strength and dilution. TNC and all TANC could effectively bind to the membranes of HEK293cells. The cellular uptake level of TANC1(without any crosslinking agents) was2.1-folds of that of TNC, demonstrating that arginine conjugation increased the cellular uptake level of nanocomplexes. The uptake levels of TANC with different composition of crosslinking agents were decreased with the increased amount of TPP. TNC was mainly internalized through the clathrin-mediated pathway. TANC1and TANC4were internalized through clathrin-mediated pathway, while TANC2and TANC3adopted the caveolae-mediated one, indicating that the addition of y-PGA changed from clathrin-mediated endocytosis to caveolae-mediated one. The increment of TPP amount in TANC was responsible for their reduced binding affinity to pEGFP and rapid pEGFP release, which was related to their subcellular distribution and transfection patterns in vitro and in vivo. The pEGFP amount in the nuclear of TANC1at12h was1.3-folds of that of TNC, due to the nuclear localization ability of arginine residues. The composition of crosslinking agents affected the nuclear distribution of pEGFP in TANC. The faster the pEGFP released from TANC, the sooner it reached the summit. The transfection efficiency of TANC1was remarkably higher than that of TNC, which was1.3-fold of that of TNC at96h, possibly due to the membrane permeability and nuclear localization capacity of arginine residues. The composition of crosslinking agents affected the transfection efficiency and kinetics of TANC. The transfection efficiency of TANC3at48h reached35.1%, which was the highest among all TANC. The transfection efficiency of TANC1increased steadily in the duration of96h, while that of TANC2, TANC3, and TANC4arrived at the summit at72h,48h, and24h, respectively. TANC3mediated the most efficient Green Fluorescence Protein (GFP) expression at3days post intramuscular administration in mice, which were2.6-folds,1.7-folds,3.9-folds and3.2-folds of that of TANC1, TANC2, TANC4and Lipofectamine2000/pEGFP nanocomplexe, respectively. Compared to the level of GFP expression of3days post administration, that of7days of TANC1and TANC2augmented for2.2-folds and1.5-folds, respectively.