Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes

A series of five poly[(ethylene imine)-co-N-(2-hydroxyethyl-ethylene imine)] copolymers with similar molecular weights and different degrees of branching was established to study structure-function relationship with regard to physicochemical and biological properties as gene delivery systems. Copolymers were synthesized by acid-catalyzed ring-opening copolymerization of aziridine and N-(2-hydroxyethyl)-aziridine in aqueous solution and characterized by GPC-MALLS, (1)H- and (13)C NMR, IR, potentiometric titration, and ion exchange chromatography. Complexation of DNA was determined by agarose gel electrophoresis, and complex sizes were quantitated by PCS. Cytotoxicity of the copolymers in fibroblasts was assessed by MTT-assay, LDH-assay, and hemolysis. The transfection efficiency was determined using the reporter plasmid pGL3 in 3T3 mouse fibroblasts. The copolymers obtained by solution polymerization had relatively low molecular weights of about 2000 Da, and the degree of branching increased with increasing ethylene imine ratio. The pK(a) as well as the buffer capacity increased proportional to the number of primary and secondary amines. Higher branched polymers showed stronger complexation and condensation of DNA, formed smaller polymer/DNA complexes, and induced the expression of plasmids to a higher extent than less branched polymers. In vitro cytotoxic effects and the hemolysis of erythrocytes decreased with decreased branching. Our results indicate that the basicity and degree of protonation of the polymers depending on their amount of primary and secondary amines seem to be important factors both for their transfection efficiency and for their cytotoxicity in gene transfer.     

Structural characterization and buffering capacity in relation to the transfection efficiency of biodegradable polyurethane

Inefficient release of polymer/DNA complexes from endocytic vesicles into the cytoplasm and the cytotoxic nature of cationic polymers are two of the primary causes of poor gene delivery. EG-polyurethane [poly(ethylene glycol)-PU, Poly 1], EGDM-polyurethane [poly(ethylene glycol), 2-(dimethylamino)ethylamine-PU, Poly 2], and MDEADM-polyurethane [N-methyldiethanolamine, 2-(dimethylamino)ethylamine-PU, Poly 3] were designed in this study to overcome these obstacles. The structural characteristics of polyurethanes and physicochemical properties of their formed complexes with DNA were determined to correlate their transfection efficiency. The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than those of Poly 2 and Poly 1. This study reveals that the amine groups in the polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery. Also the size and binding properties of DNA and polymeric transfectants can be in correlation to the transfection efficiency of resulting DNA/polymer complexes.     

miR-126报告基因载体的构建及其功能鉴定

目的:根据miR-126的预测靶点构建荧光素酶报告基因重组质粒,并进行功能鉴定。方法:利用sanger数据库提供的miR-126靶序列设计引物,PCR扩增目的微小RNA(microRNAs,miRNAs)靶基因3'非编码区(three-prime untranslated regions,3'UTRs)序列,PCR产物双酶切,后连入经过同样双酶切的pGL3-control载体中,连接产物转化大肠杆菌DH5α,进行阳性克隆鉴定。同样,将候选靶基因3'UTRs突变,突变型3'UTR克隆入pGL3-control报告载体,构建野生型和突变型的报告基因重组质粒。将野生型和突变型的报告基因载体分别和化学合成的microRNA以及内参质粒共转染293TN细胞,进行双荧光素酶检测。结果:成功构建miR-126报告基因野生型和突变型重组质粒pGL3-VEGF-A-3'UTR和pGL3-VEGF-A-3'UTR,质粒测序及酶切结果完全正确。瞬时转染实验显示,过表达miR-126能直接抑制VEGF-A-3'UTRs报告基因活性。结论:miR-126对VEGF-A具有靶向调节功能。     

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.     

Synthesis, characterization, and evaluation as transfection reagents of ampholytic star copolymers: effect of star architecture

Five star polymers based on the positively ionizable hydrophilic 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the hydrophobic but hydrolyzable tetrahydropyranyl methacrylate (THPMA) were prepared by group-transfer polymerization (GTP) using ethylene glycol dimethacrylate (EGDMA) as the coupling agent. In particular, four isomeric star copolymers (one heteroarm, two star block, and the statistical star), all with a 3:1 DMAEMA:THPMA molar ratio, plus one star homopolymer of DMAEMA, with degrees of polymerization of the arms equal to 15, were synthesized. After star polymer preparation and preliminary characterization, the THPMA units were hydrolyzed to negatively ionizable hydrophilic methacrylic acid (MAA) untis, thus yielding star polyampholytes. All the star polyampholytes as well as the commercially available transfection reagent SuperFect were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 bearing the enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was affected by star architecture. The DMAEMA15-star-MAA5 polyampholyte presented the highest transfection efficiency of all the star polymers tested but lower than that of SuperFect at its optimum conditions. All four star copolymers showed decreased toxicity compared to the DMAEMA star homopolymer for the same amounts of star polymer tested and also compared to the SuperFect at its optimum conditions.     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

