Optimization of DNA Transfection Mediated by Linear Polyethylenimine (LPEI)

Xing-yu MA; Guo-geng YE; Bing-ke QIAO; Jian-jun ZHAO; An-fang LIU

doi:10.13718/j.cnki.xdzk.2019.06.005

Message Board

Dear readers, authors and reviewers,you can add a message on this page. We will reply to you as soon as possible!

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.

Corresponding author: An-fang LIU

Received Date: 20/12/2018

Available Online: 20/06/2019

Key words:

Abstract: 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.

HTML

核酸传递是生物学研究、转基因和基因治疗必经的重要关键环节.由于核酸酶的广泛存在和核酸的高分子量及生理生化特性，导致外源核酸很难独立地被递送进靶细胞，而少量递送入胞质的核酸又易被溶酶体-内涵体系统降解^[1].因此，开发利用核酸运载体克服核酸转染屏障是生物学研究的重要问题.目前核酸运载体主要分为病毒类和非病毒类，尽管前者的转染效率高，但后者在安全性、稳定性、成本上要优于前者^[2-3].

聚乙烯亚胺(Polyethylenimine，PEI)是一种水溶性阳离子高分子聚合物，化学结构中大量胺基正电氮原子使其携带高密度正电荷，早在1995年被用于体外核酸转染^[4-5].带正电荷的PEI利用静电交互作用缩聚带负电的核酸并形成易被细胞摄取的正电小粒径复合体，这种凝聚核酸的能力极大地影响了转染效率^[6]. PEI所含的大量胺基具有质子海绵效应，能促使PEI-核酸复合物逃离出内涵体和溶酶体继而在胞内释放核酸使目的基因得以表达，同时PEI可与细胞膜结构上的蛋白或磷脂相互作用影响膜的完整性，产生细胞毒性并影响细胞的转染效率^[7-9].转染效率的提高及细胞毒性的控制是PEI介导核酸转染应用的关键. PEI的转染效率和细胞毒性受到PEI中的N元素与质粒DNA中P元素的摩尔数比(N/P)、PEI和核酸聚合及转染条件、PEI的分子量和分枝化等多种因素的影响^[10-11].低分子量的PEI不能有效压缩聚合核酸，导致复合物表面电荷低，难以被细胞摄取^[12].尽管PEI分子量的升高转染效率会升高，但毒性也会增强^[13].大量研究最终确认25 kDa PEI是阳离子转染领域中的“黄金标准”^[14-15]. PEI含分支化PEI (Branched PEI，BPEI)和线性PEI(Linear PEI，LPEI)两大类^[16-17].研究表明BPEI虽然有着相对LPEI更强的核酸凝聚能力，但核酸释放能力仍很弱，进入胞内无法再有效释放核酸，同时其毒性也更大^[16]. 25 kDa LPEI是一种相对于传统PEI毒性更小、转染效率更高的核酸阳离子载体^[17]. N/P是影响PEI转染效率的最主要因素，但25 kDa LPEI N/P筛选研究差异较大，筛选的N/P范围(1~20)也较小^[18-19]. PEI的转染效率也与LPEI-DNA复合体聚合程度和转染条件密切相关，研究表明不同PEI溶剂、盐离子浓度、冻融、血清、细胞密度等均会影响PEI的转运核酸能力^[20-23]. LPEI作为核酸传递载体具有生物安全性高、操作简便、安全廉价等优点，具有作为基因治疗中核酸传递工具的应用潜力.但25 kDa LPEI介导核酸转染的条件缺乏系统性研究，其介导核酸转染的条件需要进一步筛选优化，以提高其携带核酸进行细胞内传递的效率.因此，本研究在前人研究的基础上，以适用性和认可度更好的25 kDa LPEI作为试验材料，探究N/P比，LPEI与DNA聚合时间，聚合pH对LPEI-DNA复合体聚合的影响，优化其对细胞转染的能力，为LPEI转染条件的完善及利用提供理论基础.

