Native PEI Research Articles

Polyethyleneimine-polyethylene glycol copolymer targeted by anti-HER2 nanobody for specific delivery of transcriptionally targeted tBid containing construct

The present research seeks to investigate the process of mixing targeted gene delivery and transcriptional targeting. We have conjugated Polyethylenimine polymers (PEI) and molecules of poly (ethylene glycol). The next step was covalent attachment of anti-HER2 variables domains of camelid heavy chains antibodies (VHHs) or nanobodies (Nbs) to the distal terminals of NHS-PEG3500 in PEI-PEG nanoparticles. The whole procedure yielded PEI-PEG-Nb immunoconjugates. Having determined the properties of polyplexes, steps were taken to investigate the most efficient ratio of PEI polymers to pDNA molecules (N/P) so that the greatest rate of transfection may be obtained. This immune targeted nano biopolymer could condense the gene constructs that coded a transcriptionally targeted truncated –Bid (tBid) killer gene which was controlled by the breast cancer-specific MUC1 promoter. The favourable physicochemical properties matching both the size and zeta potential were observed in engineered polyplexes. Elevated transfection efficiency in HER2 positive cell lines using Nb-modified polyplexes were shown by the results of flow cytometry as compared against non-modified particles. 1.6 and 4.8 fold higher transfection efficiencies were observed in in vitro gene expression researches which used PEI-PEG-Nb/pGL4.50 compared to the situation when native PEI polymers were utilized in both BT-474 and SK-BR-3, respectively. A 2.22 and 3.62 fold rise in the level of tBid gene expression in BT-474 and SK-BR-3 cell lines relative to unmodified PEI treated cells was the result of transfection with PEI-PEG-Nb/pMUC1-tBid, respectively. In those HER2-positive cells which were transfected by targeted polyplexes, higher levels of cell death were observed. This fact points not only to the effective targeted delivery, but it is also indicative of transcriptional targeting efficiency of tBid killer gene when its expression is controlled by MUC1 promoter.

Nuclease-responsive DNA-PEI hollow microcapsules for bio-stimuli controlled release.

In this paper, a DNA-PEI microcapsule with novel nuclease-responsive controlled release properties was demonstrated. This microcapsule was constructed by the self-assembly of native DNA and PEI, and can work as a dual carrier for both DNA in the capsule shell and drugs inside the capsule. The controlled release behavior of the model drug BSA-FITC was studied using fluorescence microscopy and UV spectroscopy, and the results show that the synergetic controlled release can be triggered very well by a bio-stimulus, DNA nuclease recognition capsule decomposition, as well as the pH value and salt concentration. The therapeutic effects of the drug are enhanced due to the bio-stimuli responsive properties and the synergetic release of DNA and the drug together.

Ternary complexes DNA–polyethylenimine–Fe(iii) with linear and branched polycations: implications on condensation, size, charge and in vitro biocompatibility

The successful use of nucleic acids in medicine requires efficient DNA condensation and protection, as well as a low cytotoxicity of the vector. In previous work, we have used a ternary DNA–PEI–Fe(III) complex and have shown that the addition of Fe(III) to the DNA–PEI complexes enhances DNA condensation, whereas allows an easier DNA release from the complexes, in the presence of a decompacting agent. The focus of the present work was to investigate the influence of the polycation architecture on the DNA–PEI–Fe(III) ternary system, and also to develop a potential gene delivery system with reduced toxicity. Herein, lPEI (2.5 and 25 kDa) and bPEI (1.2 and 10 kDa) were used in conjunction with Fe(III), and the complexes formed were physico-chemically characterized using precipitation and gel electrophoresis assays, and size and zeta potential determinations. It was observed that the addition of Fe(III) ions equalizes the charge ratios for the onset of DNA condensation irrespective of the PEI used, although a more pronounced condensation effect is still visible with the polycations with a lower Mw. At low N/P ratios, the presence of Fe(III) caused the zeta potential to change from negative to positive values. Also, a reduction in polyplex size is visible before aggregation takes place. The hemolytic activity of the condensing agents was found to be dependent on the pH of the medium, but no perturbation of erythrocytes integrity was detected. Furthermore, the cytotoxicity of each condensing agent individually and of the complexes towards 3T3 fibroblast cell line and HeLa cell line was monitored using the MTT and LDH assays. The magnitude of the cytotoxic effects of the polycations and Fe(III) was found to be dependent on the concentration of the condensing agents, the molecular weight of the polycations, and the pH of the culture medium. The combination of Fe(III) with PEI determines higher IC50 values than in the absence of the metal ion. Additionally, a lower cytotoxicity of DNA–PEI–Fe(III) complexes was observed, when compared to native PEI–DNA complexes, at the same degree of condensation and values of zeta potential. The results show that Fe(III) reinforces the DNA–PEI condensation in both PEI architectures. It allows the cytotoxicity of PEIs to be mitigated and the concentration of the latter needed to obtain stable polyplexes to be decreased.