pH敏脂质体对反义寡核苷酸抗流感病毒活性的影响

为了研究具有临床应用前景的 Ａ Ｓ Ｏ Ｄ Ｎ 脂质体转运系统,以临床药用大豆磷脂为主要原料制备了ｐ Ｈ 敏脂质体,并测定了脂质体体外转染活性、ｐ Ｈ 敏特性、细胞毒性和对 Ａ Ｓ Ｏ Ｄ Ｎ 抗流感病毒活性的影响 结果发现,批号为 ９８０５１９０３,９８０５１１０２ 和 ９８０５１２０２ 的脂质体具有较高转染活性,但只有ｌｉｐｏｆｅｃｔｉｎ 转染活性的 １／５０～１／１００当质粒／脂质体（ Ｗ ／ Ｗ ）为 １∶４～１∶８,转染时间为 ３～５ ｈ,质粒量为 ０５ μｇ,转染后 ２４～４８ ｈ 内检测时转染活性最高 脂质体 ９８０５１２０２ 表现明显 ｐ Ｈ值依赖溶解红细胞膜特性,而脂质体 ９８０５１１０２ 和 ９８０５１９０３ 的 ｐ Ｈ 敏特性不明显 脂质体细胞毒性明显降低,如 ９８０５１９０３、９８０５１１０２ 和 ９８０５１２０２ 的毒性分别是 ｌｉｐｏｆｅｃｔｉｎ 毒性的 １／１６、１／８ 和 １／４ｐ Ｈ 敏脂质体 ９８０５１２０２ 具有促进 Ａ Ｓ Ｏ Ｄ Ｎ 抗流感病毒作用,当 Ａ Ｓ Ｏ Ｄ Ｎ 浓度为 ０２ μｍ ｏｌ／ Ｌ 时,ｐ Ｈ 敏脂质体 ９８０５１２０２ 使其抗病毒活性提高 ５ 倍,但 Ａ Ｓ Ｏ Ｄ Ｎ 浓度较高时ｐ Ｈ 敏脂质体对 Ａ Ｓ Ｏ Ｄ Ｎ抗     

Poly(DMAEMA-NVP)-b-PEG-galactose as gene delivery vector for hepatocytes

A block copolymer composed of cationic polymer and poly(ethylene glycol) (PEG) was used as a DNA carrier. Poly(2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-N-vinyl-2-pyrrolidone (NVP)) having a terminal carboxylic group was synthesized by free radical polymerization using an initiator, 4,4'-azobis(4-cyanovaleric acid). The terminal carboxylic acid was activated by N-hydroxysuccinimide (NHS) with dicyclohexylcarbodiimide (DCC) and then conjugated with PEG-bis(amine). For specific gene targeting to asialoglycoprotein receptor of hepatocytes, a galactose moiety was incorporated into the PEG terminal end of poly(DMAEMA-NVP)-b-PEG by reductive coupling using lactose and sodium cyanoborohydride. RSV luciferase plasmid was used as a reporter gene, and in vitro gene transfection efficiency was measured in HepG2 human hepatocarcinoma cells. Poly(DMAEMA-NVP)-b-PEG-galactose/DNA complexes formed at 0.5-2 polymer/plasmid weight ratio had compacted structures around 200 nm particle size and exhibited slightly negative surface charge. These complexes were coated with a cationic, pH sensitive, endosomolytic peptide, KALA, to generate positively charged poly(DMAEMA-NVP)-b-PEG-galactose/DNA/KALA complex particles. In the presence of serum proteins, both the PEG block and the galactose moiety of poly(DMAEMA-NVP)-b-PEG-galactose greatly enhanced the gene transfection efficiency, which was very close to that of Lipofectamine plus. Irrespective of the presence of serum proteins, as the KALA/DNA weight ratio increased, the transfection efficiency of poly(DMAEMA-NVP)-b-PEG-galactose was enhanced due to the pH dependent endosomal disruptive property of KALA. This study demonstrates that sufficient transfection efficiency as high as that of commercial agent could be attained by judicious formulation of molecular engineered poly(DMAEMA-NVP)-b-PEG-galactose in combination with an endosomolytic peptide, KALA.     

Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery

A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1–2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of > 10–25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations.     