1. 材料与方法

1.1. 细胞与试剂

HEK293 T细胞由中国科学院干细胞库提供；LPEI(25 kDa)，Santa Cruz公司；MTT，Sigma公司；DMEM和F12细胞培养基，Opti-MEM，胎牛血清，Gibco公司；胰酶细胞消化液，Beyotime公司；100x青霉素-链霉素溶液，Biosharp公司；绿色荧光基因质粒pEGFP由笔者所在实验室在pCDNA3.1载体中插入EGFP基因构建而成；DMSO，Sigma公司；HBS缓冲液(20 mmol/L)，Hepes；150 mmol/L NaCl(pH值为7.1)，其余试剂均为国产分析纯试剂.

1.2. 核酸阻碍试验

聚合N/P比优化：按照核苷酸中平均330 Da含1个P，LPEI中平均43 Da含1个N，以此计算不同N/P所需的LPEI和核酸量^[24].用HBS缓冲液配制特定N/P(0，1，2，3，4，6，8，10，20)的LPEI，经0.22 μm滤膜过滤后与质粒混合，配制总体积为20 μL的LPEI/DNA混合液，每份含1 μg质粒pEGFP.上述混合液在室温下静置30 min后，各取5 μL在1%琼脂糖凝胶上进行电泳，电泳条件为1×TAE电泳缓冲液，电压5 V/cm，电泳10 min用凝胶成像仪观察DNA条带的迁移并拍照.

1.3. 聚合时间优化

用HBS缓冲液配制特定N/P(0，1，2，3，4，6，8，10，12，16，20)的LPEI与质粒混合液，分别静置10 min，30 min，1 h，2 h后按照上述相同电泳条件进行凝胶电泳阻滞试验.

1.4. 聚合pH优化

利用1 mol/mL的NaOH溶液与酸度计精确调整HBS缓冲液和LPEI溶液的pH，按pH值为6，6.5，7，7.5，8分为5个组，每组分别配置N/P为2，4，6的LPEI与质粒混合液，按照上述相同电泳条件进行凝胶电泳阻滞试验.

1.5. HEK293 T细胞培养

HEK293 T培养在含有双抗(青霉素100 U/mL和链霉素100 μg/mL)和10%胎牛血清(FBS)的DMEM/F12培养液中.细胞培养条件：37 ℃，5% CO₂，饱和湿度条件下，每2 d换液1次，待细胞生长达90%汇合状态时用胰酶消化法传代细胞.将细胞密度调整至2×10⁵个/mL，以每孔400 μL细胞悬液接种24孔培养板，培养24~36 h，细胞汇合度达到80%左右时用于转染试验.

1.6. 转染效率检测

按照N/P比(0，1，2，6，10，20，30，40，60，80，100)每个处理5个平行孔.转染前细胞培养液换为400 μL Opti-MEM培养基，将且在pH值为6聚合1 h后的无菌25 kDa LPEI和pEGFP质粒(每孔的质粒量为1 μg)混合物滴加到对应的细胞孔中，轻轻晃动使转染物分布均匀.细胞继续培养4 h后换加Opti-MEM新鲜培养基，48 h后在荧光倒置显微镜下观察荧光细胞比例并拍照.利用Image-J软件进行绿色荧光阳性细胞计数分析，计算出转染效率.

1.7. MTT检测

将HEK293 T细胞按照转染效率检测试验的操作和分组均等接种于96孔板中，培养24 h后按照不同N/P比进行LPEI转染，每孔200 ng质粒(方法同1.6)，每个处理5个平行.转染48 h后除去培养液，在每孔中加入20 μL MTT溶液(5 mg/mL)和80 μL新鲜的DMEM培养基，放入37 ℃培养箱培养4 h，移除MTT溶液，加入150 μL DMSO溶解紫色甲瓒结晶，用酶标仪在570 nm波长下测定每个孔的吸光度，计算细胞存活率：

1.8. 统计学分析

数据统计使用SPSS 19，方差齐性通过Levene方法检测，并进行单因素方差分析(one-way ANOVA)，Tukey's检验进行事后多重检验. p＜0.05表示差异有统计学意义，数据表示方法均为平均值±标准差.

3. 讨论

25 kDa LPEI是一种有效的核酸和药物传递载体，制备方便，价格低廉，其核酸转染功能被广泛应用^[25-26].为了更有效地利用25 kDa LPEI这一核酸转染工具，本研究通过凝胶阻滞试验研究N/P，LPEI与DNA聚合pH，聚合时间对25 kDa LPEI聚合DNA能力的影响，并通过细胞转染试验研究了大范围N/P对LPEI介导的DNA转染效率及细胞毒性的影响.

