线性聚乙烯亚胺(LPEI)介导DNA转染的优化研究
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摘要
线性聚乙烯亚胺(LPEI)是一类广泛用于核酸转染或药物传递的阳离子聚合物.为了进一步探究25 kDa LPEI的转染条件,通过凝胶电泳阻滞试验评估N/P,聚合pH及聚合时间对LPEI-DNA复合物形成的影响,继而利用大范围N/P的25 kDa LPEI对HEK293 T细胞进行绿色荧光蛋白表达质粒转染,转染48 h后通过绿色荧光细胞比例及MTT的统计分析,评估转染效率与LPEI造成的细胞毒性.结果表明, N/P的上升能增强LPEI-DNA的电泳阻滞;随着共孵育pH值从6～8升高, LPEI-DNA聚合逐渐减弱;随着共孵育时间延长, LPEI-DNA聚合逐渐增强,孵育1 h时达到饱和.随着体外转染HEK293 T细胞的N/P升高,转染阳性细胞比例逐渐升高,当N/P达到40～60时转染效率达到最高,进一步提高N/P后转染效率极显著下降(p<0.01);随着N/P升高,细胞活力逐渐下降.结果表明, N/P, pH,聚合时间均可影响LPEI聚合核酸能力;综合考虑N/P对HEK293 T细胞转染效率及细胞毒性的影响,确定25 kDa LPEI转染细胞适宜的条件为N/P为40～60,聚合pH值为6.0,聚合时间为1 h.
Linear polyethylenimine(LPEI) is a cationic polymer which is widely used for nucleic acid transfection and drug transport. In order to further explore the transfection conditions of 25 kDa LPEI, gel retardation assay was used to investigate the combined effect of N/P, co-incubation time of LPEI and DNA and co-incubation pH of LPEI-DNA on the formation of LPEI-DNA complex in this research. Then HEK293 T cell was used to transfect the 25 kDa LPEI with a GFP expression plasmid in a wide range of N/P, and the transfection efficiency and cytotoxicity were assessed with statistic analysis of fluorescent cell percent and MTT after 48-h transfection. The results showed that the gel retardation of LPEI-DNA increased with N/P, LPEI-DNA polymerization gradually decreased with the increase of co-incubation pH from 6 to 8, and LPEI-DNA polymerization gradually increased with the extension of co-incubation time and reached saturation at 1 h. With the increase of N/P in in vitro transfected HEK293 T cells, the proportion of transfected positive cells gradually increased. When N/P reached 40-60, the transfection efficiency reached the maxximum. With further increase in N/P, the transfection efficiency sharply decreased(p<0.01). The cell vabiliity gradually decreased with increasing N/P. Conclusion: N/P, polymerization pH and polymerization time all affected the ability of 25 kDa LPEI to polymerize nucleic acid. Considering the effect of N/P on HEK293 T cell transfection efficiency and cytotoxicity, the optimal conditions for 25 kDa LPEI transfection were N/P=40-60, polymerization pH=6.0, and polymerization time =1 h.
Linear polyethylenimine(LPEI) is a cationic polymer which is widely used for nucleic acid transfection and drug transport. In order to further explore the transfection conditions of 25 kDa LPEI, gel retardation assay was used to investigate the combined effect of N/P, co-incubation time of LPEI and DNA and co-incubation pH of LPEI-DNA on the formation of LPEI-DNA complex in this research. Then HEK293 T cell was used to transfect the 25 kDa LPEI with a GFP expression plasmid in a wide range of N/P, and the transfection efficiency and cytotoxicity were assessed with statistic analysis of fluorescent cell percent and MTT after 48-h transfection. The results showed that the gel retardation of LPEI-DNA increased with N/P, LPEI-DNA polymerization gradually decreased with the increase of co-incubation pH from 6 to 8, and LPEI-DNA polymerization gradually increased with the extension of co-incubation time and reached saturation at 1 h. With the increase of N/P in in vitro transfected HEK293 T cells, the proportion of transfected positive cells gradually increased. When N/P reached 40-60, the transfection efficiency reached the maxximum. With further increase in N/P, the transfection efficiency sharply decreased(p<0.01). The cell vabiliity gradually decreased with increasing N/P. Conclusion: N/P, polymerization pH and polymerization time all affected the ability of 25 kDa LPEI to polymerize nucleic acid. Considering the effect of N/P on HEK293 T cell transfection efficiency and cytotoxicity, the optimal conditions for 25 kDa LPEI transfection were N/P=40-60, polymerization pH=6.0, and polymerization time =1 h.
