双向基因调控黑素细胞凋亡的聚阳离子纳米复合物靶向给药系统的研究
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摘要
白癜风（Vitiligo）是一种由于黑素细胞特异性损害而致色素脱失的获得性皮肤病，症状为皮肤出现局限性或泛发性色素脱失斑，此病由皮肤和毛囊的黑色素脱失所引起，是一种容易诊断而难于治疗的皮肤病。近期研究发现，黑素细胞生长存活缺陷，已成为白癜风皮损中黑素细胞丢失的一个主要原因，因此，抑制黑素细胞凋亡是白癜风治疗的一个重要潜在靶点。研究发现，在黑素细胞中，Bcl-2是最主要的细胞凋亡抑制基因，Bax（Bcl-2associated X protein，Bcl-2相关蛋白X）则是最主要的细胞凋亡促进基因，因此，将Bcl-2质粒与Bax的小干扰RNA片段以一定的比例导入到黑素细胞内，可有效地控制黑素细胞凋亡。
本课题的研究目标是利用RNA干扰/DNA表达的双向基因调控技术，依靠α-黑素细胞刺激（α-melanocyte-stimulating hormone，α-MSH）与黑素细胞表面的黑皮素-1受体（melanocortin1receptor，MC-1R）的特异性结合作用，以低分子量聚乙烯亚胺（low molecular weight polyethyleneimine，LMW PEI）为基因载体，构建一个靶向于黑素细胞的聚阳离子基因纳米复合物给药系统。主要研究内容分为两部分：一方面，利用具有亲水亲脂两性基团的普朗尼克（Pluronic）连接低分子量聚乙烯亚胺，形成高分子聚合物，探讨其分子量大小、结构特征、水解能力、细胞毒性及转染效率等性质，以期得到一个更加安全、有效的阳离子聚合物基因载体；同时，在该载体上连接黑皮素-1受体的配体，作为靶向头基，实现黑素细胞的靶向；另一方面，利用RNA干扰/DNA表达的双向基因调控技术，将小干扰RNA片段/质粒包裹于上述载体，在抑制bax过表达的同时促进bcl-2表达，从而达到抑制黑素细胞调亡的目的，为白癜风等因黑素细胞特异性损害而致的疾病提供一条新的有效的治疗途径。本文主要从以下几部分进行研究：
第一部分，低分子量聚乙烯亚胺阳离子聚合物的合成及聚阳离子/DNA纳米复合物的理化性质、体外转染和毒性评价。使用三光气＋N-羟基琥珀亚酰胺法将不同种类Pluronic双端羟基活化并与低分子量PEI反应，利用凝胶渗透层析色谱法（GPC）对所得的产物进行分子量测定，利用核磁共振（NMR）对其结构进行测定，测定聚合物在外界环境下的水解能力，制备不同质量比的聚阳离子/DNA纳米复合物，利用马尔文微粒测定仪等测定影响复合物粒径、Zeta电位和稳定性的因素；使用琼脂糖凝胶电泳考察不同质量比条件下阳离子聚合物对DNA的浓缩能力，考察聚阳离子/DNA复合物抗酶解能力，选择Hela细胞株进行体外转染实验；采用CCK-8法测定细胞毒性。
第二部分，Bcl-2/Bax的双向基因策略调节细胞凋亡的体外评价。构建Bcl-2表达质粒，验证其体外表达效率；参考文献进行小干扰RNA片段合成并验证，获得有效的Bax干扰小RNA；采用优化比例的混合核酸，经聚阳离子纳米复合物包裹后体外转染黑素细胞，进行基因表达与凋亡评价。
第三部分，使用α-黑素细胞刺激素修饰第一部分所得聚合物，得到含有靶向头基的载体材料；将包裹了DNA和RNA的聚阳离子纳米复合物与黑素细胞在体外培育，对其粒径及Zeta电位进行测定，并考察其稳定性，体内外靶向性，以及促进黑素细胞增殖的效果。
第四部分，双向基因调控的聚阳离子纳米复合物抗凋亡作用研究及安全性评价。构建白癜风的小鼠动物模型；通过皮肤亮度效果评价和黑素细胞数量变化评价其治疗效果；通过动物血常规及肝、肾功能检测，病理组织的免疫组化检测，给药系统溶血性考察等评价该复合物的安全性。
综上所述，为了抑制黑素细胞的凋亡，本课题通过基因治疗的方法，利用RNA干扰/DNA表达的双向基因调控技术，以黑皮素-1受体介导的靶向于黑素细胞的低分子量聚乙烯亚胺阳离子聚合物为载体，将外源的基因导入生物细胞内，使Bcl-2的表达提高的同时，抑制Bax基因的表达，从而起到防止黑素细胞凋亡的作用，保证其在体内发挥正常的生理作用。这种利用双向基因调控来抑制黑素细胞凋亡的研究在国外未见到相关报道，同时以α-黑素细胞刺激素作为配体，起到黑素细胞靶向作用的研究亦未见报道，因此，本项目的研究，有望为白癜风等皮肤病的临床治疗开辟一条新的途径。
Vitiligo is an acquired depigmentation caused by melanocytes' specific injury,depigmentation of the skin and hair follicles to be more specific, characterized bydepigmented macules. It's readily diagnosable but recalcitrant to cure. Recent studiesindicate that defective melanocytes have become a leading cause for melanocytes loss onvitiligo legions. Therefore, suppressing the apoptosis of melanocyte is a critical potentialtarget for vitiligo treatment. Studies show that Bcl-2in melanocytes is the principalapoptosis suppressing gene, while Bax (Bcl-2associated X protein) is the principalapoptosis promoting gene. The apoptosis regulation of Bcl-2and Bax depends on the ratioof homodimer to heterodimer which they integrate into. Therefore, Bcl-2plasmid andsmall interfering RNA fragment of Bax on a proper proportion introduced into melanocytescould effectively contain melanocytes apoptosis.