PEI聚合核酸的能力与其化学骨架中携带阳离子的胺基量密切相关.有研究表明，胺基相对于核酸的量，即N/P升高时，PEI与核酸形成的正电复合体表面电势变大，粒径变小，PEI对核酸的吸附和压缩能力增强^[24].本研究中不同N/P的25 kDa LPEI-DNA复合物凝胶阻滞试验表明，随着N/P的增加，核酸在电泳中受到的阻滞作用越强.当N/P较高时，核酸在琼脂糖凝胶中的迁移被完全抑制.表明N/P的增加有助于25 kDa LPEI对核酸的聚合及阳离子化，这符合大多数PEI或修饰性LPEI传递核酸的规律^[27-28].

本研究中不同聚合时间下25 kDa LPEI-DNA复合物凝胶阻滞试验表明，25 kDa LPEI与DNA共孵育10 min无法有效形成稳定的LPEI-DNA复合物，而在共孵育60 min时LPEI与DNA聚合达到饱和，120 min时出现核酸逃逸现象.表明25 kDa LPEI与核酸聚合反应时间低于10 min或超过1 h均不利于形成LPEI-DNA复合物，最适聚合时间为1 h.研究表明22 kDa LPEI与DNA共孵育时间过长，LPEI-DNA复合物粒径将迅速变大，并逐渐趋于不稳定^[29-30].

PEI与核酸共孵育环境可以通过离子强度、渗透压来影响聚合物表面电势及粒径^[31-32].本研究中不同聚合pH下25 kDa LPEI和DNA复合物凝胶阻滞试验表明，较低的pH有利于LPEI对核酸聚合，最适聚合pH值为6.有研究报道酸性条件可以增加PEI电势，但对细胞转染效率无显著影响^[33].本文推测尽管pH的降低有利于LPEI对核酸的聚合，但聚合过程中LPEI的H⁺过度富集导致LPEI阳离子缓冲性减弱，最终可能影响LPEI-DNA复合物逃逸溶酶体，这一推测还需研究验证.

研究表明阳离子载体的质子缓冲能力越强其对细胞的核酸递送能力越强，而前人研究已表明LPEI的质子缓冲能力与N/P密切相关^{[1, 34]}.本研究中不同N/P对HEK293 T细胞的转染试验表明，N/P小于10时LPEI难以介导核酸转染，随N/P升高转染效率逐渐增强，在N/P为60时转染阳性细胞达到峰值，这与前人得出的PEI，BPEI，10 kDa LPEI的最佳转染N/P为6~10的结论有所不同，本文推测这可能与试验采用的LPEI分子以及聚合条件不同有关，同时前人试验中认为LPEI的毒性过强会影响转染效率导致N/P的筛选范围过小^{[28, 35-36]}.

一定浓度的游离LPEI可在细胞表面吸附和聚集引起膜结构的融合、改变，也可以渗透入核造成DNA损伤，最终导致细胞死亡，而DNA与LPEI聚合后反而会降低毒性，因此恰当的N/P选择对细胞活率尤为重要^{[34, 37]}.有研究表明，细胞毒性过大，进而造成细胞死亡降低转染效率^[38].而本研究显示，随N/P增加细胞毒性增大，当N/P大于60后转染效率下降，但细胞活率变化不明显，表明转染效率的降低与细胞毒性的增加没有相关性.本研究还推测N/P过大造成转染效率降低的原因可能是过量LPEI过度结合DNA，导致DNA无法脱离LPEI-DNA复合体，从而DNA难以进入细胞核表达，这一推测还需研究验证.本研究表明，满足转染效率的同时并结合细胞活率筛选确定N/P为40~60可能是细胞转染的最佳N/P范围.

除此之外，科学家还通过对LPEI进行修饰、交联、螯合等不同方式提高转染效率或降低细胞毒性^[39-41].随着LPEI优化研究的不断深入，多项研究表明该系统具有治疗肝癌、结肠癌、膀胱癌等重大疾病的重大潜力^[42-44].本研究对25 kDa LPEI介导核酸转染的优化以及合适转染N/P范围的筛选能为后续生物学与基因治疗研究提供参考依据以及应用基础.

