Transfection Efficiency Of Polyplexes Research Articles

Bioreducible poly(amido amine)s for gene delivery to ovarian cancer cells

Bioreducible poly(amido amine)s (SS-PAAs) with different groups in the side chain were evaluated for gene delivery to ovarian cancer cells, both in vitro and in vivo after intraperitoneal administration in mice bearing an ovarian cancer xenograft. Polyplexes of SS-PAAs with e.g. hydroxybutyl or hydroxypentyl side groups induce much higher transfection in vitro than polyplexes of branched pEI (25 kDa) as a positive control. The in vivo transfection efficiency of polyplexes of SS-PAA with hydroxybutyl side groups is similar to that of linear pEI (22 kDa).

Gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan block copolymers: Establishment of structure–activity relationships in vitro

Chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan/DNA complexes were characterized concerning physicochemical properties such as hydrodynamic diameter, condensation efficiency and DNA release. Furthermore, cytotoxicity of polymers and uptake- and transfection efficiency of polyplexes were evaluated in vitro. Under conditions found in cell culture, formation of aggregates of ∼ 1000 nm and strongly decreased DNA condensation efficiency was observed in the case of chitosan polyplexes. These characteristics resulted in only 7% cellular uptake in NIH/3T3 cells and low transfection efficiencies in 4 different cell lines. By contrast, quaternization of chitosan strongly reduced aggregation tendency and pH dependency of DNA complexation. Accordingly, cellular uptake was increased 8.5-fold compared to chitosan polyplexes resulting in up to 678-fold increased transfection efficiency in NIH/3T3 cells. Apart from reduction of the cytotoxicity, PEGylation led to improved colloidal stability of polyplexes and significantly increased cellular uptake compared to unmodified trimethyl chitosan. These improvements resulted in a significant, up to 10-fold increase of transfection efficiency in NIH/3T3, L929 and MeWo cells compared to trimethyl chitosan. This study not only highlights the importance of investigating polyplex stability under different pH- and ionic strength conditions but also elucidates correlations between physicochemical characteristics and biological efficacy of the studied polyplexes.

In vivo tumor transfection mediated by polyplexes based on biodegradable poly(DMAEA)-phosphazene

In recent years, increasing interest is being paid to the design of transfectants based on non-toxic and biodegradable polymers for gene therapy purposes. We recently reported on a novel, biodegradable polymer, poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) for use in non-viral gene delivery. In this study, the biodistribution and in vivo transfection efficiency of polyplexes composed of plasmid DNA and p(DMAEA)-ppz were investigated after intravenous administration in tumor bearing mice. Data were compared with those of polyplexes based on the non-biodegradable polyethylenimine (PEI 22kDa). Both polyplex systems were rapidly cleared from the circulation (< 7% ID, at 60 min after administration) and showed considerable disposition in the liver and the lung, all in line with earlier work on cationic polyplex systems. The lung disposition is attributed to aggregates formed by interaction of the polyplexes with blood constituents. Redistribution of the polyplexes from the lung was observed for both polyplex formulations. Importantly, both polyplex systems showed a substantial tumor accumulation of 5% and 8% ID/g for p(DMAEA)-ppz and PEI22 polyplexes, respectively, at 240 min after administration. The tumor disposition of the p(DMAEA)-ppz and PEI22 polyplexes was associated with considerable expression levels of the reporter gene. In contrast to PEI22 polyplexes, p(DMAEA)-ppz polyplexes did not display substantial gene expression in the lung or other organs (organ gene expression < 1 / 100 of tumor gene expression). The observed preferential tumor gene expression mediated by the p(DMAEA)-ppz polyplexes enables the application of this polymer to deliver therapeutic genes to tumors.

C- versus N-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of DNA polyplexes

One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency. The N-terminus of natural all-(L)- or non-immunogenic all-(D)-melittin was covalently coupled to PEI. In addition, two different all-(D)-melittin conjugates were synthesized, with PEI covalently attached to either the C-terminus (C-mel-PEI) or the N-terminus of melittin (N-mel-PEI). Melittin-PEI polyplexes with particle sizes < 150 nm were generated in HEPES-buffered glucose and tested in transfection experiments. The membrane lytic activities of conjugates and polyplexes were analyzed at neutral and endosomal pH. All-(D)-melittin conjugates mediated enhanced gene expression similar to the natural all-(L)-stereoisomer, with up to 160-fold higher luciferase activity than unmodified PEI. The site of melittin linkage strongly influenced the membrane-destabilizing activities of both conjugates and polyplexes. C-mel-PEI was highly lytic at neutral pH and therefore elevated doses of C-mel-PEI polyplexes induced high toxicity. In contrast, N-mel-PEI was less lytic at neutral pH but retained higher lytic activity than C-mel-PEI at endosomal pH. This apparently promoted better endosomal release of N-mel-PEI polyplexes resulting in efficient gene delivery in different cell lines. The high potency of C-mel-PEI to destabilize membranes at neutral pH is presumably due to a reported destabilization mechanism proceeding through membrane insertion of the peptide. In contrast, N-mel-PEI is supposed to induce lysis by insertion-independent pore formation according to the toroidal pore model.

