Poly(lactic-co-glycolic acid) nanosphere as a vehicle for gene delivery to human cord blood-derived mesenchymal stem cells: comparison with polyethylenimine

Polyethylenimine (PEI) is one of the most extensively studied non-viral vectors but its cytotoxicity limits its clinical value. PLGA nanospheres are biocompatible and can facilitate sustained release of plasmid DNA. This study compares the cytotoxicity and long-term transgene expression between PLGA nanosphere and PEI. PLGA nanospheres were significantly less cytotoxic than PEI at various concentrations. PLGA nanospheres induced significantly higher transgene expression in vitro for a longer duration (21 days) than PEI. We conclude that PLGA nanospheres have potential as gene delivery vehicles for use in gene therapy for diseases in which a long-term therapeutic gene expression regimen is necessary.     

Utility of Polycation-Treated Filters for the Assay of Receptors for VIP

Abstract

This study examined the utility of four polycationic agents for treating glass fibre filters used in the receptor binding assay for vasoactive intestinal peptide (VIP). Polyethylenimine (PEI), polybrene, protamine and methylated bovine serum albumin proved satisfactory in terms of low filter binding of free radioligand and retention of membrane-bound radioligand. Their performance was superior or comparable to untreated Millipore EGWP cellulose acetate filters which we had previously utilized but which are no longer manufactured. The results with polycations indicate the importance of ionic interactions between filter, biological membranes and radioligand in determining the performance of a filtration assay for radioligand-receptor binding. At a practical level, PEI has the disadvantage of potential toxicity. The satisfactory performance of the other polycations indicates that they provide safer alternatives to PEI for filtration assay of the VIP receptor and possibly receptors for other basic ligands.     

聚乙烯亚胺转基因影响因素的测定及其优化

聚乙烯亚胺 (PEI)为阳离子多聚物 ,可浓缩DNA形成纳米级颗粒 ,作为基因释放载体转染真核细胞 .选用Mr2 5 0 0 0 ,分枝状的聚乙烯亚胺转染质粒 ,比较多种转基因效率的影响因素 .通过MTT法测定PEI对COS 7细胞的细胞毒性 .利用电泳阻滞实验测定PEI与DNA形成复合物时所需的比例 .通过PEI转染增强型绿色荧光蛋白的pEGFP质粒、编码β 半乳糖苷酶的pSVβ质粒 ,探索氯喹、白蛋白、血清、盐离子浓度、质粒剂量、细胞数量等对聚乙烯亚胺转基因效率的影响 .实验发现 ,PEI对细胞的毒性作用与剂量相关 .PEI DNA的N P比在 3 0以上方可完全结合DNA .溶酶体抑制剂氯喹可增加转染效率 .培养液中的白蛋白、血清会降低转染效率 .生理盐溶液作为配制PEI DNA复合物的溶媒 ,转染效率高于 5 %葡萄糖作为溶媒 .随着转染质粒剂量的增加 ,转染效率呈剂量依赖正效应 .聚乙烯亚胺是有效的体外真核细胞转染剂 ,可用于合成更复杂的基因释放载体 .     

Crosslinked polyethylenimine: An enzyme carrier with spacers of various lengths introduced in crosslinking reaction

Polyethylenimine (PEI) reacted with epichlorhydrin to various degrees of crosslinking was further activated with thiophosgene or with succinic anhydride; the carboxyl derivatives obtained were converted to hydrazide derivatives. d-Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4), glucoamylase (exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) and cholinesterase (acetyl-, butyryl-) [acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7), butyryl cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8)] were bound in their native forms to the first two types of carriers, while in the case of the hydrazide derivative of crosslinked PEI this occurred just after periodate oxidation of a glycoenzyme. The PEI derivatives prepared from the crosslinked PEI with the lowest degree of crosslinking, i.e. from that having the longest mean length of the spacer (～ 11.1 Å), revealed the best binding properties towards low-molecular thiols, amino acids and proteins/enzymes. After enzyme coupling, the isothiocyanate derivatives of crosslinked PEI gave preparations with the highest residual activities.     

