Transfection Efficiency Of Polyethylenimine Research Articles

Improving the gene transfection efficiency of polyethylenimine (PEI) based polyplex nanoparticles by cell cycle regulation

Synthesis and characterization of phosphate structured dendrimers as drug delivery carriers Zhen Zhang, Jianbin Tang, Xiangrui Liu, Youqing Shen, Center for Bionanoengineering and Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China ⁎Corresponding author. E-mail address: shenyq@zju.edu.cn (Y. Shen)

Polyethylenimine derivate conjugated with RGD-TAT-NLS as a novel gene vector.

To solve the contradiction between the cell toxicity and transfection efficiency of polyethylenimine (PEI) derivate in non-viral gene therapy, a novel gene vector, P123-PEI-R18 was synthesized by using biodegradable PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS. The particle size of P123-PEI-R18/DNA was around 100-250 nm. The gene vector could condense DNA at the weight ratio of 2 and protect plasmid DNA from being dissolved in the blood circulation. Importantly, the complexes exhibited lower cell toxicity and higher transfection efficiency contrasted with PEI 25 kDa in vitro. P123-PEI-R18 holds high potential as a safe and efficient gene vector.

The effect of cationic charge density change on transfection efficiency of polyethylenimine.

Polyethylenimine (PEI) is a potent non-viral gene delivery carrier. PEI surface charge plays a major role in its condensation ability which in turn is a necessary requirement for high transfection efficiency. As the PEI charge density is dependent on pH, the effect of pH changing PEI on PEI condensation ability, transfection efficiency, and cytotoxicity was investigated. In order to compare the different values of pH, 25 kDa PEI solutions were prepared at the pH values of 5, 7 and 10, afterward complexed with two plasmids encoding reporter genes of luciferase and enhanced green fluorescence protein to evaluate the capability of polymers in plasmid delivery at two incubation time (4 versus 24 hr). The condensation ability of PEI in acidic, neutral and basic environments was similar. At low pH value, the polyplex had negative surface charge whereas at a higher pH value, the surface charge increased and became positive. The higher transfection efficiency and lower cytotoxicity were achieved by the polyplexes prepared at the pH value of 5 with longer incubation time of 24 hr. The results showed the impact of pH value of PEI on biological and biophysical properties of polyplexes. Although the proton sponge effect can justify several properties of PEI, these results suggest that the proton sponge hypothesis may not be applicable for polymers with low buffering capacity at the pH values around 5.

Polyethylenimine-mediated transfection of bone marrow mesenchymal stem cells

Objective To study transfection efficiency of polyethylenimine (PEI) into bone marrow mesenchymal stem cells (BMSCs) in order to optimize the conditions that can be used for transfection experiments.Methods Cell counting kit-8 (CCK-8) assay was used to test the cytotoxicity of 0.2％-2.0％ PEI.Flow cytometry and fluorescence microscope were employed to determine the transfection efficiency by N/P 3.97-37.75 and test the effects of chloroquine (0-100 μmol/L),10％ albumin,5％ serum and 0-20 mmol/L Mg2+ in the culture on the transfection efficiency of BMSCs.Results When PEI in the complete culture medium was below 0.8％,the survival rate of BMSCs was 70％.After optimizing the N/P charge ratio,the PEI/DNA microcapsule delivered plasmid into BMSCs and obtained optimum transfection efficiency N/P 15.Chloroquine enhanced the transfection efficiency of PEI.10％ albumin,5％ serum and Mg2+ in the culture decreased the transfection efficiency.Conclusion PEI is a kind of effective non-viral vectors for BMSCs. Key words: Non-viral gene vectors ; Transfection; Polyethylenimine ; Bone marrow mesenchymal stem cells

Design, Synthesis and Evaluation of Cationic Poly(N-substituent acrylamide)s for In Vitro Gene Delivery

