Polyethylenimines for in vivo gene delivery

Branched and linear polyethylenimines (PEIs) are proving to be efficient, non-toxic and versatile agents for in vivo gene delivery by a number of routes. A major factor in the successful use of PEIs seems to be the small size of PEI/DNA complexes which can be achieved under controlled conditions of formulation, mainly by using PEIs of low molecular weights. This review considers the in vivo use of PEI, from formulation to delivery and analysis of gene expression. PEI delivery is already used for the analysis of numerous physiological processes. It is hoped that scrutiny of the mechanisms involved with PEI-based gene delivery at different levels of the transfection process and in different in vivo contexts will aid the transition towards its use in therapeutic situations.     

Efficient gene delivery into human dendritic cells by adenovirus polyethylenimine and mannose polyethylenimine transfection

Gene-modified human dendritic cells (DCs) were generated by transfection with adenovirus polyethylenimine DNA (Ad/PEI/DNA) and mannose polyethylenimine DNA (ManPEI/DNA) complexes. Ad/PEI/DNA complexes have plasmid DNA bound to adenovirus particles by PEI and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yield high transduction levels and sustained expression of luciferase and green fluorescent protein reporter genes and were almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor-mediated endocytosis via mannose receptor, which is highly expressed on DCs. While gene delivery by ManPEI/DNA complexes was less efficient than by Ad/PEI transfection, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhanced transduction efficiencies and transgene expression. We also demonstrate that Ad/PEI-transfected DCs are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions, and in activating T cells from T cell receptor (TCR)-transgenic mice in an antigen-specific manner. Thus, the present study establishes the following relative order of transduction efficiencies of viral and nonviral gene delivery systems for primary human DCs: recombinant adenovirus > Ad/PEI = Ad/ManPEI > ManPEI > PEI. Ad/PEI and ManPEI transfection modes represent particularly versatile transduction systems for DCs, with ManPEI being built up exclusively of synthetic compounds.     

Characterization of a novel diolein-based LPDII vector for gene delivery.

LPDII vectors are non-viral vehicles for gene delivery comprised of polycation-condensed plasmid DNA (polyplexes) complexed with anionic pH-sensitive liposomes. Here, we describe a novel LPDII formulation containing polyethylenimine (PEI) polyplexes complexed with anionic pH-sensitive liposomes composed of diolein/cholesteryl hemisuccinate (CHEMS) (6:4 mol/mol). The pH-sensitivity of diolein/CHEMS liposomes was evaluated through quantitative fluorescence measurements of calcein release and particle size analysis. The results indicated that diolein/CHEMS liposomes are stable at physiological pH, but undergo rapid aggregation and fluorescence dequenching at pH values < or =5.0. Using a luciferase reporter gene, in vitro transfection of KB oral cancer cells showed that the transfection efficiency of LPDII vectors was superior to other well-characterized polyplexes and lipoplexes. Results further showed that gene delivery using diolein-containing LPDII vectors was dependent on the PEI nitrogen/DNA phosphate (N/P) ratio, the lipid/DNA weight ratio and the cell line being transfected. Replacing PEI with poly-L-lysine as the DNA condensing agent resulted in only a moderate reduction in transfection activity. Moreover, in contrast to LPDII formulations incorporating dioleoylphosphatidylethanolamine (DOPE), the transfection efficiency of diolein-based LPDII vectors was sustained in media containing up to 50% fetal bovine serum. Since diolein-based LPDII vectors mediate efficient gene transfer and retain their transfection activity in the presence of serum, diolein may be a promising alternative to DOPE for the construction of non-viral vectors for in vivo gene delivery.     

Overcoming the Nuclear Barrier: Cell Cycle Independent Nonviral Gene Transfer with Linear Polyethylenimine or Electroporation

In many cases, nonviral particle-mediated gene delivery is highly dependent on the cell cycle status of transfected cells. Here we compare particle-mediated delivery with linear polyethylenimine (PEI) and physical transfer of DNA by electroporation with branched PEI and lipofection for their ability to transfect cells at different stages of the cell cycle. In contrast to other particle-mediated delivery methods (using Lipofectamine or branched PEI) linear PEI led to only small differences (within 1 log unit) in gene transfer between HeLa cells transfected in G1 and those in S/G2. Parallel transfections (lipofection or branched PEI) resulted in 2 to > 3 log-unit differ-ences in luciferase expression between cells transfected in G1 and S/G2. Gene transfer by elec-troporation also revealed hardly any cell cycle dependence and displayed completely different expression kinetics. Reporter gene expression is already very high 3 hours after electroporation with roughly the same level of reporter gene expression in all cell cycle phases. We suggest that DNA electroporation and DNA transfection with linear PEI particles have improved nuclear import characteristics relative to the other tested DNA delivery systems.     

