新型阳离子基因传递载体PEI-PBLG的细胞转染研究

对新型阳离子聚合物PEI(10kD)-PBLG进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。通过粒径分析及扫描电镜(SEM)观察PEI(10kD)-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径,预测其进入细胞的可能性。使用MTT比色法分析PEI(10kD)-PBLG、PEI(25kD)-PBLG、PEI(10kD)和PEI(25kD)的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型,将其与PEI(10kD)-PBLG自组装后,分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304,应用流式细胞术检测细胞转染效率,并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI(10kD)-PBLG可包裹质粒形成粒径100~120nm的纳米复合物,适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低,并能够在10%血清存在的条件下,转染全部实验用细胞株,尤其对Hela的转染效率最高,其次是COS-7、Vero-E6和ECV304;其中PEI-PBLG(10kD)/pEGFP复合物转染Hela细胞的比率为45.02%,高于PEI(10kD)/pEGFP的29.16%;PEI(10kD)-PBLG的细胞毒性作用显著低于PEI(25kD)、PEI(10kD)和PEI(25kD)-PBLG。新型阳离子多聚物PEI(10kD)-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性,提高了生物相容性,有望成为基因转移的有效载体。     

Self-assembled terplexes for targeted gene delivery with improved transfection

To improve transfection efficiency and to incorporate target ligands to the gene delivery systems, heparin and heparin-biotin were introduced to complexes of polyamidoamine dendrimer and DNA (PAMAM/DNA) via electrostatic interactions to form self-assembled PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes, respectively. The self-assembled terplexes were characterized by agarose gel electrophoresis and particle size analysis. The MTT assay indicated that, after incorporation of heparin and heparin-biotin, the terplexes exhibited decreased cytotoxicity. In addition, as compared with PAMAM/DNA and PAMAM/DNA/heparin complexes, the PAMAM/DNA/heparin-biotin complexes exhibited much higher cellular uptake into HeLa cells due to the specific interactions between biotin and biotin receptors on HeLa cells, which led to the enhanced transfection activity. The PAMAM/DNA/heparin-biotin complexes would be a promising targeting gene delivery system.     

PEI-Et输送特异性shRNA对大鼠肝细胞AGT基因表达的影响

目的:研究交联小分子量聚乙烯亚胺衍生物PEI-Et对大鼠肝细胞(BRL-3A)的细胞毒性、转染效率和携带高血压相关基因血管紧张素原(AGT)短发卡RNA(shRNA)沉默AGT表达的能力。方法:MTT法检测PEI-Et/shRNA复合物对BRL-3A细胞的毒性,流式细胞术检测PEI-Et/shRNA复合物对BRL-3A细胞的转染效率,RT-PCR和Western blot检测PEI-Et/shRNA对AGT的基因沉默效果。结果:在相同质量比(w/w)时PEI-Et/shRNA的细胞毒性小于PEI 25kDa/shRNA(P0.01),PEI-Et/shRNA在w/w为30时达到最高转染效率,高于PEI 25 kDa(P0.01),PEI-Et/shRNA能高效沉默BRL-3A细胞中AGT基因的表达。结论:PEI-Et在BRL-3A细胞中是一种低细胞毒性、高转染效率的非病毒基因载体(与商业化的PEI 25kDa比较),能携带AGT shRNA高效沉默BRL-3A细胞中AGT基因的表达,通过用PEI-Et/AGT shRNA来抑制AGT的表达将为高血压的基因治疗提供一种新的思路。     

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.     

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.     

Cardiac Usage of Reducible Poly(oligo-D-arginine) As a Gene Carrier for Vascular Endothelial Growth Factor Expression

Developments of non-viral carriers have headed toward reducing cytotoxicity, which results from the use of conventional gene carriers, and enhancing gene delivery efficiency. Cys-(d-R9)-Cys repeated reducible poly(oligo-D-arginine) (rPOA) is one of the most efficient non-viral carriers for gene therapy; however, while its efficiency has been verified in the lung and brain, it is necessary to confirm its activity in each organ or tissue since there are differences of gene carrier susceptibility to among tissue types. We therefore tested the compatibility of rPOA in cardiac tissue by in vitro or in vivo experiments and confirmed its high transfection efficiency and low cytotoxicity. Moreover, substantial regenerative effects were observed following transfection with rPOA/pVEGF expression vector complexes (79% decreased infarct size) compared to polyethyleneimine (PEI) (34% decreased infarct size) in a rat myocardial infarction (MI) model. These findings suggest that rPOA efficiently enables DNA transfection in cardiac tissue and can be used as a useful non-viral therapeutic gene carrier for gene therapy in ischemic heart disease.     

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.     

PEG修饰的聚阳离子基因载体PEG-Polycarbam-SP在COS-7细胞中转染和毒性的研究

目的：寻找一种转染效率高,细胞毒性低的非病毒基因载体,研究以人体内源性精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以聚乙二醇（PEG）作为亲水基团连接剂合成亲水修饰聚阳离子载体PEG-Polycarbam-SP的基因担载效率,以及对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：琼脂糖凝胶电泳方法考察复合物的基因担载效率,检测基因复合物的粒径和电位,以荧光素酶质粒为报告基因,研究PEG-Polycarbam-SP/DNA的复合物在COS-7细胞的转染活性,用MTT方法研究PEG-Polycarbam-SP对COS-7细胞的毒性。结果：聚合物与质粒在质量比5以后形成的复合物粒径稳定在50nm左右,Zate电位在20mV左右。COS-7细胞实验显示PEG-Polycarbam-SP具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：PEG-Polycarbam-SP是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

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.     