A series of poly(N-substituent acrylamide)s (PAms) that differ in alkylamine side-chain was synthesized via free radical polymerization. The PAms were designed to examine the effects of the methylene numbers (from 2 to 12) in the alkylamine side-chain on cytotoxicity, plasmid DNA (pDNA) binding affinity, cellular uptake efficiency and gene expression. The cytotoxicity of PAms evaluated in HEK293 cells using the MTT assay showed a trend of decreasing toxicity with increasing side-chain length and the IC50 values of all PAms were lower than that of polyethylenimine (PEI) control. The primary amine-based polymers were able to efficiently condense pDNA to form complexes with size ranging from 100 to 350 nm. The gene transfection ability of PAms is dominantly determined by the specific side-chain length that P8Am (with an octylamine side-chain) reveals higher gene expression than other PAms containing the same backbone structure. Although the gene transfection efficiency of PEI was better than all of PAms, PAms were found not to be uptake-limited. This was supported by the effect of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. Especially, complexes formed from P8Am displayed high uptake level relative to PEI, which was attributed to the proper structure of P8Am to compact pDNA to form stable nanoparticles in the heparin replacement assay. The present study offers the understanding to polymer structure that influences the transfection ability and gives useful information when designs efficient polymeric gene carrier.

Effects of cell‐penetrating peptides and pegylation on transfection efficiency of polyethylenimine in mouse lungs

Cell-penetrating peptides (CPPs) could potentially be used as vectors for intracellular delivery of proteins, peptides and nucleic acids. The present study examined different CPPs, such as TAT-derived and arginine rich sequences, as well as model amphiphilic peptide, with respect to transfection efficiency of pegylated polyethylenimine (PEI) in A549, Calu-3 cells and in mice after intra-tracheal administration. The conjugates were prepared by the coupling of CPPs to PEI via a heterobifunctional polyethylene glycol (PEG) linker, resulting in the bioconjugates CPP-PEG-PEI. Structures were successfully confirmed by (1)H-nuclear magnetic resonance and diffusion-ordered spectroscopy. Unmodified PEI 25 kDa was compared with pegylated PEI, and aggregation tendency in cell culture medium, interaction with mucin and stability against heparin was assayed. After evaluating transfection efficiency of the polymers in two different lung cell lines, luciferase reporter gene expression was determined in mouse lungs. All conjugates showed superior transfection efficiency compared to unmodified PEI 25 kDa. The conjugates sizes were generally < 300 nm, thus enabling them to penetrate through the mucus lining of the lung and reach the target cells. Coupling of CPPs to PEG-PEI, however, did not significantly improve transfection efficiency in A549 cells, calu-3 cells and in mouse lungs. We show that small and stable polyplex size achieved by pegylation is favourable for successful pulmonary gene delivery. Compared to PEI 25 kDa, pegylated PEI and CPP-PEG-PEI displayed enhanced transfection efficiency both in vitro and in vivo.

Pseudomonas aeruginosaInfection Destroys the Barrier Function of Lung Epithelium and Enhances Polyplex-Mediated Transfection

Challenged by the lack of success of experimental gene therapy of cystic fibrosis, we set out to investigate one of the potential causes of this failure, the barrier function of the airway epithelium and the way this is affected by bacterial infection. In an in vitro model of the airway epithelium we determined the effect of Pseudomonas aeruginosa or Escherichia coli on the transfection efficiency of polyethylenimine (PEI)-plasmid DNA complexes, carrying a luciferase gene, as well as on the barrier function of the epithelial cell layer, using transepithelial resistance (TER), cytotoxicity, bacterial transmigration, and morphological appearance as parameters. The level of luciferase expression was more than one order of magnitude higher in the cells which, before transfection, were incubated with P. aeruginosa. TER was strongly reduced by P. aeruginosa, whereas E. coli had no effect. Pseudomonas aeruginosa also effectively destroyed the structure of the tight junctions, as visualized by immunostaining of the zonula occludens. By the same token, small but significant numbers of P. aeruginosa cells were found to migrate through the epithelial layer, whereas no E. coli cells were observed at the transcompartment of the wells. Release of lactate dehydrogenase from the epithelial cells, a parameter of cell damage, occurred in a dose-dependent manner on incubation with P. aeruginosa, but not with E. coli. To evaluate the relevance of these results for the in vivo situation, we infected C57BL/6 mice with P. aeruginosa or E. coli 48 hr before transfecting them intratracheally with PEI-DNA polyplexes. Infection with P. aeruginosa caused a 5-fold increase in luciferase expression whereas infection with E. coli had no effect. Fluorescence microscopy of lung sections, after administration of fluorescein isothiocyanate-labeled polyplexes, showed that prior treatment with P. aeruginosa effectuated penetration of the complexes deeper into the epithelium than in untreated animals. In P. aeruginosa-treated animals fluorescence was detected not only in the airway epithelium itself but also in the parenchyma. We conclude that infection with P. aeruginosa causes disruption of the tight junctions between the cells and thus of the barrier function of the epithelium. As a consequence, PEI-DNA complexes injected intratracheally into infected animals gain access to the basolateral side of the cells and to spaces across the epithelial lining, giving rise to substantially increased transfection efficiency.

