八聚精氨酸修饰的载ASODN脂质体的构建及体外评价  

The formulation and in vitro evaluation of R8-modified liposome for carrying antisense oligodeoxynucleotide

作　　者：王文喜[1] 代凯[1] 唐岚[1] 

机构地区：[1]浙江工业大学药学院,浙江杭州310014

出　　处：《浙江工业大学学报》2014年第3期334-337,共4页Journal of Zhejiang University of Technology

基　　金：国家自然科学基金资助项目(81102396)

摘　　要：用硬脂酰八聚精氨酸(R8)对普通脂质体进行修饰,以期获得高效低毒的反义寡核苷酸(Antisense oligodeoxynucleotide,ASODN)载体.修饰后的脂质体(R8-Lip)与反义寡核苷酸孵育,用琼脂糖凝胶电泳法考察复合物的形成情况;在肝癌细胞(HepG2)、肺癌细胞(A549)和口腔上皮癌细胞(KB)中,用MTT法评估R8-Lip的细胞毒性;同时考察在这三种细胞株中,R8的浓度与摄取效率的关系.当R8-Lip/ASODN复合物中正负电荷比(+/-)超过4∶1时,ASODN已与R8-Lip完全结合成带正电的复合物;当R8-Lip中R8的质量浓度小于10μg/mL时,细胞存活率大于75%;R8-Lip对ASODN细胞摄取的促进作用与R8的浓度有关,当R8的质量浓度由0.3μg/mL增加到9.6μg/mL(+/-由1∶2增加到16∶1),细胞内荧光强度逐渐增加;当R8-Lip/ASODN复合物中+/-=16∶1时,R8对ASODN摄取的促进作用强于市售的转染试剂Lipfectamine2000.因此,R8-Lip作为ASODN的载体,具备了高效低毒的优势,值得进一步深入研究.In order to obtain effective and less toxic carriers of antisense oligodeoxynucleotide,conventional liposome is modified with stearyl octaarginine （R8）.The modified liposome （R8-Lip） is incubated with antisense oligodeoxynucleotide and the efficiency of complex formation is investigated by agarose gel electrophoresis.The cell toxicity of R8-Lip is evaluated by the method of MTT in liver cancer cells （HepG2）,lung cancer cells （A549） and oral epithelial cells （KB）.The relationship between the concentration of R8 and the efficiency of cellular uptake is researched in the three cell lines also.When the ratio of ＋/-in complex is more than 4 ∶ 1,R8-Lip/ASODN has been combined into a positively charged complex.When the R8 concentration is less than 10μg/mL,the cell survival rate is more than 75％.The efficiency of cellar uptake is dependent on the concentration of the R8.When the concentration of the R8 increase from 0.3 μg/mL to 9.6 μg/mL（＋/-from 1 ∶ 2 to 16 ∶ 1）,intracellular fluorescence intensity increases gradually.When the ＋/-ratio of R8-Lip/ASODN complex is 16 ∶ 1,the efficient of cellar uptake is better than the commercially available reagents Lipfectamine2000.So,the R8-Lip as the carrier of ASODN,has the advantages of high efficiency and low toxicity and is worthy of further research.

关 键 词：八聚精氨酸 反义寡核苷酸 细胞摄取 非病毒载体 脂质体 

分 类 号：R944[医药卫生—药剂学]