Alginate-Graft-PEI as a Gene Delivery Vector with High Efficiency and Low Cytotoxicity

To overcome the efficiency–cytotoxicity dilemma of native PEI and incorporate the advantages of alginate, we designed a novel gene vector by grafting PEI 2000 onto alginate, an anionic polysaccharide with excellent biocompatibility. The alginate-graft-PEI (Alg-g-PEI) was successfully synthesized and then characterized by elemental analysis, 1H-NMR and 13C-NMR. The M w of Alg-g-PEI is ca. 17 000. Acid–base titration confirmed that Alg-g-PEI retained the buffering capacity of native PEI. The DNA binding ability of the polymer was confirmed by gel retardation assay. DSL analysis showed that Alg-g-PEI had a particle size and zeta-potential similar to PEI 25K. AFM detected a clear and well-shaped morphology of the complexes. Additionally, Alg-g-PEI exhibited lower cytotoxicity than PEI 25K in BEL7402, MSC and RVMSC cells. Compared with PEI 25K, Alg-g-PEI had comparable or even higher transfection efficiency. Similarly, Alg-g-PEI-mediated VEGF expression was significantly higher compared with PEI 25K-mediated VEGF expression. All together, our results suggest that Alg-g-PEI has a potential to be a safe and efficient agent for gene therapy.

EFFICIENT DELIVERY OF NUCLEIC ACIDS BY USING MODIFIED POLYETHYLENIMINE-BASED NANOPARTICLES

Branched polyethylenimine, (b PEI, 25 kDa), was converted into nanoparticles by using three different crosslinkers, 1,6-hexanebisphosphate (HP), adipic acid (AA), and 1,4-butane dialdehyde (BA), having same carbon chain length (C-6), and the effect of ionic and covalent crosslinking on the transfection efficiency was studied. The synthesized nanoparticles were characterized by 1H-NMR, IR, DLS, AFM, and TEM. The entire series of nanoparticles was tested for their toxicity and ability to deliver genes in COS-1 and CHO cell lines. It was observed that, AAP-3 nanoparticle/DNA complex exhibited highest transfection efficiency (~2.1–10.7 folds) than the native PEI and commercially available transfection reagents, such as GenePORTER 2TM, LipofectamineTM, and SuperfectTM, with cell viability >80%. Among the series, the highest cell viability was observed with BAP nanoparticles (>96%). These nanoparticles were able to deliver GFP-siRNA very efficiently inside the cells with ~76%–90% suppression of EGFP expression. Cellular trafficking studies showed that these nanoparticles carry pDNA inside the nucleus of the cells within 2 h of the addition to the cells.

Polyethylenimine-polyethyleneglycol-bis(aminoethylphosphate) nanoparticles mediated efficient DNA and siRNA transfection in mammalian cells

In an attempt to circumvent toxic effects of branched polyethylenimine (bPEI, 25 kDa), it was crosslinked with varying proportions of a novel linker, PEG600-bis(aminoethylphosphate) (PaP), which resulted in the formation of nanoparticles (PPaP) in the range of 61–99 nm. These nanoparticles were found to have significantly lower toxicity in vitro than the native PEI. GFP expression in cells mediated by PPaP (8.1%)/DNA complex was found to be ∼1.1–4.8 folds higher compared to GenePORTER 2™, Lipofectamine™, Superfect™ and native PEI in HeLa, HEK293 and CHO cells. FACS analysis on HeLa cells revealed ∼62% transfected cells, whereas, in the case of the GenePORTER 2™ transfection reagent, transfected cells were found to be ∼36%. Intracellular trafficking in HeLa cells showed a significant population of PPaP (8.1%) nanoparticles and their DNA complex in nucleus after 1 h of treatment. Also, efficient delivery of GFP specific siRNA resulted in ∼71% suppression of the target gene. DNase protection assay revealed that ∼78% of complexed DNA was protected by PPaP(8.1%) nanoparticles even after 2 h of treatment. In vivo transgene expression studies in Balb/c mice showed significantly higher expression in the spleen. The results advocate the potential of PPaP nanoparticles as efficient carriers of nucleic acids in vivo.

