Transient gene expression in mammalian cells grown in serum-free suspension culture

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Triple interpolyelectrolyte complexes DNA-polycation-polyanion: a new approach to optimization of mixtures for transfection in eukaryotic cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M 30-40 kDa as polycation and polyacrylic acid (PA) with M 20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA-PEG incorporation into a ternary complex (by itself or as a 1:1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA-PEG (as a 1:9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a beta-galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1:1 mixture of PA and PA-PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.     

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25 kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.     

瞬时基因表达可溶性的VEGFR2: I-IV

通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。     

瞬时基因表达可溶性的VEGFR2: I-IV

通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。     

Ternary Interpolyelectrolyte Complexes DNA–Polycation–Polyanion: A New Approach to Optimization of Mixtures for Transfection of Eukaryotic Cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M30–40 kDa as polycation and polyacrylic acid (PA) with M20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA–PEG incorporation into a ternary complex (by itself or as a 1 : 1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA–PEG (as a 1 : 9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a -galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1 : 1 mixture of PA and PA–PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.     

Cyclodextrin-polyethylenimine conjugates for targeted in vitro gene delivery

Many human gene therapies will require cell-specific targeting. Though recombinant viruses are much more efficient than nonviral vectors, the latter, especially polymers, have the advantage of being targetable via conjugation of cell-specific ligands, including sugars, peptides, and antibodies, which can be covalently attached to the polymer using a variety of chemistries. Cyclodextrin, which forms inclusion complexes with small hydrophobic molecules, has been incorporated into a gene-delivery polymer and may provide a facile and versatile attachment site for targeting ligands. Polyethylenimine (PEI) was derivatized with beta-cyclodextrin on approximately 10% of the polymer's amines (termed CD-PEI). Human insulin was also derivatized with a hydrophobic palmitate group (pal-HI), which could anchor the protein to CD-PEI/DNA polyplexes. CD-PEI was essentially nontoxic to HEK293 cells at concentrations optimal for gene delivery and mediated nearly 4-fold higher gene expression than unmodified PEI, which is relatively toxic to these cells. More importantly, addition of the pal-HI to CD-PEI enhanced gene expression by more than an order of magnitude compared to unmodified PEI, either with or without the pal-HI. Because of the relative ease with which CD-binding moieties may be attached to various types of ligands, CD-PEI may be a generally useful material for testing novel cell-specific targeting compounds.     

Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

Quaternary complexes composed of plasmid DNA/protamine/fish sperm DNA/stearic acid grafted chitosan oligosaccharide micelles for gene delivery

Quaternary complexes with condensed core of plasmid DNA, protamine, fish sperm DNA and shell of stearic acid grafted chitosan oligosaccharide (CSO-SA), were prepared. The CSO-SA could self-assemble to form nano-sized micelles in aqueous solution and demonstrated excellent internalization ability of tumor cells. Dynamic light scattering (DLS) measurement and transmission electrostatic microscope (TEM) images showed that quaternary complexes had spherical shape with about 25 nm number average diameter, and the size of quaternary complexes was smaller than that of CSO-SA micelles and CSO-SA micelles/plasmid DNA binary complexes. The transfection efficiencies of quaternary complexes on HEK293 and MCF-7 cells increased with incubation time, and were significantly higher than that of CSO-SA micelles/plasmid DNA binary complexes. The optimal transfection efficiency of quaternary complexes on HEK293 and MCF-7 cells measured by flow cytometer after 96 h was 23.82% and 41.43%, respectively. Whereas, the transfection efficiency of Lipofectamine? 2000 on HEK293 and MCF-7 cells after 96 h was 32.45% and 33.23%, respectively. The data of luciferease activity measurement showed that the optimal ratio of plasmid DNA:fish sperm DNA:protamine:CSO-SA was 1:1:5:5. The results indicated that the present quaternary complexes were potential non-viral gene delivery system.     

