| 摘要: |
| 目的 制备一种应用于基因递送系统的硫辛酸修饰的聚精氨酸多肽纳米复合物，并考察其对人胚肾细胞系HEK293细胞的转染效率及细胞毒性。方法 以不同量的半胱氨酸作为交联剂，合成4种不同交联度的还原性硫辛酸修饰的交联聚精氨酸组氨酸（LHRss），利用1H NMR和凝胶色谱鉴定合成的LHRss。取质粒DNA（pDNA）和LHRss以不同氮磷比（N/P）自组装形成纳米复合物，用粒度测定仪测定复合物的粒径和zeta电位，凝胶阻滞电泳测定载体LHRss对pDNA的包裹能力。用LHRss/pDNA纳米复合物与HEK293细胞共同培养，考察不同交联度复合物的细胞摄取情况及相关基因转染情况，并测定不同纳米复合物对HEK293细胞的细胞毒性。结果 通过结构鉴定确定LHRss多肽合成成功。组装形成的纳米复合物粒径分布均匀，N/P≥40时LHRss3/pDNA及LHRss4/pDNA复合物的zeta电位均大于30 mV。凝胶阻滞电泳结果显示N/P值为5时，LHRss3可完全包裹pDNA。当N/P值为40时，HEK293细胞对LHRss3/pDNA的摄取及转染效率高于其他3种复合物及单体硫辛酸修饰的聚精氨酸组氨酸（LHR）；其中LHRss3/pGL3复合物的平均荧光强度约为LHR/pGL3复合物的3.98倍，差异具有统计学意义（P<0.05）。细胞毒性实验显示LHR/pGL3及不同交联度LHRss/pGL3转染HEK293细胞24 h后，细胞存活率均在80%以上，其毒性作用均低于bPEI-25K（20 μg/mL bPEI-25K转染细胞24 h后细胞存活率为25%左右，P<0.05）。结论 制备的硫辛酸修饰的聚精氨酸多肽纳米复合物有望成为一种高效的基因载体。 |
| 关键词: 聚精氨酸多肽 质粒DNA 转染 基因转移技术 |
| DOI：10.16781/j.0258-879x.2016.11.1353 |
| 投稿时间：2016-04-21修订日期：2016-09-01 |
| 基金项目:国家自然科学基金（81172514，81372762）. |
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| Preparation and in vitro evaluation of biodegradable polypeptide gene vector |
| HU Chu-ling1,GU Fen-fen1,TAI Zong-guang1,FANG Ji-wei2,GAO Yuan1,GAO Shen1* |
(1. Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
2. Department of Pharmacy, Zhoupu Hospital of Shanghai Pudong New District, Shanghai 201318, China
*Corresponding author.) |
| Abstract: |
| Objective To prepare a lipoic acid modified polyarginine polypeptide nanocomplex for gene delivery system, and to observe its transfection efficiency and cytotoxicity on HEK293 cells. Methods We synthesized four disulfide cross-linked lipoic acid modified polyarginine peptide and histidine (LHRss) at different cross-linked degrees using different mol fraction of L-cysteine as cross-linking agent. The construction of LHRss was characterized by 1H nuclear magnetic resonance (1H NMR) and gel permeation chromatography. The LHRss/plasmid DNA (pDNA) nanocomplexes were self-assembled with LHRss and pDNA at different nitrogen/phosphorus (N/P) ratios. The size and zeta potential of LHRss/pDNA nanocomplexes were characterized by particle size analyzer, and the pDNA enrichment capability was determined by electrophoretic mobility shift assay (EMAS). Then, the intracellular uptake and gene transfection efficiency of LHRss/pDNA nanocomplexes in HEK293 cells were investigated. CCK-8 method was used to determine the cytotoxicity of LHRss/pDNA nanocomplexes on HEK 293 cells. Results 1H NMR results showed that LHRss was successfully synthesized. The nanocomplexes had a uniform distribution of particle size, and the zeta potential of LHRss3/pDNA and LHRss4/pDNA nanocomplexes were more than 30 mV when N/P≥40. EMAS results showed that pDNA could be completely wrapped by LHRss3 when N/P=5. When N/P=40, the intracellular uptake and transfection efficiency of LHRss3/pDNA nanocomplex by HEK293 cells was significantly higher than that of other three nanocomplexes and lipoic acid modified polyarginine peptide and histidine (LHR)/pDNA; the mean fluorescence intensity of LHRss3/pGL3 nanocomplexes was approximately 3.98 times that of the LHR/pGL3 nanocomplex (P<0.05). Cytotoxicity results showed that the cell survival rates were more than 80% at 24 h after transfected with LHR/pGL3 and LHRss/pGL3, and its toxicity was significantly lower than that of bPEI-25K, with the cell survival rates being about 25% at 24 h after transfected with 20 μg/mL bPEI-25K (P<0.05). Conclusion The prepared lipoic acid modified polyarginine polypeptide nanocomplex is expected to become an efficient gene vector. |
| Key words: polyarginine polypeptide plasmid DNA transfection gene transfer techniques |