Surface modification of crosslinked dextran nanoparticles influences transfection efficiency of dextran–polyethylenimine nanocomposites

Dextran–PEI grafted nanoparticles as transfection agents have been reported, however, with limited success due to their large size, poor buffering capacity and inadequate availability of charge for competent binding to DNA. To address these concerns, dextran was first crosslinked with 1,4-butanediol diglycidyl ether (BDE), a commercially available homobifunctional crosslinker, to form its nanoparticles (DN), which were then partially oxidized with sodium periodate to generate aldehyde functionalities on them. The resultant nanoparticles were grafted with branched polyethylenimine (bPEI, 25 kDa) to obtain a series of DN–PEI nanocomposites (DP), having comparatively smaller size, enhanced buffering capacity and competent binding ability with DNA. Subsequent to biophysical characterization, these nanocomposites were evaluated for their transfection efficiency in serum and serum-free environments and toxicity in various mammalian cell lines. A significantly (p < 0.05) improved transfection efficiency of these nanocomposites concurrent with their minimal cytotoxicity as compared to the selected commercial transfection agents and native PEI was observed. These results were further validated by intracellular trafficking, wherein DP4 (the best working system in the series) was able to carry the DNA inside the nucleus after 1 h of the addition of the complex. The in vivo gene expression profile of the DP4/DNA complex in male Balb/c mice exhibited maximum expression in their spleen tissue raising promise of using these nanocomposites as improved non-viral transfection agents.

Polyethylenimine–PEG coated albumin nanoparticles for BMP-2 delivery

Bone Morphogenetic Protein-2 (BMP-2) plays an important role in stimulating new bone formation, and has been utilized in clinical bone repair by implantation. In this study, we report a nanoparticulate (NP) system for BMP-2 delivery based on bovine serum albumin (BSA) NPs stabilized with a poly(ethylene glycol) modified polyethylenimine (PEI–PEG) coating. PEI–PEG with different PEG substitutions were synthesized, and the cell viability assay showed PEG substitution greatly reduced the cytotoxicity of the native PEI. Furthermore, PEI–PEG coated BSA NPs demonstrated smaller size and decreased zeta potential compared to PEI-coated NPs. The bioactivity of the encapsulated BMP-2 and the toxicity of PEI–PEG coated NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using human C2C12 cells. The results indicated that BMP-2 remained bioactive in NPs and PEI–PEG coating was advantageous in reducing the NP toxicity as compared to PEI. A 7-day pharmacokinetics study showed the BMP-2 retention in PEI–PEG coated NPs was similar to the uncoated NPs, but lower than that of the PEI-coated NPs. The osteoinductivity of BMP-2 delivered in NPs was determined by subcutaneous implantation in rats, and the results revealed that PEI–PEG coated BSA NPs induced significant de novo bone formation after implantation, while PEI-coated NPs demonstrated much less bone formation. We conclude that BMP-2 delivered by PEGylated PEI-coated BSA NPs displays favorable biocompatibility and promotes new bone formation after implantation.

Polyethylenimine derived nanoparticles for efficient gene delivery

Introduction of therapeutic genes into the cells of an organism in a safe and efficient way has become a challenging task in non-viral mediated gene therapy. Here, branched polyethylenimine (bPEI, 25 kDa) was converted into nanoparticles through electrostatic interactions with anionic polysaccharides (e.g. alginic acid, Al and hyaluronic acid, HA). A small library of PEI-Al and PEI-HA nanoparticles was synthesized by varying the amounts of anionic polysaccharides and evaluated in terms of their size, surface charge, cytotoxicity, transfection efficiency, etc. Both the series of nanoparticles exhibited higher cell viability and transfection efficiency as compared to native PEI and the standard transfection reagents. In vivo targeting efficacy of PEI-HA(4.6%) nanoparticles was examined in tumor induced mice.

Influence of acyl chain length on transfection mediated by acylated PEI nanoparticles

Polyethylenimine (750 kDa) has been derivatized to influence the proton sponge mechanism and hydrophobic–hydrophilic balance. The polymer was acylated using acid anhydrides of varying carbon chain length, followed by cross-linking with PEG-bis-P to form compact nanoparticles. The chemical linkages in the particles were characterized by FTIR and NMR spectroscopy. The hydrodynamic diameter of nanoparticles was found to be in the range of 83.5–124 nm. AFM imaging of native and DNA-loaded nanoparticles revealed highly compact and spherical shape. The positive surface charge on particles decreased with the increase in percentage of acylation and also on complexing with DNA. The buffering capacity of PEI was reduced considerably on preparing acylated nanoparticles. The nanoparticles formed stable complexes with DNA and higher weight ratios were required for formation of electro-neutral complexes. Further, these nanoparticles were investigated for their gene delivery efficacy on COS-1 cells. It was found that acylated PEI nanoparticles were 5–12-fold more efficient transfecting agents as compared to native PEI (750 kDa) and commercially available transfecting agent lipofectin. The MTT colorimetric assay revealed of considerable reduction in toxicity of acylated PEI nanoparticles as compared PEI. Of all the systems prepared, nanoparticles with 30% acylation using propionic anhydride were found to be the most efficient in in vitro transfection.

