Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency
Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency
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DOI：
10.1016/s0168-3659(00)00361-8
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发表时间：
2001-02-23
影响因子：
10.8
通讯作者：
Leong, KW
中科院分区：
文献类型：
--
作者：
Mao, HQ;Roy, K;Leong, KW
采用复凝聚法制备了壳聚糖-DNA纳米粒。研究了DNA、壳聚糖和硫酸钠的浓度、溶液的温度、缓冲液的pH值以及壳聚糖和DNA的分子量等影响纳米颗粒合成的重要参数。在氨基与磷酸基团的比率（NIP比率）在3和8之间并且壳聚糖浓度为100 μ g/ml时，颗粒的尺寸被优化为类似于100-250 nm，具有窄的分布，其中DNA和壳聚糖的组成分别为35.6和64.4重量%。这些颗粒的表面电荷为轻微的正性，在pH低于6.0时zeta电位为+12至+18 mV，在pH 7.2时变为接近中性。电泳迁移率分析表明，壳聚糖-DNA纳米颗粒可以部分保护包裹的质粒DNA免受核酸酶的降解。壳聚糖-DNA纳米粒的转染效率具有细胞类型依赖性。通常，以相对光单位计，其比HEK 293细胞中的背景水平高3至4个数量级，并且比LipofectAMINE（TM）-DNA复合物所实现的低2至10倍。10%胎牛血清的存在不干扰它们的转染能力。氯喹可以以5.2%共包封在纳米颗粒中，但尽管壳聚糖与PEI相比仅显示出有限的缓冲能力，但具有可忽略的增强效果。本研究还开发了三种不同的方案将转铁蛋白或KNOB蛋白缀合到纳米颗粒表面。转铁蛋白缀合物在HEK 293细胞和HeLa细胞中的转染效率最多仅增加4倍，而KNOB缀合的纳米颗粒可以将HeLa细胞中的基因表达水平提高130倍。PEG在纳米颗粒上的缀合允许冻干而不聚集，并且在储存中至少1个月不损失生物活性。静脉内给药后，PEG化纳米颗粒在小鼠中的清除在15分钟时比未修饰的纳米颗粒慢，并且在肾脏和肝脏中具有更高的沉积。然而，在I-h时间点未观察到差异。(C)出版社：Elsevier Science B. V.
Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (NIP ratio) between 3 and 8 and a chitosan concentration of 100 mug/ml, the size of particles was optimized to similar to 100-250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +12 to +18 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by LipofectAMINE(TM)-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI.. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the I-h time point. (C) 2001 Published by Elsevier Science B.V.