引文
[1] ELSABAHY M,NAZARALI A,FOLDVARI M.Non-Viral Nucleic Acid Delivery:Key Challenges and Future Directions [J].Curr Drug Deliv,2011,8(3):235-244.
[2] WILLIFORD J,ARCHANG M,MINN I,et al.Critical Length of PEG Grafts on LPEI/DNA Nanoparticles for Efficient in Vivo Delivery [J].Acs Biomater Sci Eng,2016,2(4):567-578.
[3] GOYAL R,TRIPATHI S K,TYAGI S,et al.Linear PEI Nanoparticles:Efficient pDNA/siRNA Carriers in Vitro and in Vivo [J].Nanomedicine-Uk,2012,8(2):167-175.
[4] TANG M X,REDEMANN C T,SZOKA F J.In Vitro Gene Delivery by Degraded Polyamidoamine Dendrimers [J].Bioconjug Chem,1996,7(6):703-714.
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[6] GODBEY W T,WU K K,MIKOS A G.Poly(Ethylenimine) and Its Role in Gene Delivery [J].J Control Release,1999,60(2-3):149-160.
[7] PATTI A,JENNIFER M,MIN-SUNG K,et al.Transient Mammalian Cell Transfection with Polyethylenimine (PEI) [J].Methods Enzymol,2013,529:227-240.
[8] PACK D W,HOFFMAN A S,PUN S,et al.Design and Development of Polymers for Gene Delivery [J].Nat Rev Drug Discov,2005,4(7):581-593.
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[11] 曹慧玲,曹慧玲,滕凤猛,等.PEI与脂质体介导基因转染的比较研究 [J].国际检验医学杂志,2015(3):328-330.
[12] GODBEY W T,WU K K,MIKOS A G.Size Matters:Molecular Weight Affects the Efficiency of Poly(Ethylenimine) as a Gene Delivery Vehicle [J].J Biomed Mater Res,1999,45(3):268-275.
[13] NEU M,FISCHER D,KISSEL T.Recent Advances in Rational Gene Transfer Vector Design Based on Poly(Ethylene Imine) and Its Derivatives [J].J Gene Med,2005,7(8):992-1009.
[14] GODBEY W T,WU K K,MIKOS A G.Size Matters:Molecular Weight Affects the Efficiency of Poly(Ethylenimine) as a Gene Delivery Vehicle [J].J Biomed Mater Res,1999,45(3):268-275.
[15] NEU M,FISCHER D,KISSEL T.Recent Advances in Rational Gene Transfer Vector Design Based on Poly(Ethylene Imine) and Its Derivatives [J].J Gene Med,2005,7(8):992-1009.
[16] BRISSAULT B,KICHLER A,GUIS C,et al.Synthesis of Linear Polyethylenimine Derivatives for DNA Transfection [J].Bioconjug Chem,2003,14(3):581-587.
[17] VON HARPE A,PETERSEN H,LI Y,et al.Characterization of Commercially Available and Synthesized Polyethylenimines for Gene Delivery [J].J Control Release,2000,69(2):309-322.
[18] 余冰菲,张垲,陈瑞川,等.线性与分枝状聚乙烯亚胺介导基因体外转染的比较与研究 [J].厦门大学学报(自然版),2008,47(2):36-39.
[19] 王春晓,林超,赵鹏,等.生物还原响应的葡聚糖修饰的线性聚乙烯亚胺基因释放载体的研究 [J].青岛科技大学学报(自然科学版),2015,36(6):605-611.
[20] 谈娟,郝朋,张喜慧,等.非病毒载体聚乙烯亚胺介导DNA转染的研究 [J].医学分子生物学杂志,2007(3):229-233.
[21] MERKEL O M,ZHENG M,DEBUS H,et al.Pulmonary Gene Delivery Using Polymeric Nonviral Vectors [J].Bioconjug Chem,2012,23(1):3-20.
[22] 李经忠,王青青,余海,等.聚乙烯亚胺转基因影响因素的测定及其优化 [J].中国生物化学与分子生物学报,2004(2):234-240.
[23] MERKEL O M,ZHENG M,DEBUS H,et al.Pulmonary Gene Delivery Using Polymeric Nonviral Vectors [J].Bioconjug Chem,2012,23(1):3-20.
[24] AHN C H,CHAE S Y,BAE Y H,et al.Biodegradable Poly(Ethylenimine) for Plasmid DNA Delivery [J].J Control Release,2002,80(1-3):273-282.
[25] 黄红辉,朱世诚,董国萍,等.运用CRISPR/Cas9技术对斑马鱼前肾表达基因的敲除研究 [J].西南大学学报(自然科学版),2017,39(5):54-61.