Galactosylated polyethylenimine-graft-poly(vinyl pyrrolidone) as a hepatocyte-targeting gene carrier.

Polyethylenimine (PEI) has been used for the gene delivery system in vitro and in vivo since it has high transfection efficiency owing to proton buffer capacity. However, the use of PEI for gene delivery is limited due to cytotoxicity, non-specificity and unnecessary interaction with serum components. To overcome cytotoxicity and non-specificity, PEI was coupled with poly(vinyl pyrrolidone) (PVP) as the hydrophilic group to reduce cytotoxicity and lactose bearing galactose group for hepatocyte targeting. The galactosylated-PEI-graft-PVP (GPP) was complexed with DNA, and GPP/DNA complexes were characterized. GPP showed good DNA binding ability, high protection of DNA from nuclease attack. The sizes of DNA complexes show tendency to decrease with an increase of charge ratio and had a minimum value around 59 nm at the charge ratio of 40 for the GPP-1/DNA complex (PVP content: 4.1 mol%). The GPP showed low cytotoxicity. And GPP/DNA complexes were mediated by asialoglycoprotein receptors (ASGP-R)-mediated endocytosis. Also, the transfection efficiency of GPP-1/DNA complex at charge ratio of 40 in the HepG2 was higher than that of PEI/DNA one.     

A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo

Polyethylenimine (PEI), a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. We have defined the optimal conditions for PEI-based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore Cl(-) channel activity in a CF mouse model. Three forms of PEI, a linear 22 kDa (ExGen 500) form and branched 25 or 50 kDa forms were evaluated. All forms of PEI significantly increased luciferase reporter gene expression compared to the liposome DCChol/DOPE in a human bronchial epithelial cell line (16HBE) irrespective of the extent of cell confluency. With subconfluent cells, gene expression was around 1000-, 200- and 25-fold higher than liposomes using linear 22, 25 and 50 kDa PEI, respectively. The transfection efficiency was reduced in confluent and polarized epithelial cells but linear 22 kDa PEI showed the smallest decrease and gave 8000-fold better transfection in polarized cells compared to liposomes. A comparison of linear 22 or 25 kDa PEI with DCChol/DOPE for airway delivery in vivo via intranasal instillation was also performed. Linear 22 kDa PEI gave significantly better luciferase reporter gene expression of 350-fold in the lung, 180-fold in the nose and 85-fold in the trachea compared to liposome. In contrast, the 25 kDa form of PEI was no better than DCChol/DOPE. Repeat dosing with linear 22 kDa PEI failed to give reporter gene delivery comparable to the initial dose. To establish that PEI can be used to deliver a physiologically relevent gene in vivo, we used it to restore Cl(-) secretion by CFTR gene delivery in the airways of a CF mouse model.     

Synergistic effect of poly(ethylenimine) on the transfection efficiency of galactosylated chitosan/DNA complexes.

The use of chitosan for gene delivery is limited due to the low transfection efficiency and difficulty in transfecting into a variety of cell types, especially the hepatoma cells. In order to solve this problem, lactobionic acid (LA) bearing galactose group was coupled with water-soluble chitosan (WSC) for liver specificity and poly(ethylenimine) (PEI) was combined to galactosylated chitosan (GC)/DNA complexes to enhance the transfection efficiency. For initial study, the effect of PEI on the transfection efficiency of WSC/DNA complex was studied in HeLa, A549 and 293 T cells, and bafilomycin A1 was used to ascertain the mechanism of synergistic effect. Transfection efficiency, cytotoxicity, and physicochemical properties of GC/DNA complex combined with PEI were investigated to determine the potential for the hepatocyte-targeting. The combination of PEI with WSC/DNA and GC/DNA complex dramatically increased the luciferase expression 10- to 1000-fold in various cell lines, and the synergistic effect was proved to be induced by proton sponge effect of PEI. The transfection of GC/DNA complex in HepG2 was much higher than that of WSC/DNA even after combination with PEI, and was highly inhibited in the presence of galactose. Cytotoxicity of PEI was much decreased by combination with GC/DNA complex. And PEI was proved to be coated on the surface of GC/DNA complex through the ionic interaction.     