The goal of this study is to build a polycation gene nanocomposite drug deliverysystem with low molecular weight polyethyleneimine as gene carrier by using bidirectionalgene regulation technology of RNA interference and DNA expression and integratingspecifically α-melanocyte stimulating hormone and melanocortin-1receptor onmelanocytes surface. Intervention of melanocytes apoptosis by using bidirectional generegulation technology of RNA interference and DNA expression has two parts. The firstpart is to generate high molecular polymer by connecting lipohydrophilic prownic with lowmolecular weight polyethyleneimine. By discussing its molecular weight, structuralfeatures, modification and hydrolysis in different biological circumstances, a safer andmore effective polycation gene carriers can be created. At the same time, connect theligand of the melanocortin-1receptor with the carrier as the targeting group to realize thetargeting at the melanocyte. On the other hand, using the bidirectional gene regulationtechnology of RNA interference/DNA expression, the small RNA interference fragment/plasmid is to be encapsulated in the above carrier to inhibit the bax overexpression whilepromoting the bcl-2expression, thus preventing the melanocyte apoptosis.
Part one, synthetization of low molecular weight polyethyleneimine and evaluation of the property, in vitro transfection and toxicity of polycation nanocomposite. Usetriphosgene plus N-hydroxy-succinamide (NHS) to activate hydroxy on double ends ofdifferent types of pluronic, which interacts with low molecular weight PEI. Determine themolecular weight of the products by gel permeation tomographic chromatography. UseMRI to asertain its structure, in an effort to determine the modificatory degree of PEI.Hydrolysis of the polymers in different biological circumstances will also be tested inorder to create polycation nanocomposite with different N/P ratio. Malvernmicromerigraph is to be used to determine the factors against diameter of polycation, Zetapotential and its stability. Use agarose gel electrophoresis to test the polycation'sconcentrating capacity on different N/P ratio condition for DNA/RNA, and DNA/RNApolycation anti-enzymatic ability will also be tested. We will choose mice melanoma cellline B16to do the transfection experiment. Two nucleic acids will be picked (pEGFP,green fluorescence protein reporter gene plasmid and small RNA fragment marked byFITC); use CCK-8to test toxicity of melanoma cells.
Part two, in vitro evaluation of bidirectional regulation of Bax/Bcl-2apoptosis gene.Build Bcl-2expression plasmid to prove its in vitro expression degree; Generate and provesmall interference RNA fragment by reference literature in order to gain effective smallBax interference RNA; Optimize the feeding ratio of small interference RNA andexpression plasmid to figure out the optimum ratio of expression of Bax/Bcl-2affectedjointly by both. Melanoma cells will be in vitro transfected by mixed nucleic acids onoptimum ratio, wrapped up by polycation nanocomposite, to evaluate gene expression andapoptosis.