[26] ZHOU Z ,MURDOCH W J ,SHEN Y.A Linear Polyethylenimine (LPEI) Drug Conjugate with Reversible Charge to Overcome Multidrug Resistance in Cancer Cells [J].Polymer,2015,76:150-158.
[27] ZHANG B ,XINPENG M ,SUI M ,et al.Guanidinoamidized Linear Polyethyleneimine for Gene Delivery [J].Chinese Journal of Polymer Science,2015,33(6):908-919.
[28] 李经忠,王青青,余海.新型非病毒载体聚乙烯亚胺介导基因转染参数的研究 [J].中国生物医学工程学报,2006(4):481-487.
[29] 王春晓,林超,赵鹏,等.生物还原响应的葡聚糖修饰的线性聚乙烯亚胺基因释放载体的研究 [J].青岛科技大学学报(自然科学版),2015,36(6):605-611.
[30] 谈娟,郝朋,张喜慧,等.非病毒载体聚乙烯亚胺介导DNA转染的研究 [J].医学分子生物学杂志,2007,4(3):229-233.
[31] 刘少娇,李巍巍,杨黎明,等.PEI介导的大规模基因瞬时转染研究进展 [J].现代生物医学进展,2015,15(1):145-148.
[32] STATHOPULOS P B,ZHENG L,LI G Y,et al.Structural and Mechanistic Insights Into STIM1-Mediated Initiation of Store-Operated Calcium Entry [J].Cell,2008,135(1):110-122.
[33] BOUSSIF O,LEZOUALC'H F,ZANTA M A,et al.A Versatile Vector for Gene and Oligonucleotide Transfer Into Cells in Culture and in Vivo:Polyethylenimine [J].Proc Natl Acad Sci U S A,1995,92(16):7297-7301.
[34] GODBEY W T,WU K K,MIKOS A G.Poly(Ethylenimine) and Its Role in Gene Delivery [J].Netherlands:Elsevier B V,1999,60:149-160.
[35] GOYAL R,TRIPATHI S K,TYAGI S,et al.Linear PEI Nanoparticles:Efficient pDNA/siRNA Carriers in Vitro and in Vivo [J].Nanomedicine:Nanotechnology,Biology and Medicine,2012,8(2):167-175.
[36] ZHANG B,MA X,SUI M,et al.Guanidinoamidized Linear Polyethyleneimine for Gene Delivery [J].Chinese J Polym Sci,2015,33(6):908-919.
[37] FISCHER D,BIEBER T,LI Y,et al.A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight,Branched Polyethylenimine:Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity [J].Pharm Res,1999,16(8):1273-1279.
[38] OMIDI Y,KAFIL V.Cytotoxic Impacts of Linear and Branched Polyethylenimine Nanostructures in A431 Cells [J].BioImpacts,2011,1(1):23-30.
[39] SUN Q,RADOSZ M,SHEN Y.Challenges in Design of Translational Nanocarriers [J].J Control Release,2012,164(2):156-169.
[40] CADETE A,ALONSO M J.Targeting Cancer with Hyaluronic Acid-Based Nanocarriers:Recent Advances and Translational Perspectives [J].Nanomedicine (Lond),2016,11(17):2341-2357.
[41] PINNAPIREDDY S R,DUSE L,STREHLOW B,et al.Composite Liposome-PEI/Nucleic Acid Lipopolyplexes for Safe and Efficient Gene Delivery and Gene Knockdown [J].Colloids Surf B Biointerfaces,2017,158:93-101.
[42] URNAUER S,MULLER A M,SCHUG C,et al.EGFR-Targeted Nonviral NIS Gene Transfer for Bioimaging and Therapy of Disseminated Colon Cancer Metastases [J].Oncotarget,2017,8(54):92195-92208.
[43] URNAUER S ,KLUTZ K ,GRüNWALD,GEOFFREY K,et al.Systemic Tumor-Targeted Sodium Iodide Symporter (NIS) Gene Therapy of Hepatocellular Carcinoma Mediated by B6 Peptide Polyplexes [J].The Journal of Gene Medicine,2017.
[44] SIDI AA,OHANA P,BENJAMIN S,et al.Phase I/II Marker Lesion Study of Intravesical BC-819 DNA Plasmid in H19 Over Expressing Superficial Bladder Cancer Refractory to Bacillus Calmette-Guerin [J].J Urol,2008,180(6):2379-2383.
[2] WILLIFORD J,ARCHANG M,MINN I,et al.Critical Length of PEG Grafts on LPEI/DNA Nanoparticles for Efficient in Vivo Delivery [J].Acs Biomater Sci Eng,2016,2(4):567-578.