Modified polyethylenimines as non viral gene delivery systems for aerosol therapy: effects of nebulization on cellular uptake and transfection efficiency

This study examined the effect of nebulization on the cellular uptake and transfection efficiency of polyplexes from four polyethylenimine (PEI) modifications: branched 25 kDa PEI (bPEI), linear 22 kDa PEI (linPEI), pegylated PEI (pegPEI) and biodegradable PEI (bioPEI). Polyplexes were aerosolized with air-jet and ultrasonic nebulizers. The aerosol was collected and used to determine complex size and zeta potential. Fluorescence-assisted cell sorting (FACS) was used to quantify the cellular association of polyplexes in primary alveolar cells (AEC), A549 cells and primary bronchial cells (BEC). Confocal laser scanning microscopic images provided information about the internalization of polyplexes. Transfection efficiencies of polyplexes were quantified via measurement of luciferase expression. All polymers were stable during nebulization, although size increases were observed after air-jet nebulization. FACS studies showed a two- to three-fold increase in polyplex association with BEC compared to A549 cells, while polyplex association with AEC was negligible. BPEI, linPEI and bioPEI polyplexes were internalized, while pegPEI polyplexes remained predominately attached to the cellular membrane. Luciferase expression was detected only in BEC and A549 cells with transfection efficiencies approximately one order of magnitude higher in BEC. All PEI modifications investigated were suitable for aerosol therapy, although cell type and polymer structure significantly influenced the uptake and transfection efficiency of the polyplexes.

Cationic polymethacrylates with covalently linked membrane destabilizing peptides as gene delivery vectors

A membrane-disrupting peptide derived from the influenza virus was covalently linked to different polymethacrylates (pDMAEMA, pDAMA and the degradable pHPMA–DMAE, monomers depicted in Fig. 1) using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as coupling agent to increase the transfection efficiency of polyplexes based on these polymers. It was shown by circular dichroism (CD) measurements that the polymer-conjugated peptide was, as the free peptide, able to undergo a conformational change of a random coil to an alpha helix upon lowering the pH to 5.0. This indicates that the property of the peptide to destabilize the endosomal membrane was preserved after its conjugation to the cationic polymers. In line herewith, a liposome leakage assay revealed that the polymer-bound peptide has comparable activity as the free peptide. The DNA condensing properties of the synthesized polymer–peptide conjugates were studied with dynamic light scattering and ζ-potential measurements, and it was shown that small (100 to 250 nm), positively charged (+15 to +20 mV) particles were formed. In vitro transfection and toxicity was tested in COS-7 cells, and these experiments showed that the polyplexes with grafted peptide had a substantially higher transfection activity than the control polyplexes, while the toxicity remained unchanged. Cellular uptake of the polyplexes was visualized with confocal laser scanning microscopy, and no differences in cellular uptake could be determined between the peptide containing systems and the control formulation. This shows that the increased transfection activity is indeed due to a better endosomal escape of the peptide grafted polyplexes. This study demonstrates that it is possible to covalently conjugate an endosome disruptive peptide to cationic gene delivery polymers with preservation of its membrane destabilization activity, making these conjugates suitable for in vivo DNA delivery.

Identification of an Internalising Peptide in Differentiated Calu-3 Cells by Phage Display Technology; Application to Gene Delivery to the Airways

Differentiated, human submucosal-gland carcinoma, Calu-3 cell monolayers were used as in vitro model for the airway epithelium. Internalised phage were selected from a recombinant pComb8 phage library by repetitive cycles of bio-panning on Calu-3 monolayers, protease K degradation, cell-lysis and amplification. After four selection rounds, sequence analysis of 15 enriched phage colonies revealed two clones of 73 and 27% abundancy, named IB1 and IB2, respectively. The IB2 sequence was eliminated due to a frame shift. IB1-phage internalisation at 4°C was significantly lower (P<0.05)than at 37°C, suggesting involvement of a receptor-mediated endocytosis pathway. The IB1 peptide was synthesised, biotinylated and complexed to streptavidin. IB1/streptavidin-complexes co-administrated with PEI/DNA-polyplexes, enhanced polyplex transfection efficiency, dose dependently, by 6- and 4-fold in Calu-3 cells. IB1/Alexa488-streptavidin complexes were used for confocal laser-scanning microscopy (CLSM) visualisation and showed basolateral localisation in membrane associated and internalising vesicles. This study demonstrates the potential of phage display technology for identification of internalising peptide–epitopes that can enhance gene delivery efficiency in differentiated airway epithelial cells.