Carbamate-linked cationic lipids for gene delivery

Series of cationic lipids 1a-p, with variable length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, as well as different anion combined with them, were synthesized for liposome-mediated gene delivery. Two plasmid DNAs, pGL3-control and pGFP-N2, were transferred by cationic liposomes formed from the above cationic lipids into five mammalian cell lines, and the transfection efficiency of some of the cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and Sofast.     

活化素抑制大鼠垂体GH_3细胞中人生长激素基因启动子的活性

本研究旨在探讨活化素(activin)对大鼠垂体GH3细胞中人生长激素(hGH)基因启动子活性的影响及其可能的调节机制。采用荧光素酶报告基因方法。首先建立含hGH基因启动子(-484～+30bp)和荧光素酶融合基因的稳定转染GH3细胞株,然后加入活化素或同时加入活化素与相关信号转导途径的激动剂,通过检测细胞培养液和细胞裂解液中GH的含量,以及GH3细胞内荧光素酶的变化,反映活化素对GH分泌、合成和hGH基因启动子活性的影响。将含不同长度hGH基因启动子序列的荧光素酶表达质粒分别转染GH3细胞,观察它们对活化素的反应,寻找活化素影响hGH基因启动子活性的关键DNA序列。结果表明,活化素(5,50nmol/L)能抑制大鼠垂体GH3细胞中GH的分泌和合成,活化素(5,50nmol/L)还能够抑制GH3细胞中hGH基因启动子的活性,使之仅达对照组的77%和69%;在胞内信号转导激动剂中,丝裂原活化蛋白激酶激酶(MAPKK/MEK)特异性激动剂C6ceramide(1μmol/L)完全取消了活化素对hGH基因启动子活性的抑制作用;活化素发挥抑制作用所需要的hGH基因启动子关键序列位于-132～-66bp之间。上述研究表明,活化素能抑制大鼠垂体GH3中hGH基因启动子的活性,它可能是通过抑制细胞内依赖MAPK的信号转导途径来完成的,同时hGH启动子上-132～-66bp的序列在其中发挥重要的作用。     

Nanoscopic cationic methacrylate star homopolymers: synthesis by group transfer polymerization, characterization and evaluation as transfection reagents

Seven star polymers with degrees of polymerization (DPs) of the arms from 10 to 100 and dimensions in the nanometer range were prepared using sequential group transfer polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA, hydrophilic positively ionizable monomer) and ethylene glycol dimethacrylate (hydrophobic neutral cross-linker). The polymers were characterized in tetrahydrofuran by gel permeation chromatography and static light scattering to determine the molecular weights and the weight-average number of arms for each sample. The number of arms of the star polymers varied from 20 to 72. Aqueous solutions of the star polymers were studied by turbidimetry, hydrogen ion titration, and dynamic light scattering to determine their cloud points, pKs, and hydrodynamic diameters. The cloud points of the larger star polymers, with arm DP 30-100, were found to be 29-34 degrees C, almost independent of the DP of the arms. Similarly, the pKs of all star polymers were calculated to range between 6.7 and 7.0, again independent of the arm DP. In contrast, the hydrodynamic diameters of the star polymers strongly depended on the DP of the arms. In particular, by increasing the DP of the arms from 20 to 100, the hydrodynamic diameters in water increased from 7 to 31 nm. All star polymers were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 with the enhanced green fluorescent protein as the reporter gene. Our results showed that as the DP of the arms of the DMAEMA star homopolymers increased from 10 to 100, the overall transfection efficiency decreased, with the star polymer with DP of the arms of 10 emerging as the best transfection reagent. Systematic variation of the amounts of star polymer and plasmid DNA used in the transfections led to an optimization of the performance of this star polymer, yielding overall transfection efficiencies of 15%, comparable to the optimum overall transfection efficiency of the commercially available transfection reagent SuperFect of 13%.     

