Single arginine modified chitosan as gene carriers
10.3760/cma.j.issn.1673-4181.2018.02.003
- VernacularTitle:单精氨酸修饰壳聚糖作为基因载体的研究
- Author:
Xiuxiang TAN
1
;
Ning WANG
;
Ying YANG
;
Mei YU
;
Hailing ZHANG
;
Xigang LENG
Author Information
1. 300192天津,中国医学科学院北京协和医学院生物医学工程研究所,天津市生物医学材料重点实验室
- Keywords:
Chitosan;
Single arginine modified chitosan;
Gene carrier;
Nanoparticles
- From:
International Journal of Biomedical Engineering
2018;41(2):118-124
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore a new method for synthesizing arginine modified chitosan ( AC ) with mono-arginine substitution and high degree of substitution, and to evaluate the biological effect of AC as gene carriers. Methods The single arginine modified chitosan (sAC) was synthesized by means of protecting and de-protecting the arginine amino group before and after chitosan modified arginine reaction. Liu's arginine-modified chitosan ( LAC ) was prepared according to the methods reported in the literature . The conjunction of arginine to chitosan was detected by infrared spectroscopy and nuclear magnetic resonance spectroscopy. Three kinds of chitosan gene nanoparticles were respectively prepared by complex coacervation and characterized, including sAC gene nanoparticles (sACGNs), the LAC gene nanoparticles (LACGNs) and the chitosan gene nanoparticles (CGNs). A10 rat vascular smooth cells transfected with sACGNs were used to estimate the in vitro cellular uptake, internalization mechanisms and transfection efficiency. Thiazolyl blue tetrazolium blue (MTT) assay was used to measure the cytotoxicity. Results The infrared spectrum analysis confirmed that sAC was obtained via the conjunction of arginine to chitosan. Nuclear magnetic resonance spectrum analysis showed that the degree of substitution of arginine in sAC and LAC was 21.3％and 6.4％, respectively. When the ratio of nitrogen to phosphorus (N/P ratio) was 2:1, the particle sizes of CGN, LACGN, and sACGN were (94.81±2.93) nm, (124.53±2.55) nm, and (128.53±2.04) nm, respectively, and the Zeta potentials were (3.50±1.61) mV, (5.74±0.41) mV, and (6.04±1.39) mV, respectively. For the cellular uptake, CGNs were mainly through the clathrin-mediated endocytic pathway, and LACGNs and sACGN were mainly through the caveolin-mediated endocytic pathway. Compared with CGNs, LACGNs and sACGNs showed higher cellular uptake and transfection efficiency , and the differences were statistically significant ( all P<0 . 05 ) . sACGNs achieved the highest transfection efficiency in the near-neutral pH environment. MTT results showed that when the mass concentration of sACGNs reached 100μg/ml, the survival rate of A10 cells was still higher than 90％, indicating the non-cytotoxicity of sACGNs. Conclusion The new method successfully synthesized single arginine-modified chitosan. As a gene carrier, sACGNs show higher gene transfection efficiency and lower cytotoxicity than CGNs and LACGNs in near neutral pH environment.