Pegylated polyethylenimine-Fab' antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells

Specific targeting of ovarian carcinoma cells using pegylated polyethylenimine (PEG-PEI) conjugated to the antigen binding fragment (Fab') of the OV-TL16 antibody, which is directed to the OA3 surface antigen, was the objective of this study. OA3 is expressed by a majority of human ovarian carcinoma cell lines. To demonstrate the ability of the PEG-PEI-Fab' to efficiently complex DNA, an ethidium bromide exclusion assay was performed. Comparison with PEG-PEI or PEI 25 kDa showed only minor differences in the ability to condense DNA. Since conjugation of Fab' to PEG-PEI might influence complex stability, this issue was addressed by incubating the complexes with increasing amounts of heparin. This assay revealed stability similar to that of unmodified PEG-PEI/DNA or PEI 25 kDa/DNA complexes. Complexes displayed a size of approximately 150 nm with a zeta potential close to neutral. The latter property is of particular interest for potential in vivo use, since a neutral surface charge reduces nonspecific interactions. Binding studies using flow cytometry and fluorescently labeled DNA revealed a more than 6-fold higher degree of binding of PEG-PEI-Fab'/DNA complexes to epitope-expressing cell lines compared to unmodified PEG-PEI/DNA complexes. In OA3-expressing OVCAR-3 cells, luciferase reporter gene expression was elevated up to 80-fold compared to PEG-PEI and was even higher than that of PEI 25 kDa. The advantage of this system is its specificity, which was demonstrated by competition experiments with free Fab' in the cell culture media during transfection experiments and by using OA3-negative cells. In the latter case, only a low level of reporter gene expression could be achieved with PEG-PEI-Fab'.     

Opposing roles of syndecan-1 and syndecan-2 in polyethyleneimine-mediated gene delivery

Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI.DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1.polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2.polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection.     

Poly(ethyleneimine)-mediated large-scale transient gene expression: influence of molecular weight, polydispersity and N-propionyl groups

Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI "Max") regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI "Max" binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-M(n) PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells.     

1,4-Butanediol diglycidyl ether (BDE)-crosslinked PEI-g-imidazole nanoparticles as nucleic acid-carriers in vitro and in vivo

Previously reported 1,4-butanediol diglycidyl ether (BDE) crosslinked PEI (branched polyethylenimine, 25 k) nanoparticles (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) (PN NPs) were reacted with varying proportions of a novel linker, 2-(N-1-tritylimidazol-4-yl)-N-(6-glycidyloxyhexyl)-acetamide (IGA linker, 3), to yield PN-g-imidazolyl nanoparticles (PNIm) with improved transfection efficiency. Here, the IGA linker (3) reacted through an epoxy ring to partially convert the residual 1° and 2° amines present in PN NPs to 2° and 3°, respectively, without altering the total number of amines and additionally incorporating the delocalized positive charge of the imidazolyl moiety. The resulting particles were characterized for their size, zeta potential and DNA complexing ability. PNIm/DNA nanoplexes, in the size range of 120-400 nm, were evaluated for transfection efficiency in HeLa, HEK293 and CHO cell lines, which was found to be ～11, ～2-3 and ～2-17 folds higher than PEI, PN-2 (the best working sample of the PN series) (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) and commercial transfection reagents tested in this study, respectively. Also, flow cytometric analysis showed ～78% (ca.～43% in PN-2) cells transfected with the PNIm 10(6)/DNA complex (the best working sample of the PNIm series) in HEK293 cells. Transfection of GFP specific siRNA in HEK293 cells suppressed the gene expression by ～90% (ca.～70% in PN-2). All the cell lines treated with PNIm/DNA nanoplexes showed >90% viability. In vivo gene expression of luciferase enzyme in Balb/c mice showed highest expression in spleen after seven days.     

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.     

Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights, such as PEI25K (m.w. 25 kd) and PEI2K (m.w. 2 kd). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.