小鼠Lrp5基因5′端调控区域的功能

为研究小鼠低密度脂蛋白(LDL)受体相关蛋白5(LRP5)基因5′端调控序列的功能,PCR扩增小鼠Lrp5基因翻译起始位点上游3041bp(-2909bp~+132bp)DNA序列.PCR产物定向克隆到pGL3-basic载体上,重组质粒命名为pGL3-2909.以pGL3-2909质粒为模板,以不同的引物扩增出不同长短的DNA片段,分别定向克隆到含小鼠Lrp5基因基本启动子并含有荧光素酶报道基因的pGL3-103载体上,构建了12种荧光素酶报告基因表达体系:pGL3-267,pGL3-513,pGL3-535,pGL3-560,pGL3-575,pGL3-623,pGL3-645,pGL3-719,pGL3-770,pGL3-1032,pGL3-1330,pGL3-1619.以pRL-TK为内参照质粒,瞬时转染COS-7细胞,48h后收集细胞测定荧光素酶相对表达活性,pGL3-575(-2909bp~-2334bp)活性是pGL3-513(-2909bp~-2396bp)的20%,pGL3-535(-2909bp~-2374bp)的活性是pGL3-513的44%,pGL3-575的活性是pGL3-560(-2909bp~-2349bp)的48%,均有显著性差异.结果表明,在-2396bp与-2374bp之间的22bp区域内以及-2349bp与-2334bp之间的15bp区域内存在负调控元件.软件分析表明,此区域含有IK2,LYF1及MZF1调控元件.     

Human Serum Albumin-Based Nanoparticle-Mediated In Vitro Gene Delivery

The genetic treatment of neurodegenerative diseases still remains a challenging task since many approaches fail to deliver the therapeutic material in relevant concentrations into the brain. As viral vectors comprise the risk of immune and inflammatory responses, human serum albumin (HSA) nanoparticles were found to represent a safer and more convenient alternative. Their ability to cross the blood-brain barrier (BBB) and deliver drugs into the brain in order to enhance gene-based therapy has been previously demonstrated. The present study deals with the development of pGL3-PEI-coated HSA nanoparticles and subsequent in vitro testing in cerebellar granular and HeLa cells. The luciferase control vector pGL3 was chosen as reporter plasmid encoding for the firefly luciferase protein, linear polyethylenimine (22 kDa) as endosomolytic agent for enhancing the cells’ transfection. Studies on particle characteristics, their cellular uptake into aforementioned cell lines and on subcellular localisation, and transfection efficiency in the cerebellar cells proved the feasibility of nanoparticle-based gene delivery.     

人Tudor-SN基因启动子荧光素酶报告基因表达质粒的构建及活性检测

目的 将人类Tudor-SN基因的启动子序列片段定向连入pGL3-Basic质粒载体,并进行鉴定和启动子活性检测.方法 以HeLa细胞全基因组DNA为模板,PCR法扩增出目的基因,利用XhoⅠ和HindⅢ双酶切法将目的片段连接到pGL3-Basic载体上.再将构建成功的pGL3-Basic-Tudor-SN-promoter重组质粒和内参质粒β-gal瞬时共转染入宫颈癌HeLa细胞,培养48 h后检测萤火虫荧光素酶活性.结果 双酶切和基因测序法鉴定构建的重组质粒无误,转染重组质粒后可检测到萤火虫荧光素酶活性.结论 成功构建了人类Tudor-SN基因启动子重组质粒,为Tudor-SN蛋白基因调控机制的研究奠定基础.     

人端粒酶催化亚基基因启动子调控的肿瘤细胞特异表达载体

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗 ,克隆并构建了人端粒酶催化亚基 (hTERT)基因启动子调控的萤光素酶报告载体 .用脂质体转染法将其分别转染肿瘤细胞和正常细胞 ,检测其在肿瘤细胞和正常细胞中的转录活性 .hTERT启动子在所检测的 4种端粒酶阳性的肿瘤细胞中具有明显的转录活性 ,平均为阳性对照的 4 4 3% ;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性 .提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调 ,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体     

Transfection using synthetic peptides: comparison of three DNA-compacting peptides and effect of centrifugation

Positively charged peptides have been shown to allow efficient transfection in vitro, especially when mixed with lipids. We have compared the ability of three positively charged peptides both to compact DNA and to increase the transfection efficiency of the cationic lipid DOTAP. The peptides are: a polymer of 17 lysines (pK17), YKAWK8WK (peptide K8) and SPKRSPKRSPKR (peptide P2). Peptides pK17 and K8 compact DNA efficiently in a gel retardation assay and protect DNA efficiently against DNase I degradation. Peptide P2, on the other hand, interacts weakly with DNA and provides poor protection. In order to compare their transfection efficiency, the three peptides were mixed with DNA (plasmid pEGFP-N1) at different charge ratios (+/-) and DOTAP (at a charge ratio of 2). The transfection efficiency was measured by FACS analysis at different times post-transfection. With NIH-3T3 cells, peptide P2 provides the highest transfection efficiency (about 40%), when compared with peptides pK17 (29%) and K8 (31%) and DOTAP alone (21%) under optimal conditions. Finally, we showed that centrifugation of the complexes onto the cells increased the transfection efficiency by a factor 1.5 to 2 with the various cell lines tested (ECV, primary human keratinocyte, CFT-2, NT-1).