Part three, modify the product polymer of the first part with α-melanocyte stimulatinghormone to gain carrier materials with targeting group; in vitro cultivate polycation andmelanoma wrapped by DNA and RNA to observe the integration of polycation andmelanoma, and evaluate primarily its targeting; generate frozen section by subcutaneousinduction of the DNA/RNA compound marked by quantum dot after corneum damaged bymicropins. use fluorescent confocal microscope to observe the layout of melanocytes marked by Melanoma Specific Fluorescent Antibody, and its perienchyma on dermis.
Part four, apoptosis suppression of bidirectional gene regulated polycationnanocomposite. Establish vitiligo mouse model; apply drugs onto the localized vitiligo ofpathological model induced by adjuvant, and then test the melanocytes Bax/Bcl-2expression, and changes of melanocytes apoptosis on the pathological tissue by TUNELmethod.
To sum up, in order to suppress melanocytes apoptosis, using RNA interference/DNAexpression bidirectional gene regulation technology, taking melanocortin-1receptormediated low molecular weight Polyethyleneimine targeting melanocytes as carrier,foreign gene is induced into biological cell to suppress Bax expression while boostingBcl-2expression, in an effort to prevent melanocytes apoptosis, and guaranteemelanocytes' biological role in vivo. So far, no report of study on suppressing melanocytesapoptosis by bidirectional gene regulation is seen abroad, and report of study onα-melanocyte stimulating hormone targeting melanocytes as ligand is also unseen.Therefore, this study is expected to provide a new perspective for treatment of vitiligo andother skin diseases.
本课题的研究目标是利用RNA干扰/DNA表达的双向基因调控技术，依靠α-黑素细胞刺激（α-melanocyte-stimulating hormone，α-MSH）与黑素细胞表面的黑皮素-1受体（melanocortin1receptor，MC-1R）的特异性结合作用，以低分子量聚乙烯亚胺（low molecular weight polyethyleneimine，LMW PEI）为基因载体，构建一个靶向于黑素细胞的聚阳离子基因纳米复合物给药系统。主要研究内容分为两部分：一方面，利用具有亲水亲脂两性基团的普朗尼克（Pluronic）连接低分子量聚乙烯亚胺，形成高分子聚合物，探讨其分子量大小、结构特征、水解能力、细胞毒性及转染效率等性质，以期得到一个更加安全、有效的阳离子聚合物基因载体；同时，在该载体上连接黑皮素-1受体的配体，作为靶向头基，实现黑素细胞的靶向；另一方面，利用RNA干扰/DNA表达的双向基因调控技术，将小干扰RNA片段/质粒包裹于上述载体，在抑制bax过表达的同时促进bcl-2表达，从而达到抑制黑素细胞调亡的目的，为白癜风等因黑素细胞特异性损害而致的疾病提供一条新的有效的治疗途径。本文主要从以下几部分进行研究：
第一部分，低分子量聚乙烯亚胺阳离子聚合物的合成及聚阳离子/DNA纳米复合物的理化性质、体外转染和毒性评价。使用三光气＋N-羟基琥珀亚酰胺法将不同种类Pluronic双端羟基活化并与低分子量PEI反应，利用凝胶渗透层析色谱法（GPC）对所得的产物进行分子量测定，利用核磁共振（NMR）对其结构进行测定，测定聚合物在外界环境下的水解能力，制备不同质量比的聚阳离子/DNA纳米复合物，利用马尔文微粒测定仪等测定影响复合物粒径、Zeta电位和稳定性的因素；使用琼脂糖凝胶电泳考察不同质量比条件下阳离子聚合物对DNA的浓缩能力，考察聚阳离子/DNA复合物抗酶解能力，选择Hela细胞株进行体外转染实验；采用CCK-8法测定细胞毒性。
第二部分，Bcl-2/Bax的双向基因策略调节细胞凋亡的体外评价。构建Bcl-2表达质粒，验证其体外表达效率；参考文献进行小干扰RNA片段合成并验证，获得有效的Bax干扰小RNA；采用优化比例的混合核酸，经聚阳离子纳米复合物包裹后体外转染黑素细胞，进行基因表达与凋亡评价。
第三部分，使用α-黑素细胞刺激素修饰第一部分所得聚合物，得到含有靶向头基的载体材料；将包裹了DNA和RNA的聚阳离子纳米复合物与黑素细胞在体外培育，对其粒径及Zeta电位进行测定，并考察其稳定性，体内外靶向性，以及促进黑素细胞增殖的效果。
第四部分，双向基因调控的聚阳离子纳米复合物抗凋亡作用研究及安全性评价。构建白癜风的小鼠动物模型；通过皮肤亮度效果评价和黑素细胞数量变化评价其治疗效果；通过动物血常规及肝、肾功能检测，病理组织的免疫组化检测，给药系统溶血性考察等评价该复合物的安全性。
综上所述，为了抑制黑素细胞的凋亡，本课题通过基因治疗的方法，利用RNA干扰/DNA表达的双向基因调控技术，以黑皮素-1受体介导的靶向于黑素细胞的低分子量聚乙烯亚胺阳离子聚合物为载体，将外源的基因导入生物细胞内，使Bcl-2的表达提高的同时，抑制Bax基因的表达，从而起到防止黑素细胞凋亡的作用，保证其在体内发挥正常的生理作用。这种利用双向基因调控来抑制黑素细胞凋亡的研究在国外未见到相关报道，同时以α-黑素细胞刺激素作为配体，起到黑素细胞靶向作用的研究亦未见报道，因此，本项目的研究，有望为白癜风等皮肤病的临床治疗开辟一条新的途径。