Figure (4) Reference (44)

Name
	Name cannot be empty!
E-mail
	Mailbox cannot be empty! Mailbox cannot be empty!
Telephone
	Mobile number cannot be empty! Please enter a valid mobile number!
Title

Content
Verification Code

[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. doi: 10.2174/156720111795256174 CrossRef Google Scholar
[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. doi: 10.1021/acsbiomaterials.5b00551 CrossRef Google Scholar
[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. Google Scholar
[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. doi: 10.1021/bc9600630 CrossRef Google Scholar
[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. doi: 10.1073/pnas.92.16.7297 CrossRef Google Scholar
[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. doi: 10.1016/S0168-3659(99)00090-5 CrossRef Google Scholar
[7]	PATTI A, JENNIFER M, MIN-SUNG K, et al. Transient Mammalian Cell Transfection with Polyethylenimine (PEI)[J]. Methods Enzymol, 2013, 529:227-240. doi: 10.1016/B978-0-12-418687-3.00018-5 CrossRef Google Scholar
[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. doi: 10.1038/nrd1775 CrossRef Google Scholar
[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. doi: 10.1016/j.jconrel.2011.01.003 CrossRef Google Scholar
[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. doi: 10.1023/A:1014861900478 CrossRef Google Scholar
[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. doi: 10.3969/j.issn.1673-4130.2015.03.018 CrossRef Google Scholar
[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. doi: 10.1002/(ISSN)1097-4636 CrossRef Google Scholar
[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. doi: 10.1002/(ISSN)1521-2254 CrossRef Google Scholar
[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. doi: 10.1002/(ISSN)1097-4636 CrossRef Google Scholar
[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. doi: 10.1002/(ISSN)1521-2254 CrossRef Google Scholar
[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. Google Scholar
[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. doi: 10.1016/S0168-3659(00)00317-5 CrossRef Google Scholar
[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. Google Scholar
[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. Google Scholar
[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. doi: 10.3870/j.issn.1672-8009.2007.03.009 CrossRef Google Scholar
[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. Google Scholar
[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. doi: 10.3969/j.issn.1007-7626.2004.02.017 CrossRef Google Scholar
[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. Google Scholar
[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. doi: 10.1016/S0168-3659(01)00547-8 CrossRef Google Scholar
[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. Google Scholar
[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. doi: 10.1016/j.polymer.2015.08.061 CrossRef Google Scholar
[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. doi: 10.1007/s10118-015-1644-9 CrossRef Google Scholar
[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. doi: 10.3969/j.issn.0258-8021.2006.04.019 CrossRef Google Scholar
[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. Google Scholar
[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. doi: 10.3870/j.issn.1672-8009.2007.03.009 CrossRef Google Scholar
[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. Google Scholar
[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. doi: 10.1016/j.cell.2008.08.006 CrossRef Google Scholar
[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. doi: 10.1073/pnas.92.16.7297 CrossRef Google Scholar
[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. Google Scholar
[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. Google Scholar
[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. doi: 10.1007/s10118-015-1644-9 CrossRef Google Scholar
[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. doi: 10.1023/A:1014861900478 CrossRef Google Scholar
[38]	OMIDI Y, KAFIL V. Cytotoxic Impacts of Linear and Branched Polyethylenimine Nanostructures in A431 Cells[J]. BioImpacts, 2011, 1(1):23-30. Google Scholar
[39]	SUN Q, RADOSZ M, SHEN Y. Challenges in Design of Translational Nanocarriers[J]. J Control Release, 2012, 164(2):156-169. doi: 10.1016/j.jconrel.2012.05.042 CrossRef Google Scholar
[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. doi: 10.2217/nnm-2016-0117 CrossRef Google Scholar
[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. doi: 10.1016/j.colsurfb.2017.06.022 CrossRef Google Scholar
[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. Google Scholar
[43]	URNAUER S, KLUTZ K, GRVNWALD, 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. Google Scholar
[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. doi: 10.1016/j.juro.2008.08.006 CrossRef Google Scholar

Message Board

Optimization of DNA Transfection Mediated by Linear Polyethylenimine (LPEI)

Abstract

References

Access History

通讯作者: 陈斌, bchen63@163.com

Article Metrics

Access History

Other Articles By Authors

Optimization of DNA Transfection Mediated by Linear Polyethylenimine (LPEI)

Corresponding author: An-fang LIU