RETRACTED: Imidazolyl-PEI modified nanoparticles for enhanced gene delivery

The derivatives of polyethylenimine (PEI 25 and 750kDa) were synthesized by partially substituting their amino groups with imidazolyl moieties. The series of imidazolyl-PEIs thus obtained were cross-linked with polyethylene glycol (PEG) to get imidazolyl-PEI-PEG nanoparticles (IPP). The component of hydrophobicity was introduced by grafting the lauryl groups in the maximal substituted IPP nanoparticles (IPPL). The nanoparticles were characterized with respect to DNA interaction, hydrodynamic diameter, zeta potential, in vitro cytotoxicity and transfection efficiency on model cell lines. The IPP and IPPL nanoparticles formed a loose complex with DNA compared to the corresponding native PEI, leading to more efficient unpackaging of DNA. The DNA loading capacity of IPP and IPPL nanoparticles was also lower compared to PEI. The imidazolyl substitution improved the gene delivery efficiency of PEI (750kDa) by nine- to ten-fold and PEI (25kDa) by three- to four-fold. At maximum transfection efficiency, the zeta potential of nanoparticles was positive after forming a complex with DNA. The maximum level of reporter gene expression was mediated by IPPL nanoparticles in both the series. The cytotoxicity, another pertinent problem with cationic polymers, was also negligible in case of IPP and IPPL nanoparticles.

PEI-alginate nanocomposites as efficient in vitro gene transfection agents

The positive charge on PEI was partially shielded by forming ionic nanocomposites with a polysaccharide, alginic acid, in aqueous solution, bypassing tedious chemical synthesis. The content of alginic acid was varied systematically to obtain a series of nanocomposites. The nanocomposites were first characterized by assessing the surface charge (zeta potential), size (DLS) and morphology (AFM) followed by evaluation for their DNA interaction ability, cytotoxicity and transfection efficiency on various cell lines. The transfection efficiency of PEI-alginate (6.26%) nanocomposites improved dramatically (2–16-fold over native PEI) in all the cell lines studied. However, a decrease in transfection efficiency was observed on deviating from this optimal concentration of alginic acid in nanocomposites. Cytotoxicity of PEI-alginate/DNA complexes was nearly abolished on increasing the concentration of alginic acid in nanocomposites. PEI-alginate (6.26%) nanocomposites also delivered SiRNAs efficiently into mammalian cells, resulting in 80% suppression of GFP expression. The cellular uptake and endosomal escape of PEI-alginate nanocomposites and PEI were found to follow a similar route when transfection was carried out in presence of chloroquine, bafilomycin A1, cytochalasin B and methyl-β-cyclodextrin. The results demonstrate a versatile vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids.

Analysis of Hepatic Disposition of Native and Galactosylated Polyethylenimine Complexed with Plasmid DNA in Perfused Rat Liver

We studied the intrahepatic disposition characteristics of galactosylated polyethylenimine (Gal-PEI)/plasmid DNA (pDNA) complexes using rat liver perfusion experiment. After intraportal administration, transfection activity in liver of Gal-PEI complexes was approximately 26-fold higher than that of native PEI complexes. To evaluate the relationship between hepatic gene expression and disposition profiles, hepatic disposition of Gal-PEI complexes were pharmacokinetically analyzed by use of perfused rat liver, which enables uptake characteristics intrinsic to the liver to be elucidated. Moment analysis revealed that both complexes exhibited very high single-pass extraction. To characterize each kinetic process in hepatic uptake of Gal-PEI complexes, their outflow profiles were analyzed based on a two-compartment dispersion model. Consequently, the tissue binding affinity of Gal-PEI complexes was 3.0-fold larger than that of native PEI complexes, suggesting the increasing of hepatic binding affinity much enhanced the hepatic gene transfection efficiency. In contrast, galactosylation of PEI did not affected internalization (and/or sequestration) rate.