Vitiligo is an acquired depigmentation caused by melanocytes' specific injury,depigmentation of the skin and hair follicles to be more specific, characterized bydepigmented macules. It's readily diagnosable but recalcitrant to cure. Recent studiesindicate that defective melanocytes have become a leading cause for melanocytes loss onvitiligo legions. Therefore, suppressing the apoptosis of melanocyte is a critical potentialtarget for vitiligo treatment. Studies show that Bcl-2in melanocytes is the principalapoptosis suppressing gene, while Bax (Bcl-2associated X protein) is the principalapoptosis promoting gene. The apoptosis regulation of Bcl-2and Bax depends on the ratioof homodimer to heterodimer which they integrate into. Therefore, Bcl-2plasmid andsmall interfering RNA fragment of Bax on a proper proportion introduced into melanocytescould effectively contain melanocytes apoptosis.
The goal of this study is to build a polycation gene nanocomposite drug deliverysystem with low molecular weight polyethyleneimine as gene carrier by using bidirectionalgene regulation technology of RNA interference and DNA expression and integratingspecifically α-melanocyte stimulating hormone and melanocortin-1receptor onmelanocytes surface. Intervention of melanocytes apoptosis by using bidirectional generegulation technology of RNA interference and DNA expression has two parts. The firstpart is to generate high molecular polymer by connecting lipohydrophilic prownic with lowmolecular weight polyethyleneimine. By discussing its molecular weight, structuralfeatures, modification and hydrolysis in different biological circumstances, a safer andmore effective polycation gene carriers can be created. At the same time, connect theligand of the melanocortin-1receptor with the carrier as the targeting group to realize thetargeting at the melanocyte. On the other hand, using the bidirectional gene regulationtechnology of RNA interference/DNA expression, the small RNA interference fragment/plasmid is to be encapsulated in the above carrier to inhibit the bax overexpression whilepromoting the bcl-2expression, thus preventing the melanocyte apoptosis.
Part one, synthetization of low molecular weight polyethyleneimine and evaluation of the property, in vitro transfection and toxicity of polycation nanocomposite. Usetriphosgene plus N-hydroxy-succinamide (NHS) to activate hydroxy on double ends ofdifferent types of pluronic, which interacts with low molecular weight PEI. Determine themolecular weight of the products by gel permeation tomographic chromatography. UseMRI to asertain its structure, in an effort to determine the modificatory degree of PEI.Hydrolysis of the polymers in different biological circumstances will also be tested inorder to create polycation nanocomposite with different N/P ratio. Malvernmicromerigraph is to be used to determine the factors against diameter of polycation, Zetapotential and its stability. Use agarose gel electrophoresis to test the polycation'sconcentrating capacity on different N/P ratio condition for DNA/RNA, and DNA/RNApolycation anti-enzymatic ability will also be tested. We will choose mice melanoma cellline B16to do the transfection experiment. Two nucleic acids will be picked (pEGFP,green fluorescence protein reporter gene plasmid and small RNA fragment marked byFITC); use CCK-8to test toxicity of melanoma cells.