Effects of cationic vectors complexed with plasmid DNA on the phagosome-lysosome fusion in murine peritoneal macrophages and J744 macrophages

Polyethylenimine (PEI) and cationic polypeptides complexed with plasmid DNA are the most efficient nonviral vectors for gene therapy. It is believed that endocytosis is the major pathway for cell entering by PEI/DNA or cationic peptides/DNA complexes. Effects of plasmid DNA complexed with PEI, poly-L-lysine (PLL), poly-D-lysine (PDL) and polyarginine (PA) on the phagosome-lysosome fusion (P-LF) were studied in murine peritoneal macrophages and J774 macrophages. Cationic polypeptide PLL can be hydrolysed by cellular peptidases, but its stereoisomer, PDL, cannot be split by these enzymes. PEI, PDL, and PA have been shown to inhibit P-LF. PLL showed a low effect on the P-LF. On the basis of these studies, we assume that lysosomotropic agents able to change functions of lysosomes in the cell may affect transfection efficiency and thus be used for gene therapy.     

Simultaneous filtration and immobilization of cells from a flowing suspension using a bioreactor containing polyethylenimine coated cotton threads: Application in the continuous inversion of concentrated sucrose syrups

Polyethylenimine(PEI)-coated cotton threads were shown to have potential for reducing microbial load from a flowing suspension. Turbid cell suspensions perfused through the PEI column appeared as totally clear in the effluent. The adhesion efficiency of the matrix was found to depend on the concentration of PEI used to treat the threads. Threads coated with 2.5% PEI were found to show optimal retention of cells. A considerable amount of binding was seen over a broad range of ionic concentration (0–0.3 M) and pH (3.6–10.3). Under similar conditions control threads did not show any filtration capacity. Saccharomyces cerevisiae, Saccharomyces fragilis, Escherichia coli and an Acetobacter species could be effectively filtered using PEI-coated threads. This technique can find potential for the simultaneous filtration and immobilization of cells in a bioreactor to be used in continuous bioprocessing as exemplified for the inversion of sucrose syrups using baker's yeast. The bioreactor could continuously hydrolyse 60% (w/v) sucrose syrups with a productivity of 2.25 kg/day for over a month without loss in efficiency.     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.     

交联聚乙烯亚胺衍生物的构建及其转染COS-7 细胞的研究

目的:优化构建交联聚乙烯亚胺(Polyethylenemine,PEI)衍生物PEI-Bu,研究其对非洲绿猴肾成纤维细胞系(COS-7)的转染活性和细胞毒性。方法:以PEI 800Da为骨架,1,4-丁二醇二氯甲酸酯为连接剂制备聚合物PEI-Bu,琼脂糖凝胶电泳考察其复合质粒DNA的能力,MTT法检测PEI-Bu对COS-7的毒性,以荧光素酶质粒作为报告基因,测定PEI-Bu/DNA复合物在COS-7细胞的转染活性。结果:凝胶电泳表明PEI-Bu/DNA在质量比大于1时即具有复合DNA的能力,PEI-Bu的细胞毒性随浓度增大而增大,在同一浓度下PEI-Bu的细胞毒性小于PEI 25kDa,(P<0.05),PEI-Bu/DNA在质量比为5时达到最高转染活性,高于PEI 25kDa(P<0.01),并与Lipofectamine2000相当(P>0.05)。结论:PEI-Bu在COS-7细胞中是一种低细胞毒性、高转染活性的非病毒基因载体(与商业化的PEI 25kDa比较),其在基因治疗领域中具有潜在的应用前景。     

Novel anticoagulant polyethylenimine: inhibition of thrombin-catalyzed fibrin formation