[3] GOYAL R,TRIPATHI S K,TYAGI S,et al.Linear PEI Nanoparticles:Efficient pDNA/siRNA Carriers in Vitro and in Vivo [J].Nanomedicine-Uk,2012,8(2):167-175.
[4] TANG M X,REDEMANN C T,SZOKA F J.In Vitro Gene Delivery by Degraded Polyamidoamine Dendrimers [J].Bioconjug Chem,1996,7(6):703-714.
[5] BOUSSIF O,LEZOUALC'H F,ZANTA M A,et al.A Versatile Vector for Gene and Oligonucleotide Transfer Into Cells in Culture and in Vivo:Polyethylenimine [J].Proc Natl Acad Sci U S A,1995,92(16):7297-7301.
[6] GODBEY W T,WU K K,MIKOS A G.Poly(Ethylenimine) and Its Role in Gene Delivery [J].J Control Release,1999,60(2-3):149-160.
[7] PATTI A,JENNIFER M,MIN-SUNG K,et al.Transient Mammalian Cell Transfection with Polyethylenimine (PEI) [J].Methods Enzymol,2013,529:227-240.
[8] PACK D W,HOFFMAN A S,PUN S,et al.Design and Development of Polymers for Gene Delivery [J].Nat Rev Drug Discov,2005,4(7):581-593.
[9] KASPER J C,SCHAFFERT D,OGRIS M,et al.Development of a Lyophilized Plasmid/LPEI Polyplex Formulation with Long-Term Stability-A Step Closer from Promising Technology to Application [J].J Control Release,2011,151(3):246-255.
[10] FISCHER D,BIEBER T,LI Y,et al.A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight,Branched Polyethylenimine:Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity [J].Pharm Res,1999,16(8):1273-1279.
[11] 曹慧玲,曹慧玲,滕凤猛,等.PEI与脂质体介导基因转染的比较研究 [J].国际检验医学杂志,2015(3):328-330.
[12] GODBEY W T,WU K K,MIKOS A G.Size Matters:Molecular Weight Affects the Efficiency of Poly(Ethylenimine) as a Gene Delivery Vehicle [J].J Biomed Mater Res,1999,45(3):268-275.
[13] NEU M,FISCHER D,KISSEL T.Recent Advances in Rational Gene Transfer Vector Design Based on Poly(Ethylene Imine) and Its Derivatives [J].J Gene Med,2005,7(8):992-1009.
[14] GODBEY W T,WU K K,MIKOS A G.Size Matters:Molecular Weight Affects the Efficiency of Poly(Ethylenimine) as a Gene Delivery Vehicle [J].J Biomed Mater Res,1999,45(3):268-275.
[15] NEU M,FISCHER D,KISSEL T.Recent Advances in Rational Gene Transfer Vector Design Based on Poly(Ethylene Imine) and Its Derivatives [J].J Gene Med,2005,7(8):992-1009.
[16] BRISSAULT B,KICHLER A,GUIS C,et al.Synthesis of Linear Polyethylenimine Derivatives for DNA Transfection [J].Bioconjug Chem,2003,14(3):581-587.
[17] VON HARPE A,PETERSEN H,LI Y,et al.Characterization of Commercially Available and Synthesized Polyethylenimines for Gene Delivery [J].J Control Release,2000,69(2):309-322.
[18] 余冰菲,张垲,陈瑞川,等.线性与分枝状聚乙烯亚胺介导基因体外转染的比较与研究 [J].厦门大学学报(自然版),2008,47(2):36-39.
[19] 王春晓,林超,赵鹏,等.生物还原响应的葡聚糖修饰的线性聚乙烯亚胺基因释放载体的研究 [J].青岛科技大学学报(自然科学版),2015,36(6):605-611.
[20] 谈娟,郝朋,张喜慧,等.非病毒载体聚乙烯亚胺介导DNA转染的研究 [J].医学分子生物学杂志,2007(3):229-233.
[21] MERKEL O M,ZHENG M,DEBUS H,et al.Pulmonary Gene Delivery Using Polymeric Nonviral Vectors [J].Bioconjug Chem,2012,23(1):3-20.
[22] 李经忠,王青青,余海,等.聚乙烯亚胺转基因影响因素的测定及其优化 [J].中国生物化学与分子生物学报,2004(2):234-240.
[23] MERKEL O M,ZHENG M,DEBUS H,et al.Pulmonary Gene Delivery Using Polymeric Nonviral Vectors [J].Bioconjug Chem,2012,23(1):3-20.
[24] AHN C H,CHAE S Y,BAE Y H,et al.Biodegradable Poly(Ethylenimine) for Plasmid DNA Delivery [J].J Control Release,2002,80(1-3):273-282.