New polyphosphoramidate with a spermidine side chain as a gene carrier.

A new cationic polymer (PPA-SP), polyphosphoramidate bearing spermidine side chain, was prepared as a non-viral vector for gene delivery. PPA-SP was synthesized from poly(1,2-propylene H-phosphonate) by the Atherton-Todd reaction. The weight average molecular weight of PPA-SP was 3.44x10(4) with a number average degree of polymerization of 90, as determined by GPC/LS/RI method. The average net positive charge per polymer chain was 102. PPA-SP was able to condense plasmid DNA efficiently and formed complexes at an N/P ratio (free amino groups in polymer to phosphate groups in DNA) of 2 and above, as determined by agarose gel electrophoresis. This new gene carrier offered significant protection to DNA against nuclease degradation at N/P ratios above 2, and showed lower cytotoxicity than PLL and PEI in cell culture. The LD(50) of PPA-SP was 85 microg/ml in COS-7 cells, in contrast to 20 and 42 microg/ml for PLL and PEI, respectively. The complexes prepared in saline at N/P ratios of 5 approximately 10 had an average size of 250 nm and zeta-potential of 26 mV. PPA-SP mediated efficient gene transfection in a number of cell lines, and the transfection protocol was optimized in HEK293 cells using a luciferase plasmid as a marker gene. Gene expression mediated by PPA-SP was greatly enhanced when chloroquine was used in conjunction at a concentration of 100 microM. Under the optimized condition, PPA-SP/DNA complexes yield a luciferase expression level closed to PEI/DNA complexes or Transfast mediated transfection. In a non-invasive CNS gene delivery model, PPA-SP/DNA complexes yielded comparable bcl-2 expression as PEI/DNA complexes in mouse brain stem following injection of the complexes in the tongue.     

Evaluation of transportan 10 in PEI mediated plasmid delivery assay.

Cell-penetrating peptides (CPPs) are novel high-capacity delivery vectors for different bioactive cargoes. We have evaluated the CPP transportan 10 (TP10) as a delivery vector in different in vitro plasmid delivery assays. Tested methods include: TP10 crosslinked to a plasmid via a peptide nucleic acid (PNA) oligomer, TP10 conjugation with polyethyleneimine (PEI), and addition of unconjugated TP10 to standard PEI transfection assay. We found that without additional DNA condensing agents, TP10 has poor transfection abilities. However, the presence of TP10 increases the transfection efficiency several folds compared to PEI alone. At as low concentrations as 0.6 nM, TP10-PNA constructs were found to enhance plasmid delivery up to 3.7-fold in Neuro-2a cells. Interestingly, the transfection efficiency was most significant at low PEI concentrations, allowing reduced PEI concentration without loss of gene delivery. No increase in cytotoxicity due to TP10 was observed and the uptake mechanism was determined to be endocytosis, as previously reported for PEI mediated transfection. In conclusion, TP10 can enhance PEI mediated transfection at relatively low concentrations and may help to develop future gene delivery systems with reduced toxicity.     

Cationic microparticles consisting of poly(lactide-co-glycolide) and polyethylenimine as carriers systems for parental DNA vaccination.

Cationic microparticles for DNA adsorption were formulated by blending poly(lactide-co-glycolide) (PLGA) (50:50), with different cationic agents, either PEI 25 kDa (polyethylenimine) or CTAB (cetyl-trimethyl-ammonium-bromide). The aim was to create adjuvant delivery systems increasing the efficiency of DNA vaccines. Microparticles formulated with 10% PEI exhibited a highly positive zeta-potential, small particle sizes, in contrast to particles prepared with CTAB, which revealed highly aggregated structures in scanning electron micrographs. PEI 10% microparticles efficiently adsorbed DNA and protected DNA from enzymatic degradation. Microparticles with up to 10% PEI did not affect membrane integrity whereas CTAB particles showed higher LDH release. Transfection efficiencies were assessed using a luciferase reporter gene assay compared to naked DNA and PEI/DNA polyplexes. DNA adsorbed onto microspheres with 10% or 50% PEI generally had higher transfection efficiencies than CTAB but reached lower expression levels than PEI/DNA polyplexes alone. This documented the intact release of DNA. The mechanism of gene delivery to non-phagocytic cells was studied via covalent fluorescence labeling of both the DNA and PEI by confocal microscopy and suggested uptake of DNA. Immunization of mice was performed using plasmids encoding immunodominant antigens of Listeria monocytogenes adsorbed onto RG 502 H+PEI 10% microparticles. The efficiency was tested by intravenous challenge with an otherwise lethal dose of L. monocytogenes. PLGA+PEI microspheres can be used as adjuvant delivery systems for DNA but further optimization is necessary to exploit their full potential.     

