Synthesis Of Multiple-modified Chitosan Derivatives And Their Applications As Gene Vectors

Chitosan is a kind of natural cationic polymers, and it has low toxicity, biodegradability, and biocompatibility, which can be applied as efficient gene vectors. However, poor solubitlity of chitosan under physicological conditions restricts its further clinical applications. Therefore, various modifications are employed by researchers to improve chitosan’s properties like solubility and enhance its transfection efficiency.Three kinds of multiple-modified chitosan derivatives are synthesized in this work. Their structures are identified by FTIR, NMR and XRD etc. Aditionally, MTT assays and in vitro transfection assays are used to evaluate the performance of these polymers as gene vectors.The main contents are summarized as follows:(1) PEG-conjugated quaternized chitosan derivatives are prepared, and the PEGylation degrees (DP) are9%,13%and27%respectively. Their properties like DNA binding capacity, cytotoxicity and transfection efficiency are evaluated by Agarose gel retardation assays, Zeta potential and particle size measurements, MTT assays and transfection assays. The results show that PHTAC derivatives have good DNA binding capacity, and can form nanocomplexes with the minimum sizes of about200nm. All of the three PHTAC copolymers have lower cytotoxicity than HTAC, and the cytotoxicity decreases with the increment of degree of PEGylation. PHTAC-1(N/P=25, DP=9%) has the highest transfection efficiency, which is5.3-fold of that of HTAC (N/P=20).(2) Arginine modified quaternized chitosan derivatives are prepared, and the degrees of arginine substitution are5.2%,9.4%and13.0%respectively. Their properties like DNA binding capacity, cytotoxicity and transfection efficiency are evaluated. The results indicate that Arg-HTAC derivatives can bind DNA tightly, and form complexes with sizes between200-300nm. Arg-HTAC copolymers have low cytotoxicity, and the cell viabilities are more than82%(HepG2cells) or95%(293T cells) even with a concentration higher than100μg/mL. Arg-HTAC-3(N/P=25, DA=13.0%) has the highest transfection efficiency, which is27-fold of that of HTAC (N/P=25) and comparable with PEI’s (25kDa) performance.(3) Arginine modified PEI-graft-chitosan (CS-PEI-Arg) is prepared, and the properties like DNA binding capacity, complexes’size and zeta potential, cytotoxicity and transfection efficiency are evaluated. The results show that CS-PEI-Arg derivatives can bind DNA thoroughly, and form complexes with sizes less than200nm. Compared with CS-PEI and PEI, CS-PEI-Arg has significant lower cytotoxicity and higher tranfection level. The best transfection efficiency of CS-PEI-Arg (N/P=50) is2.3-fold,4.2-fold of those of CS-PEI (N/P=20) and PEI’s (N/P=10) efficiency respectively.