Novel hyperbranched dendron for gene transfer in vitro and in vivo

Novel hyperbranched dendron (HD) polymers were synthesized using a low molecular weight poly(ethyleneimine) core (BPEI). Using successive attachment of ethyleneimine moieties to the PEI core, the relative ratio of linear-to-branched structures was lowered from 1.17 to 0.70. We found that the more extensive branching of PEI enables the condensation of plasmid DNA into nanostructures with a size of 70-100 nm. The obtained complexes were stable at least for 3 weeks at 4 degrees C. The HD-DNA complexes prepared using secondary and tertiary amine-containing dendrons exerted a very low cytotoxicity in vitro during a coincubation with cells for 48 h. Using firefly luciferase as a marker of protein expression, we established that HD complexes were efficient in transfecting cells in the presence of serum. Under optimized conditions, the transfection activity at a nitrogen-to-phosphate (N/P) ratio of 6 was approximately six times higher than that of the commercially available polycationic transfection reagent. Bioluminescent imaging of in vivo gene expression using a luciferase reporter gene showed the increase of the signal in the liver and in submandibular lymph nodes in live mice. Our preliminary in vivo gene expression data demonstrates the potential of HD polymers as in vivo transfection agents that could be potentially useful for lymph node gene delivery.     

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationiccharge potential.High transfection efficiency of PEI,along with its cytotoxicity,strongly depends on itsmolecular weight.To enhance its gene delivery efficiency and minimize cytotoxicity,we have synthesizedsmall cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro.Inthis study,branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate[1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h.The efficiencies of thecross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein(EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines.Flow cytometrywas used to quantify the cellular entry efficiency of plasmid and the transgene expression level.Thecytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay.EGDMA-PEI 800-4h,atypical cross-linked PEI reported here,mediated a more efficient expression of reporter gene than thecommercially available 25-kDa branched PEI control,and resulted in a 9-fold increase in gene deliveryin B16F10 cells and a 16-fold increase in 293T cells,while no cytotoxicity was found at the optimizedcondition for gene delivery.Furthermore,the transfection activity of polyplexes was preserved in thepresence of serum proteins.     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.     

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.     

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells

AIM: It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. METHODS: Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. RESULTS: Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. CONCLUSIONS: Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.     

Self-assemble gene delivery system for molecular targeting using nucleic acid aptamer

We have developed a novel vector constructed with pDNA, polyethylenimine (PEI), and mucin 1 (MUC1) aptamer for tumor-targeted gene delivery. The MUC1 aptamer and non-specific aptamer were employed to coat the pDNA/PEI complexes electrostatically and stable nanoparticles were formed. The addition of a non-specific aptamer to the pDNA/PEI complex decreased gene expression in the human lung cancer cell line, A549 cells expressing MUC1 regularly. At the same time, the pDNA/PEI/MUC1 aptamer complex showed higher gene expression than pDNA/PEI/non-specific aptamer complex. Furthermore, the pDNA/PEI/MUC1 aptamer complex showed markedly high gene expression in tumor-bearing mice; thus, pDNA/PEI/MUC1 aptamer complexes are useful as a tumor-targeted gene delivery system with high transfection efficiency.     

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.     

Polyethylenimine‐mediated gene delivery into human bone marrow mesenchymal stem cells from patients

Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post‐infarction left ventricular (LV) dysfunction. However, age‐related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non‐viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI‐mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 μg DNA/cm². The average transfection efficiency for all tested samples, middle‐age group (<65 years), old‐age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI‐mediated therapeutic gene VEGF transfer could significantly enhance the expression level.     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Synergistic effect of polyethylenimine and cationic liposomes in nucleic acid delivery to human cancer cells

Polyethylenimine (PEI) and other polycations are good vehicles for transferring genes into the cells. In earlier reports, poly-L-lysine and protamine have been shown to improve gene delivery with cationic liposomes. In this study, PEI, combined with different cationic liposomes, was studied to determine the optimal conditions for gene delivery. The reporter genes, luciferase and green fluorescent protein, were used to transfect human HeLa, HepG2 and hepatoma 2.2.15 cells with various combinations of PEIs (0.8 and 25 kDa), poly-L-lysine (15-30 kDa), protamine and cationic liposomes. The highest expression level was achieved by using the combination of PEI 25 kDa (0.65 microg/microg of DNA, nitrogen-to-DNA phosphate (N/P) ratio=4.5) with 10 nmol of DOTAP-cholesterol (DOTAP-Chol, 1:1 w/w). This DNA complex formulation dramatically increased the luciferase expression 10- to 100-fold, which was much higher than those of other polycations alone, cationic liposomes alone or the combination. In addition, PEI/DOTAP-Chol combination had little cytotoxicity than DOTAP-Chol or other cationic liposomes alone. The effect of oligonucleotide (ODN) delivery facilitated by PEI and cationic liposomes was also studied in the hepatoma cell lines. We demonstrated an antisense ODN of p53 delivered by PEI/DOTAP-Chol combination effectively inhibited the biosynthesis of p53 protein in HepG2 (68% inhibiton) and 2.2.15 cells (43% inhibition). Thus, the large PEI could synergistically increase the transfection efficiency when combined with the cationic liposomes.