Intracellular processing and stability of DNA complexed with histidylated polylysine conjugates.

Glycosylated polylysines and histidylated polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work has been undertaken in two steps to study the uptake and the intracellular processing of pDNA, which are still poorly understood in the polyfection pathway. The kinetics of the uptake and the intracellular processing of pDNA complexed with lactosylated polylysine, histidylated polylysine or histidylated polylysine bearing lactosyl residues (polyplexes) into a CF human airway epithelial cell line were assessed by flow cytometry and confocal microscopy. Complexes formed from histidylated polylysine, even though they were less taken up by cells, show better transfection efficiency with compared with lactosylated complexes. Lactosylated polymers segregated more rapidly when compared with non-lactosylated polymers into compartments different from those containing pDNA on internalization. Intracellular location and pH measurements indicated that polymers ended up in compartments of pH approximately 6.2 while pDNA reached less acidic compartments of pH approximately 6.6. These compartments did not contain the LAMP-1 lysosomal marker. The present study exhibits that, upon internalization, pDNA and polylysine conjugates underwent segregation with a rate depending on the polylysine substitution and polymer degradation. The better transfection efficiency of polyplexes with histidylated polylysine can be ascribed to their prolonged stability inside the endocytic vesicles that likely favored the pDNA escape in the cytosol.

Histidylated polylysine as DNA vector: elevation of the imidazole protonation and reduced cellular uptake without change in the polyfection efficiency of serum stabilized negative polyplexes.

We have reported that polylysine substituted with histidyl residues (His) was suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the acetylation of the alpha-amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on their transfection efficiency. We found that the presence of free alpha-amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential to -25 mV. The acetylation of the alpha-amino groups reduced slightly the adsorption of serum proteins. The presence of the alpha-amino groups increased the pK of the imidazole protonation of histidine bound to polylysine from pH 5.8 to 6.9; in addition, the protonation was further elevated in the presence of pDNA. Serum stabilized negative histidylated polyplexes were less taken up by cells but their transfection efficiency did not decrease; depending on the cell line, His-polyplexes were more efficient than AcHis-polyplexes. The results indicate that (i) the alpha-amino groups of histidyl residues bound to polylysine favorably influence the size and the transfection efficiency of polyplexes, (ii) the alpha-amino groups also elevate the imidazole protonation of His-polyplexes, which is suited to destabilize the membrane of early endocytic vesicles in order to favor pDNA delivery in the cytosol, and (iii) the absorption of selective serum proteins on His-polyplexes could be a way for in vivo gene targeting.

Copolymers of 2-(dimethylamino)ethyl methacrylate with ethoxytriethylene glycol methacrylate or N-vinyl-pyrrolidone as gene transfer agents

Random copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) with ethoxytriethylene glycol methacrylate (triEGMA) or N-vinylpyrrolidone (NVP) of different molecular weights and compositions were synthesized, characterized and evaluated as polymeric transfectants in vitro. All synthesized copolymers (comonomer fraction up to 66 mol%) were able to bind to DNA, yielding polymer–plasmid complexes (polyplexes). However, the polymer–plasmid ratio at which small complexes (size 0.2–0.3 μm) were formed, increased with increasing mole fraction of the comonomer. ζ-Potential measurements revealed that the polymer–plasmid ratio where charge neutralization of DNA occurred, increased with increasing mole fraction of triEGMA. The cytotoxicity of the copolymers, either complexed with DNA or in the free form, decreased with increasing mole fraction of both comonomers (triEGMA and NVP). This reduction was even more than what could be expected based on the DMAEMA mole fraction in the copolymer. The copolymers with a molecular weight up to 170 000 had the same transfection capability as a homopolymer of comparable molecular weight. However, higher molecular weight copolymers showed a reduced transfection capability compared to the homopolymer, which was ascribed to the reduced capability to condense the size of plasmid. Transfection efficiency of polyplexes composed of copolymers with a low triEGMA content increased with increasing molecular weight. Although the copolymers with 50 mol% triEGMA were also better transfectants than the homopolymer, the transfection efficiency did not increase further with increasing molecular weight. Interestingly, NVP–DMAEMA copolymers synthesized by polymerization to high conversion showed both excellent DNA binding and condensing characteristics (polyplex size <0.3 μm) and transfection capabilities. This is ascribed to a synergistic effect of DMAEMA-rich copolymers and NVP-rich copolymers present in this system on the complex formation with plasmid DNA.