Hypercoagulability is often associated with a variety of disease states, leading to cardiovascular complications. Polyethylenimine (PEI) prolonged prothrombin time, demonstrating its anticoagulant potential. In vitro, PEI at low concentration (nM) significantly blocked thrombin-catalyzed fibrin formation, accounting for its mode of anticoagulation. The uncompetitive inhibition by PEI of fibrin formation was independent of the concentration of fibrinogen (FBG), thrombin, or NaCl. PEI showed no effect on thrombin amidolytic activity, suggesting that the blockade of thrombin interaction with FBG could account for the inhibition on fibrin formation. PEI drastically depressed rabbit brain thromboplastin procoagulation monitored by a single-stage clotting assay using human plasma. In a THP-1 monocytic hypercoagulation model, a 4-h exposure to bacterial endotoxin or Ca(2+) ionophore A23187, respectively, resulted in a 5- or 10-fold enhancement in monocytic tissue factor (mTF) procoagulation. mTF hypercoagulation was offset by PEI included in the assay mixture. PEI showed the potential to arrest mTF hypercoagulation with IC(50) around 1.2 nM. Using a chromogenic assay to dissect the extrinsic pathway, we further assessed whether PEI has any effect on other clotting factors. PEI was not an inhibitor for either FVIIa or FXa, having no effect on not only the amidolytic but also their corresponding functionally catalytic activities. Although PEI upregulated TF-dependent FVII activation under the low-salt condition, the effective downstream inhibition of fibrin formation readily abolished and overrode the upstream enhancement, demonstrating the overall anticoagulation. PEI could present a new class of anticoagulant.     

Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression

Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (&lt; 1 min) between DNA and PEI in 150 mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300–400 nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 10⁶ cell/mL, 2.1 μg/mL of DNA, and 9.3 μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8 μg/10⁶ transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.

     

聚乙烯亚胺包裹小环DNA形成的纳米颗粒传输外源基因的性质表征

聚乙烯亚胺(PEI)是一种具有良好生物安全性和生物相容性的非病毒载体,能高效转染肿瘤细胞。小环DNA是一种去除质粒细菌骨架,只含有目的基因表达框的环状DNA分子。与普通质粒相比,小环DNA具有表达效率高、持续时间长的优势。使用PEI包裹携带报告基因gfp和抑癌基因pten小环DNA载体,并利用各种技术手段分析了该传输系统的理化性质和生物学效应。凝胶阻滞实验、电镜实验及MTT实验分析结果表明利用PEI包裹小环DNA和质粒DNA体系性质无显著的差别,并且2种复合物对细胞毒性亦无明显差别;但是动态光散射实验结果显示由于PEI可以包裹更多数量的小环DNA,所以PEI包裹小环DNA形成的复合物粒径要略大于包裹质粒DNA形成的复合物粒径。荧光显微镜实验、real-time PCR分析和Western blotting分析结果表明,PEI包裹小环DNA形成的复合物对细胞的转染效率要远远高于PEI包裹质粒DNA所形成的复合物,并且小环所携带的外源基因的表达效率要远远高于质粒DNA所携带的外源基因的表达效率。实验结果表明,PEI包裹小环DNA形成的纳米颗粒在细胞转染过程中具有很高的表达效率,这一研究结果为PEI包裹小环DNA的非病毒载体系统在传输外源基因过程中的应用提供理论基础和技术支持。     

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.     

Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes

Polyethylenimine (PEI) is one of the most efficient nonviral vectors for gene therapy. The aim of this study was to investigate the role of endocytosis in the transfection of synchronized L929 fibroblasts by PEI/DNA complexes. This was performed by confocal microscopy and flow cytometry, using the endocytosis marker FM4-64 and PEI/DNA complexes labeled either with the DNA intercalator YOYO-1, or with fluorescein covalently linked to PEI. Endocytosis appeared as the major if not the sole mode of entry of the PEI/DNA complexes into the L929 cells. The complexes followed a typical fluid phase endocytosis pathway and were efficiently taken up in less than 10 min in endosomes that did not exceed 200 nm in diameter. Later, the localization of the complexes became perinuclear and fusion between late endosomes was shown to occur. Comparison with the intracellular trafficking of the same complexes in EA.hy 926 cells (W.T. Godbey, K. Wu, A.G. Mikos, Proc. Natl. Acad. Sci. USA 96 (1999)) revealed that endocytosis of PEI/DNA complexes is strongly cell-dependent. In L929 cells, escape of the complexes from the endosomes is a major barrier for transfection. This limited the number of transfected cells to a few percent, even though an internalization of PEI/DNA complexes was observed in most cells. In addition, the entry of the complexes into the nucleus apparently required a mitosis and did not involve the lipids of the endosome membrane. This entry seems to be a short-lived event that involves only a few complexes.     

Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells

Background

Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.

Methods

PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.

Results

PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependant, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.

Conclusion

PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.     

Visualization of the degradation of a disulfide polymer, linear poly(ethylenimine sulfide), for gene delivery

Polyethylenimine (PEI) shows high transfection efficiency and cytoxicity due to its high amine density. The new disulfide cationic polymer, linear poly(ethylenimine sulfide) (l-PEIS), was synthesized for efficient and safe gene delivery. As the amine density of l-PEIS increased, the transfection efficiency also increased. l-PEIS-6 and l-PEIS-8 show transfection efficiencies that are similar to that of PEI. However, cytotoxicity of l-PEIS was not observed due to the biodegradable disulfide bond. The disulfide bonds are stable in the oxidative extracellular condition and can be degraded rapidly in the reductive intracellular condition. The degradation of l-PEIS in HeLa cells was visualized by fluorescence microscopy using the probe-probe dequenching effect of BODIPY-FL fluorescence dye. l-PEIS was degraded completely within 3 h.     

Biodegradable cross-linked poly(amino alcohol esters) based on LMW PEI for gene delivery

Polyethylenimine (PEI, especially with M(w) of 25,000) has been known as an efficient gene carrier and a gold standard of gene transfection due to its high transfection efficiency (TE). However, high concomitant cytotoxicity limited the application of PEI. In this report, several cationic polymers derived from low molecular weight (LMW) PEI (M(w) 600) linked with diglycidyl adipate (DA-PEI) or its analogs (diglycidyl succinate, DS-PEI and diglycidyl oxalate, DO-PEI; D-PEIs for all 3 polymers) were prepared and characterized. GPC gave M(w)s of DA-PEI, DS-PEI and DO-PEI as 6861, 16,015 and 35,281, respectively. Moreover, degradation of the ester-containing DS-PEI was also confirmed by GPC. In addition, hydroxyls in these polymers could improve their water solubility. These polymers exhibited good ability to condense plasmid DNA into nanoparticles with the size of 120-250 nm. ζ-potentials of the polyplexes were found to be around +10-20 mV under weight ratios (polymer/DNA) from 0.5 to 32. Agarose gel retardation showed that DNA could be released from the polyplexes after being pre-incubated for 30 h. In vitro experiments were carried out and it was found that DS-PEI showed about 5 times of TE compared to that of the PEI/DNA polyplex under a weight ratio of 1 in A549 cells. Meanwhile, the cytotoxicity of D-PEIs assayed by MTT is lower than that of 25 kDa PEI in HEK293 cells. These results suggested that this series of PEI derivatives would be promising non-viral biodegradable vectors for gene delivery.     

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.     

Salt ions and related parameters affect PEI–DNA particle size and transfection efficiency in Chinese hamster ovary cells

Transfection efficiency is directly associated with the expression level and quantity of recombinant protein after the transient transfection of animal cells. The transfection process can be influenced by many still-unknown factors, so it is valuable to study the precise mechanism and explore these factors in gene delivery. Polyethylenimine (PEI) is considered to have high transfection efficiency and endosome-disrupting capacity. Here we aimed to investigate optimal conditions for transfection efficiency by setting different parameters, including salt ion concentration, DNA/PEI ratio, and incubation time. We examined the PEI–DNA particle size using a Malvern particle size analyzer and assessed the transfection efficiency using flow cytometry in Chinese hamster ovary-S cells. Salt ions, higher amounts of PEI tended to improve the aggregation of PEI–DNA particles and the particle size of PEI–DNA complexes and the transfection efficiency were increased. Besides, the particle size was also found to benefit from longer incubation time. However, the transfection efficiency increased to maximum of 68.92 % at an incubation time of 10 min, but decreased significantly thereafter to 23.71 %, when incubating for 120 min (P < 0.05). Besides, PEI–DNA complexes formed in salt-free condition were unstable. Our results suggest DNA and PEI incubated in 300 mM NaCl at a ratio of 1:4 for 10 min could achieve the optimal transfection efficiency. Our results might provide guidance for the optimization of transfection efficiency and the industrial production of recombinant proteins.