Part two, in vitro evaluation of bidirectional regulation of Bax/Bcl-2apoptosis gene.Build Bcl-2expression plasmid to prove its in vitro expression degree; Generate and provesmall interference RNA fragment by reference literature in order to gain effective smallBax interference RNA; Optimize the feeding ratio of small interference RNA andexpression plasmid to figure out the optimum ratio of expression of Bax/Bcl-2affectedjointly by both. Melanoma cells will be in vitro transfected by mixed nucleic acids onoptimum ratio, wrapped up by polycation nanocomposite, to evaluate gene expression andapoptosis.
Part three, modify the product polymer of the first part with α-melanocyte stimulatinghormone to gain carrier materials with targeting group; in vitro cultivate polycation andmelanoma wrapped by DNA and RNA to observe the integration of polycation andmelanoma, and evaluate primarily its targeting; generate frozen section by subcutaneousinduction of the DNA/RNA compound marked by quantum dot after corneum damaged bymicropins. use fluorescent confocal microscope to observe the layout of melanocytes marked by Melanoma Specific Fluorescent Antibody, and its perienchyma on dermis.
Part four, apoptosis suppression of bidirectional gene regulated polycationnanocomposite. Establish vitiligo mouse model; apply drugs onto the localized vitiligo ofpathological model induced by adjuvant, and then test the melanocytes Bax/Bcl-2expression, and changes of melanocytes apoptosis on the pathological tissue by TUNELmethod.
To sum up, in order to suppress melanocytes apoptosis, using RNA interference/DNAexpression bidirectional gene regulation technology, taking melanocortin-1receptormediated low molecular weight Polyethyleneimine targeting melanocytes as carrier,foreign gene is induced into biological cell to suppress Bax expression while boostingBcl-2expression, in an effort to prevent melanocytes apoptosis, and guaranteemelanocytes' biological role in vivo. So far, no report of study on suppressing melanocytesapoptosis by bidirectional gene regulation is seen abroad, and report of study onα-melanocyte stimulating hormone targeting melanocytes as ligand is also unseen.Therefore, this study is expected to provide a new perspective for treatment of vitiligo andother skin diseases.
引文
1. Van den Wijngaard RM, Aten J, Scheepmaker A, et al.Expression and modulation ofapoptosis regulatory molecules in human melanocytes significance in vitiligo. BritishJournal of Dermatology.2000;143:573-581.
2. Malmusi M， Ackerman A． A critical review of apoptosis in historicalperspective．Am J Dermatopathol，2000,22:291-293.
3. Stefanaki C, Antoniou C, Stefanaki K, et al. Bcl-2and Bax in congenital naevi. Br JDermatol.2006Jun;154(6):1175-1759.
4.李春英,高天文.白癜风与黑素细胞凋亡.中国美容医学;2005年01期;118-119.
5. Raisova M，Hossini AM，Eberle J，et al. The Bax/Bcl-2ratio determines thesusceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J InvestDermatol.200l Aug;117(2):333-340.
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7. Kingo K, Aunin E, Karelson M, et al. Gene expression analysis of melanocortin systemin vitiligo. J Dermatol Sci.2007Nov;48(2):113-122.
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[7] Milowsky MI, Nanus DM, Kostakoglu L. Vascular targeted therapy with anti-prostate-specificmembrane antigen monoclonal antibody J591in advanced solid tumors. J Clin Oncol,2007,25(5):540–547
[8] Kawasaki H, Wadhwa R, Taira K. World of small RNAs: From ribozymes to siRNA and miRNA.Differentiation.2004;72(2-3):58–64.
[9] Wagner E, Zenke M, Cotten M, Beug H, Birnstiel ML. Transferrin–polycation conjugates ascarriers for DNA uptake into cells. Proc Natl Acad Sci USA1990;87:3410-4.
[10] Wu GY, Wu CH. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system.J Biol Chem1987;262:4429-32.