Galactosylated chitosan-graft-polyethylenimine as a gene carrier for hepatocyte targeting

Chitosans have been proposed as alternative, biocompatible cationic polymers for nonviral gene delivery. However, the low transfection efficiency and low specificity of chitosan need to be addressed before clinical application. We prepared galactosylated chitosan-graft-polyethylenimine (GC-g-PEI) copolymer by an imine reaction between periodate-oxidized GC and low-molecular-weight PEI. The molecular weight and composition were characterized using gel permeation chromatography column with multi-angle laser scattering and (1)H nuclear magnetic resonance, respectively. The copolymer was complexed with plasmid DNA in various copolymer/DNA (N/P) charge ratios, and the complexes were characterized. GC-g-PEI showed good DNA-binding ability and superior protection of DNA from nuclease attack and had low cytotoxicity compared to PEI 25K. GC-g-PEI/DNA complexes showed higher transfection efficiency than PEI 25K in both HepG2 and HeLa cell lines. Transfection efficiency into HepG2, which has asialoglycoprotein receptors, was higher than that into HeLa, which does not. GC-g-PEI/DNA complexes also transfected liver cells in vivo after intraperitoneal (i.p.) administration more effectively than PEI 25K. These results suggest that GC-g-PEI can be used in gene therapy to improve transfection efficiency and hepatocyte specificity in vitro and in vivo.     

超声靶向微泡破坏联合聚乙烯亚胺增强裸鼠移植瘤基因输送的初步研究

目的 探讨超声靶向微泡破坏(UTMD)联合聚乙烯亚胺(PEI)增强裸鼠Hela细胞(人宫颈癌)移植瘤基因输送的可行性和应用价值.方法 分别将2种质粒DNA[红色荧光蛋白质粒(RFP)和荧光素酶质粒(pCMV-LUC)]与PEI以不同氮/磷酸盐比(N/P比)混合,利用凝胶阻滞实验对PEI/DNA复合物进行分析.经荷瘤裸鼠尾静脉分别注入PBS、质粒、质粒+Sono Vue微泡、PEI/DNA复合物+Sono Vue微泡,仅对一侧肿瘤行超声辐照(3 MHz、2 W/cm2、2 min、20%占空比),另一侧肿瘤作为对照,并对该转染方法 的靶向性进行分析.超声辐照3 d后处死动物,行RFP表达观察、荧光素酶活性检测和组织学检查.结果 琼脂糖凝胶电泳显示PEI可有效地缩合质粒DNA.与裸质粒组比较,UTMD(超声辐照+Sono Vue微泡)能明显提高RFP转染率.与非辐照对照侧比较,UTMD的运用使RFP表达明显增强,荧光素酶活性增加了14倍(P＜0.01).UTMD联合PEI可显著增强基因转染,受辐照移植瘤的荧光素酶活性增加了10倍(P＜0.01);与非联合PEI时比较,荧光素酶表达增加了111倍(P＜0.01).无论有否超声照射,裸鼠其他器官组织中均无明显的基因表达(P＞0.05),且未观察到明显的组织损伤.结论 UTMD联合PEI可显著增强报告基因在肿瘤组织的靶向传输和转染,是一种很有前景、新型而安全的体内基因输送方法.     

PEGylated polyethylenimine for in vivo local gene delivery based on lipiodolized emulsion system.