The Internalization Route Resulting in Successful Gene Expression Depends on both Cell Line and Polyethylenimine Polyplex Type

Understanding cellular uptake and intracellular processing of nonviral gene delivery systems is a key aspect in developing more efficient vectors. In this study, the impact of clathrin- and caveolae/lipid-raft-dependent endocytosis on cell entry and overall transfection efficiency of polyethylenimine (PEI) polyplexes was evaluated. Most remarkably, the internalization pathway mediating successful transfection depended on both cell type and polyplex type applied. Colocalization studies with transferrin and cholera toxin B revealed that at least two specific endocytosis pathways—the clathrin-dependent and the lipid-raft-dependent—mediated cellular uptake of PEI polyplexes. With the help of specific uptake inhibitors (chlorpromazine and filipin III), cell-line-dependent variations regarding the route of successful transfection were observed (HUH-7, COS-7, HeLa). In COS-7 cells, the clathrin-dependent pathway was the main contributor to the transfection process. In HUH-7 cells, gene transfer by linear PEI polyplexes succeeded mainly via the clathrin-dependent route, whereas transfection by branched PEI polyplexes was mediated by both pathways. In HeLa cells, both pathways were able to mediate successful gene delivery. However, the lipid-raft-dependent pathway was more relevant. The study also revealed that the concentration window between specific inhibitory function and nonspecific toxicity of the uptake inhibitors was very narrow.

Effect of Dexamethasone Preincubation on Polymer-Mediated Gene Delivery

Nuclear membrane is one of the main barriers in intracellular delivery of genetic materials. The previous report showed that glucocorticoid receptor dilated the nuclear pore to 60 nm in the presence of a ligand. It was also suggested that the transport of genetic material to nucleus might be facilitated by glucocorticoid. In this study, the effect of glucocorticoid preincubation in the polymeric gene delivery was investigated. The cells were preincubated with dexamethasone, a potent glucocorticoid, and transfection assays were performed with polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer. As a result, the transfection efficiency of PEI or PAMAM to the cells in the presence of dexamethasone was enhanced, compared to the cells without dexamethasone. This effect was not observed in the cells preincubated with cholesterol. The polymer/DNA complex was stable in the presence of dexamethasone. In addition, the cytotoxicities of the polymeric carriers to the cells were observed in the presence of dexamethasone. In conclusion, dexamethasone enhances the transfection efficiency of polymeric carriers and may be useful in the development of polymeric gene carriers.

Polyplexes of Polyethylenimine and per-N-methylated Polyethylenimine-Cytotoxicity and Transfection Efficiency

For non-viral gene delivery, the carriers for DNA transfer into cells must be vastly improved. The branched cationic polymer polyethylenimine has been described as an efficient gene carrier. However, polyethylenimine was demonstrated to mediate substantial cytotoxicity. Therefore, this study is aimed at investigating per-N-methylated polyethylenimine, which is thought to have a much lower cytotoxicity due to its lower charge density. Results from a gel retardation assay and laser light scattering indicated that per-N-methylated polyethylenimine condenses DNA into small and compact nanoparticles with a mean diameter <150 nm. Furthermore, polyplexes of polyethylenimine and per-N-methylated polyethylenimine with DNA had a positive zeta potential and the polymers protected DNA from nuclease-mediated digestion. The transfection efficiency of polyethylenimine and per-N-methylated polyethylenimine was tested in CHO-K1 cells. Using green fluorescent protein as reporter gene and flow cytometry analysis, we demonstrated that per-N-methylated polyethylenimine has a lower cytotoxicity, but also a significantly lower transfection efficiency. Using propidium iodide staining, we could additionally distinguish between viable and dead cells. At NP > or = 12, per-N-methylated polyethylenimine showed a much higher cell viability and the ratio of viable and transfected cells to dead and transfected cells was about 1.5 to 1.7 fold higher than for polyethylenimine. The results of cell viability from flow cytometry analysis were confirmed by the MTS assay. Using luciferase reporter gene for transfection experiments, the gene expression of per-N-methylated polyethylenimine was lower at NP 6, 12 and 18 as compared to polyethylenimine, but at NP 24 it yielded similar levels.