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[6] Karaa ZS, Iacovoni JS, Bastide A,et al. The VEGF IRESes are differentially susceptible totranslation inhibition by miR-16. Rna,2009,15:249-254
[7] Milowsky MI, Nanus DM, Kostakoglu L. Vascular targeted therapy with anti-prostate-specificmembrane antigen monoclonal antibody J591in advanced solid tumors. J Clin Oncol,2007,25(5):540–547
[8] Kawasaki H, Wadhwa R, Taira K. World of small RNAs: From ribozymes to siRNA and miRNA.Differentiation.2004;72(2-3):58–64.
[9] Wagner E, Zenke M, Cotten M, Beug H, Birnstiel ML. Transferrin–polycation conjugates ascarriers for DNA uptake into cells. Proc Natl Acad Sci USA1990;87:3410-4.
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[1] Mulligan RC. The basic science of gene therapy. Science1993;260:926-32.
[2] Hartigan-O’Connor D, Chamberlain JS. Progress toward gene therapy of Duchenne musculardystrophy. Sem Neurol1999;19:323-32.
[3] Inui K, Okada S, Dickson G. Gene therapy in Duchenne muscular dystrophy. Brain Dev1996;18:357-61.
[4] Hartigan-O’Connor D, Chamberlain JS. Developments in gene therapy for muscular dystrophy.Microsc Res Tech2000;48:223-38.
[5] Lozier JN, Brinkhous KM. Gene therapy and the hemophilias. J Am Med Assoc1994;271:47-51.
[6] Alton EWFW, Geddes DM, Gill DR, Higgins CF, Hyde SC, Innes JA, et al. Towards gene therapyfor cycstic fibrosis: a clinica progress report. Gene Ther1998;5:291-2.
[7] Barkats M, Bilang-bleuel A, Buc-caron MH, Castelbarthe MN, Corti O, Finiels F,et al. Adenovirusin the brain: recent advances of gene therapy for neurodegenerative diseases.Prog Neuorbiol1998;55:333–41.
[8] Alisky JM, Davidson BL. Gene therapy for amyotrophic lateral sclerosis and other motor neurondiseases. Hum Gene Ther2000;11:2315–29.
[9] Dunnett SB, Bjorklund A. Prospects for new restorative and neuroprotective treatments inParkinson’s disease. Nature1999;399:A32–9.
[10]Bonadio J, Goldstein SA, Levy RJ. Gene therapy for tissue repair and regeneration. Adv DrugDelivery Rev1998;33:53-69.
[11]Nabel EG. Gene therapy for cardiovascular diseases. J Nucl Cardiol1999;6:69–75.
[12]Chadwick DR, Lever AML. Gene therapy for HIV infection. Exp Opin Ther Patents1998;8:983–90.
[13]Roth JA, Cristiano RJ. Gene therapy for cancer: what have we done and where are we going? J NatlCancer Inst1997;89:21-39.
[14]Folkman J. Antiangiogenic gene therapy. Proc Natl Acad Sci USA1998;95:9064–6.
[15]Vile RG, Russell SJ, Lemoine NR. Cancer gene therapy: hard lessons and new courses. Gene Ther2000;7:2–8.
[16]Nabel EG. Gene therapy for vascular diseases. Atherosclerosis1995;118:S51–6.
[17]Donnelly JJ, Ulmer JB, Shiver JW, Liu MA. DNA vaccines. Annu Rev Immunol1997;15:617–48.
[18]Vaheri A, Pagano JS. Infectious poliovirus RNA a sensitive method of assay. Virology1965;27:434–6.
[19]Graham FL, Eb AJVD. A new technique for the assay of infectivity of human adenovirus5DNA.Virology1973;52:456–67.
[20]Marshall E. FDA halts all gene therapy trials at Penn.Science2000;287:565–7.
[21]Marshall E. Clinical trials: gene therapy death prompts review of adenovirus vector. Science1999;286:2244–5.
[22]Kaiser J. Gene therapy. Seeking the cause of induced leukemias in X-SCID trial. Science2003;299:495.
[23]Marshall E. Clinical research. Gene therapy a suspect in leukemia-like disease.Science2002;298:34–5.
[24]Lollo CP, Banaszczyk MG, Chiou HC. Obstacles and advances in non-viral gene delivery. CurrOpin Mol Ther2000;2:136–42.
[25]Ledley FD. Nonviral gene therapy: the promise of genes as Pharmaceutical products. Hum GeneTher1995;6:1129–44.
[26]Kopatz I, Remy JS, Behr JP. A model for non-viral gene delivery: through syndecan adhesionmolecules and powered by actin. J Gene Med2004;6:769–76.