[25] 黄红辉,朱世诚,董国萍,等.运用CRISPR/Cas9技术对斑马鱼前肾表达基因的敲除研究 [J].西南大学学报(自然科学版),2017,39(5):54-61.
[26] ZHOU Z ,MURDOCH W J ,SHEN Y.A Linear Polyethylenimine (LPEI) Drug Conjugate with Reversible Charge to Overcome Multidrug Resistance in Cancer Cells [J].Polymer,2015,76:150-158.
[27] ZHANG B ,XINPENG M ,SUI M ,et al.Guanidinoamidized Linear Polyethyleneimine for Gene Delivery [J].Chinese Journal of Polymer Science,2015,33(6):908-919.
[28] 李经忠,王青青,余海.新型非病毒载体聚乙烯亚胺介导基因转染参数的研究 [J].中国生物医学工程学报,2006(4):481-487.
[29] 王春晓,林超,赵鹏,等.生物还原响应的葡聚糖修饰的线性聚乙烯亚胺基因释放载体的研究 [J].青岛科技大学学报(自然科学版),2015,36(6):605-611.
[30] 谈娟,郝朋,张喜慧,等.非病毒载体聚乙烯亚胺介导DNA转染的研究 [J].医学分子生物学杂志,2007,4(3):229-233.
[31] 刘少娇,李巍巍,杨黎明,等.PEI介导的大规模基因瞬时转染研究进展 [J].现代生物医学进展,2015,15(1):145-148.
[32] STATHOPULOS P B,ZHENG L,LI G Y,et al.Structural and Mechanistic Insights Into STIM1-Mediated Initiation of Store-Operated Calcium Entry [J].Cell,2008,135(1):110-122.
[33] BOUSSIF O,LEZOUALC'H F,ZANTA M A,et al.A Versatile Vector for Gene and Oligonucleotide Transfer Into Cells in Culture and in Vivo:Polyethylenimine [J].Proc Natl Acad Sci U S A,1995,92(16):7297-7301.
[34] GODBEY W T,WU K K,MIKOS A G.Poly(Ethylenimine) and Its Role in Gene Delivery [J].Netherlands:Elsevier B V,1999,60:149-160.
[35] GOYAL R,TRIPATHI S K,TYAGI S,et al.Linear PEI Nanoparticles:Efficient pDNA/siRNA Carriers in Vitro and in Vivo [J].Nanomedicine:Nanotechnology,Biology and Medicine,2012,8(2):167-175.
[36] ZHANG B,MA X,SUI M,et al.Guanidinoamidized Linear Polyethyleneimine for Gene Delivery [J].Chinese J Polym Sci,2015,33(6):908-919.
[37] FISCHER D,BIEBER T,LI Y,et al.A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight,Branched Polyethylenimine:Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity [J].Pharm Res,1999,16(8):1273-1279.
[38] OMIDI Y,KAFIL V.Cytotoxic Impacts of Linear and Branched Polyethylenimine Nanostructures in A431 Cells [J].BioImpacts,2011,1(1):23-30.
[39] SUN Q,RADOSZ M,SHEN Y.Challenges in Design of Translational Nanocarriers [J].J Control Release,2012,164(2):156-169.
[40] CADETE A,ALONSO M J.Targeting Cancer with Hyaluronic Acid-Based Nanocarriers:Recent Advances and Translational Perspectives [J].Nanomedicine (Lond),2016,11(17):2341-2357.
[41] PINNAPIREDDY S R,DUSE L,STREHLOW B,et al.Composite Liposome-PEI/Nucleic Acid Lipopolyplexes for Safe and Efficient Gene Delivery and Gene Knockdown [J].Colloids Surf B Biointerfaces,2017,158:93-101.
[42] URNAUER S,MULLER A M,SCHUG C,et al.EGFR-Targeted Nonviral NIS Gene Transfer for Bioimaging and Therapy of Disseminated Colon Cancer Metastases [J].Oncotarget,2017,8(54):92195-92208.
[43] URNAUER S ,KLUTZ K ,GRüNWALD,GEOFFREY K,et al.Systemic Tumor-Targeted Sodium Iodide Symporter (NIS) Gene Therapy of Hepatocellular Carcinoma Mediated by B6 Peptide Polyplexes [J].The Journal of Gene Medicine,2017.
[44] SIDI AA,OHANA P,BENJAMIN S,et al.Phase I/II Marker Lesion Study of Intravesical BC-819 DNA Plasmid in H19 Over Expressing Superficial Bladder Cancer Refractory to Bacillus Calmette-Guerin [J].J Urol,2008,180(6):2379-2383.