Polyethylenimine (PEI) is one of the most efficient vectors for non-viral gene delivery, whereas its poor transfection activity, compared to viral vectors, and cytotoxicity need to be improved for in vivo applications. In this study, we prepared two PEI conjugates with 6 and 10 wt.% of poly(ethylene glycol) (PEG) grafts (referred to PEI-PEG-6 and PEI-PEG-10, respectively) in order to investigate the effects of PEGylation on cytotoxicity and transfection activity in vitro. In addition, their suitability as vectors for local gene delivery in vivo was assessed by injecting lipiodolized emulsions containing polymer/DNA complexes into the femoral artery of Sprague-Dawley (SD) rats, occluded by a surgical suture to block inflow of the blood to the leg. Both PEGylated PEIs showed significantly lower cytotoxicity and higher transfection activity in COS-1 cells than PEI taken as a control; in particular, PEI-PEG-10 produced the most promising results. The stable water-in-oil emulsion, composed of aqueous domains containing the complexes and lipiodol as an oil phase, was formed in the presence of a hydrogenated castor oil. From in vivo experiments, it was found that all the complexes, dispersed in the lipiodolized emulsion, delivered effectively gene to muscle, surrounding the injection site, rather than other organs such as liver, spleen, kidney, heart and lung. The in vivo transfection activity of PEI-PEG-10 was 3-folds higher in muscle than that of PEI. Based on these results, it can be concluded that PEGylated PEIs (based on the lipiodolized emulsion system) hold a promising potential for local gene delivery in vivo.     

Chitosan-graft-polyethylenimine as a gene carrier.

Chitosans have been proposed as biocompatible alternative cationic polymers that are suitable for non-viral delivery. However, the transfection efficiency of chitosan-DNA nanoparticles is still very low. To improve transfection efficiency, we prepared chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer by an imine reaction between periodate-oxidized chitosan and polyethylenimine (PEI). The molecular weight and composition of the CHI-g-PEI copolymer were characterized, using multi-angle laser scattering (GPC-MALS) and (1)H nuclear magnetic resonance ((1)H NMR), respectively. The copolymer was complexed with plasmid DNA (pDNA) in various copolymer/DNA (N/P) charge ratios, and the complex was characterized. CHI-g-PEI showed good DNA binding ability and high protection of DNA from nuclease attack. Also, with an increase in charge ratio, the sizes of the CHI-g-PEI/DNA complex showed a tendency to decrease, whereas the zeta potential of the complex showed an increase. The CHI-g-PEI copolymer had low cytotoxicity, compared to PEI 25K from cytotoxicity assays. At high N/P ratios, the CHI-g-PEI/DNA complex showed higher transfection efficiency than PEI 25K in HeLa, 293T and HepG2 cell lines. Our results indicate that the CHI-g-PEI copolymer has potential as a gene carrier in vitro.     

超声联合聚乙烯亚胺增强MCF-7癌细胞基因表达的转染参数优化

目的 探讨超声辐照并超声造影剂联合聚乙烯亚胺(PEI)增强MCF-7乳腺癌细胞质粒DNA转染的最优条件及协同作用.方法 制备PEI/荧光素酶质粒(pCMV-luciferase-GL3)复合物,用于MCF-7癌细胞基因转染,超声辐照前添加超声造影剂SonoVue.通过荧光素酶活性和细胞存活率的测定,对超声辐照参数进行优化,对质粒浓度、孵育时间、血清、溶媒类型、培养基体积等因素进行分析.结果 细胞活力和荧光素酶活性均受超声强度、辐照时间和占空比的影响,适当条件的超声辐照可促进PEI/DNA复合物渗透入胞内,从而提高质粒DNA的转染率.最优超声辐照条件为1 w/cm2,10%占空比,辐照3 min.超声辐照并超声造影剂联合PEI的转染效率显著高于单纯超声辐照和PEI转染(P<0.01).在超声辐照前将细胞与PEI/DNA复合物共孵育2h时,荧光素酶活性显著增强(P<0.01).此外,血清、培养基体积和溶媒类型也对转染效率有影响.结论 优化的超声和转染参数能显著提高MCF-7癌细胞的基因表达效率.超声辐照并超声造影剂联合PEI对DNA转染效率有协同作用,是一种增强质粒DNA基因表达简单而有应用前景的方法.     

Efficient gene delivery via non-covalent complexes of folic acid and polyethylenimine.

Polyethylenimine (PEI) is a cationic polymer capable of delivering DNA molecules into cultured mammalian cells as charge complexes. The application of PEI polyplexes in gene therapy, however, is hampered by the sensitivity of its transfection activity to the presence of serum. We found that folic acid, in a variety of cell lines, significantly enhanced PEI-mediated transfection activity in the presence of serum, whether the folic acid was added during or after PEI/DNA polyplex formation. The increase in activity could not be produced with other anionic compounds such as cholic acid, citric acid, EDTA, or glutamic acid. This novel formulation provides a reliable, low-cost, and highly efficient method for delivery of genes and may have applications in gene therapy.     

Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI.

Gene therapy aimed at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. Polyethylenimine (PEI) has been utilized for gene delivery to the airways. In this study, we describe a new modification of PEI, in which an oligopeptide related to the protein transduction domain of HIV-1 TAT was covalently coupled to 25 kDa PEI (PEI) through a heterobifunctional polyethylenglycol (PEG) spacer resulting in a TAT-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection was observed for small (approximately 90 nm) polyplexes as well as significantly improved stability against polyanions, Alveofact, bronchial alveolar lining fluid and DNase. To determine polyplex toxicity in vitro, MTT assays were performed and, for in vivo testing, the mice bronchial alveolar lavage was investigated for total cell counts, quantity of neutrophils, total protein and TNF-alpha concentration. All parameters suggest significantly lower toxicity for TAT-PEG-PEI. Transfection efficiencies of both PEI and TAT-PEG-PEI polyplexes with DNA were studied under in vitro conditions (A549) and in mice after intratracheal instillation. While luciferase expression in A549 cells was much lower for TAT-PEG-PEI (0.2 ng/mg protein) than for PEI (2 ng/mg), significantly higher transfection efficiencies for TAT-PEG-PEI were detected in mice. Reporter gene expression was distributed through bronchial and alveolar tissue. Thus, TAT-PEG-PEI represents a new approach to non-viral gene carriers for lung therapy, comprising protection for plasmid DNA, low toxicity and significantly enhanced transfection efficiency under in vivo conditions.     

Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery

The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.     

Human serum albumin-polyethylenimine nanoparticles for gene delivery.

Nanoparticles consisting of DNA, human serum albumin (HSA) and polyethylenimine (PEI) were formed and tested for transfection efficiency in vitro with the aim of generating a nonviral gene delivery vehicle. HSA-PEI-DNA nanoparticles containing the pGL3 vector coding for luciferase as reporter gene were formed by charge neutralization. The particles were characterized by gel retardation assay, dynamic light scattering (size) and electrophoretic mobility measurements (charge). Stability was determined by spectrophotometric analysis and transfection efficiency was evaluated in cell culture using human embryonic epithelial kidney 293 cells. HSA-PEI-DNA nanoparticles were prepared by co-encapsulation of PEI as a lysosomotropic agent at varying nitrogen to phosphate (N/P) ratios. An optimum transfection efficiency was achieved when the particles were prepared at N/P ratios between 4.8 and 8.4. Furthermore, they displayed a low cytotoxicity when tested in cell culture. Our results show that HSA-PEI-DNA nanoparticles are a versatile carrier for DNA that may be suitable for i.v. administration.     

Nonviral gene transfer into fetal mouse livers (a comparison between the cationic polymer PEI and naked DNA)

We investigated the efficacy and safety of the cationic polymer polyethylenimine (PEI) as a potential tool for intrauterine gene delivery into livers of fetal mice in the last trimester of pregnancy (E17.5). Using luciferase as a reporter gene, transferrin-conjugated and ligand-free PEI/DNA complexes (containing 3 microg DNA) with varying PEI-nitrogen/DNA-phosphate (N/P) ratios and different PEI forms, branched (800, 25 kDa) and linear (22 kDa), were compared with naked DNA. Transgene expression was measured 48 h after administration of PEI/DNA complexes or naked DNA. Highest luciferase activity (9.8 x 10(3) relative light units (RLU)/mg of tissue protein) was observed with ligand-free PEI22/DNA mixtures at N/P 6.0. In addition, this formulation was associated with very low toxicity as compared to the other PEI/DNA-injected groups. Using beta-galactosidase as a reporter gene, transfection of single, but also small, clusters of cells was demonstrated throughout the liver. Injection of 3 microg naked DNA resulted in an 11-fold lower transgene expression value (0.9 x 10(3) RLU/mg of tissue protein) as compared to PEI22/DNA complexes. However, the administration of higher concentrated naked DNA (9 microg) into fetal livers yielded expression levels of 3.2 x 10(4) RLU/mg of tissue protein, a more than three-fold increase compared to PEI22/DNA complexes. Furthermore, the gene transfer efficacy of concentrated naked DNA was approximately 40 times higher in fetuses than in adults (0.8 x 10(3) RLU/mg of tissue protein), indicating that fetal tissue is especially amenable to the uptake and expression of naked DNA.