[27]Goncalves C, Mennesson E, Fuchs R, Gorvel JP, Midoux P, Pichon C. Macropinocytosis ofpolyplexes and recycling of plasmid via the clathrindependent pathway impair the transfectionefficiency of human hepatocarcinoma cells. Mol Ther2004;10:373–85.
[28]Mislick KA, Baldeschwieler JD. Evidence for the role of proteoglycans in cation-mediated genetransfer. Proc Natl Acad Sci USA1996;93:12349–54.
[29]Ruponen M, Honkakoski P, Tammi M, Urtti A. Cell-surface glycosaminoglycans inhibitcation-mediated gene transfer. J Gene Med2004;6:405–14.
[30]Goncalves C, Pichon C, Guerin B, Midoux P. Intracellular processing and stability of DNAcomplexed with histidylated polylysine conjugates. J Gene Med2002;4:271–81.
[31]Wagner E, Zenke M, Cotten M, Beug H, Birnstiel ML. Transferrin–polycation conjugates ascarriers for DNA uptake into cells. Proc Natl Acad Sci USA1990;87:3410–4.
[32]Wu GY, Wu CH. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system.J Biol Chem1987;262:4429–32.
[33]Curiel DT, Agarwal S, Wagner E, Cotten M. Adenovirus enhancement oftransferrin–polylysine-mediated gene delivery. Proc Natl Acad Sci USA1991;88:8850–4.
[34]Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS. Sizedependent DNAmobility in cytoplasm and nucleus. J Biol Chem2000;275:1625–9.
[35]Brunner S, Furtbauer E, Sauer T, Kursa M, Wagner E. Overcoming the nuclear barrier cell: cycleindependent nonviral gene transfer with linear polyethylenimine or electroporation. Mol Ther2002;5:80–6.
[36]Ludtke JJ, Zhang G, Sebestyén MG, Wolff JA. A nuclear localization signal can enhance both thenuclear transport and expression of1kb DNA. J Cell Sci1999;112:2033–41.
[37]Schaffer DV, Fidelman NA, Dan N, Lauffenburger DA. Vector unpacking as a potential barrier forreceptor-mediated polyplex gene delivery. Biotechnol Bioeng2000;67:598–606.
[38]Plank C, Tang MX, Wolfe AR, Szoka FC. Branched cationic peptides for gene delivery: role of typeand number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum GeneTher1999;10:319–32.
[39]Parker AL, Oupicky D, Dash PR, Seymour LW. Methodologies for monitoring nanoparticleformation by self-assembly of DNA with poly(L-lysine). Anal Biochem2002;302:75–80.
[40]Hardy JG, Kostiainen MA, Smith DK, Gabrielson NP, Pack DW. Dendrons with spermine surfacegroups as potential building blocks for nonviral vectors in gene therapy.Bioconjug Chem2006;17:172–8.
[41]Reineke TM, Davis ME. Structural effects of carbohydrate-containing polycations on gene delivery.2. Charge center type. Bioconjug Chem2003;14:255–61.
[42]Reineke TM, Davis ME. Structural effects of carbohydrate-containing polycations on gene delivery.1. Carbohydrate size and its distance from charge centers. Bioconjug Chem2003;14:247–54.
[43]Tomalia DA, Naylor AM, Goddard WA. Starburst dendrimers: Molecularlevel control of size, shape,surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew Chem Int EdEngl1990;29:138–75.
[44]Arima H, Kihara F, Hirayama F, Uekama K. Enhancement of gene expression by polyamidoaminedendrimer conjugates with a-, b-, and c-cyclodextrins. Bioconjugate Chem2001;12:476–84.
[45]Arima H, Kihara F, Tsutsumi T, Hirayama F, Uekama K.Effects of structure of polyamidoaminedendrimer on gene transfer efficiency of the dendrimer conjugate with a-cyclodextrin. BioconjugateChem2002;13:1211–9.
[46]Haensler J, Szoka FC. Polyamidoamine cascade polymers mediate efficient transfection of cells inculture. Bioconjugate Chem1993;4:372–9.
[47]Tang MX, Redemann C, Szoka FC. In vitro gene delivery by Degraded polyamidoaminedendrimers. Bioconjugate Chem1996;7:703–14.
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