氮磷比对脂多糖胺/质粒复合物性能的影响  

Effect of N/P ratio on physicochemical properties of LPSA/pDNA complex

作　　者：关云 高彦晨 张兴 滕伟[2] GUAN Yun;GAO Yan-chen;ZHANG Xing;TENG Wei(Department of Stomatology,the Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510120,Guangdong,China;不详)

机构地区：[1]广州中医药大学第二附属医院广东省中医院口腔科,广东广州510120 [2]中山大学光华口腔医学院中山大学附属口腔医院修复科中山大学口腔医学研究所,广东广州510055

出　　处：《广东医学》2022年第10期1234-1239,共6页Guangdong Medical Journal

基　　金：广东省基础与应用基础研究基金项目(2021A1515011480)。

摘　　要：目的采用新型阳离子共聚物脂多糖胺(lipopolysaccharide-amine,LPSA)与质粒构建脂多糖胺/质粒复合物(LPSA/pDNA),探究其理化性能随氮磷比(N/P)变化规律。方法构建含骨形态生成蛋白2(bone morphogenetic protein 2,BMP2)的非融合的真核表达质粒并鉴定。配置不同N/P的LPSA/pDNA复合物,凝胶阻滞实验探究LPSA与pDNA结合能力;动态光散射仪检测其粒径及zeta电位;透射电镜及原子力显微镜下观察LPSA/pDNA复合物形态;探究LPSA/pDNA复合物耐酶解能力随时间的变化规律。结果经琼脂糖凝胶电泳及测序鉴定,真核表达载体BMP2编码序列片段与Genebank一致。随着N/P增加,LPSA/pDNA阻滞质粒DNA能力增加;当N/P>10时,LPSA可基本阻滞pDNA。LPSA/pDNA复合物的粒径随着N/P的增加先减小后增大,当N/P>3时,LPSA/pDNA粒径<150 nm;当N/P为12~25时,LPSA/pDNA粒径稳定于65 nm左右,LPSA/pDNA表面zeta电位呈正电性,电荷值为9~35 mV。当N/P为60时,随着DNaseⅠ处理时间增加,4 h内琼脂糖凝胶电泳条带亮度无明显变化,6 h时琼脂糖凝胶电泳条带亮度稍微减弱。结论N/P为12~25时,LPSA与pDNA形成直径约65 nm的阳离子复合物纳米囊泡。N/P为60时,LPSA可有效结合pDNA,LPSA/pDNA在4 h内,耐DNaseⅠ酶解。Objective To construct LPSA/pDNA complex by using a noval cationic polymer LPSA,and to investigate the physical and chemical properties of LPSA/pDNA complex with different N/P ratios.Methods A new cationic polymer LPSA was used as the gene carrier,and a plasmid containing non-fused BMP2-GFP gene fragment was prepared to form a eukaryotic expression complex and identified.LPSA/pDNA complexes with different N/P ratios were prepared,and the related physical and chemical properties were tested by gel retardation test,particle size and Zeta potential detection,morphology observation,and in vitro enzyme degradation resistance test.Results By agarose gel electrophoresis and sequencing,the encoding sequence of the eukaryotic expression vector BMP2 was consistent with Genebank.With the increase of N/P,the DNA blocking ability of LPSA/pDNA complex increased.When N/P was greater than 10,LPSA could basically block pDNA.The particle size of LPSA/pDNA complex decreased first and then increased with the increase of N/P ratio.When N/P was greater than 3,the particle size of LPSA/pDNA was less than 150 nm.When N/P was between 12 to 25,the particle size of LPSA/pDNA is stable at about 65 nm,the zeta potential on LPSA/pDNA surface was positive,and the charge value was from 9 mV to 35 mV.When N/P was 60,with the increase of DNase I treatment time,there was no significant change in the brightness of agarose gel electrophoresis bands within 4 h,but the brightness of agarose gel electrophoresis bands decreased slightly at 6 h.Conclusion When N/P ranges from 12 to 25,LPSA and pDNA form cationic complex nanovesicles with a diameter of 65 nm.When N/P is 60,LPSA binds pDNA effectively,and LPSA/pDNA is resistant to DNase I enzymatic hydrolysis within 4 h.

关 键 词：脂质体转染 骨形态生成蛋白2 脂质体 胆固醇 海藻酸钠 

分 类 号：R914.5[医药卫生—药物化学] R783.1[医药卫生—药学]