Effective and safe in vivo gene delivery based on polyglutamic acid complexes with heterocyclic amine modified-polyethylenimine (original) (raw)
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Biomaterials, 2007
High-molecular-weight polyethylenimine (25 kDa, PEI25k) is one of the most common cationic polymers utilized in non-viral gene therapy. However, its methylene backbone (-CH 2 CH 2 N x -) and high charge density can result in poor biodegradability and high toxicity to cells. We hypothesize that optimizing the polymer length and charge density of PEI analogues may result in decreased toxicity and higher transfection efficiency, and improved biocompatibility in vivo. A series of PEI analogues with controlled molecular weight and charge density were synthesized by grafting low-molecular-weight PEI800 (800 Da) to a polyaspartate peptide backbone of varying degrees of polymerization. The optimum polymer had a degree of polymerization of 65 with an average of 16 PEI800 groups conjugated to it. All of the polycations investigated in the study caused inflammation and apoptosis/necrosis in the liver and spleen of rodents 24 h post-injection; however, by day 5, the optimized poly(aspartate-g-PEI800) polymer and PEI800 did not show tissue damage or apoptosis, whereas PEI25k exhibited evidence of apoptosis/necrosis in the kidneys and spleen. Our study points to the need to optimize gene carriers to minimize toxicity, especially important for the safe delivery of therapeutic genes to explicit organs. r
A review of the developments of characteristics of PEI derivatives for gene delivery applications
Redox-active stimuli have gained a great deal of interest as an indicating factor for designing bioresponsive matrices in gene delivery. Hence, a wide range of gene carriers has been designed incorporating the redox-stimuli characteristics. The most important type of gene carriers is the class of redox responsive polymers. Among them, disulfide incorporated redox-responsive poly- ethyleneimine (PEI) and its derivatives, as a result of their outstanding DNA entrapping characteristics and their intrinsic endosomo- lytic activity, have attracted considerable attention in recent studies. The review presents the main developments of the characteristics of PEI derivatives and their applications in gene delivery. It is found that despite the uniquely stated characteristics, the noncleavable structure of conventional PEI (high molecular weight PEI: 25k), which makes it a nondegradable material, as well as the frequent inclusion of positively charged amino groups, which reduces its blood circulation period, render conventional PEI a very toxic mate- rial for gene-delivery applications. The extremely high cellular toxicity of conventional PEI has restricted its administration for real in-vivo physiological media. Recent studies have shown that employing low molecular weight PEI cross-linked by disulfide linkages (SS-PEI) and assembling low molecular weight disulfide linkages PEI (LMW SS-PEI) with bio-detachable anionic groups were two successful approaches for increasing bioavailability of the PEI-based gene carriers, while keeping outstanding cellular transfection.
Long-term efficient gene delivery using polyethylenimine with modified Tat peptide
Biomaterials, 2014
Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (z5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (z7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.
A mannosylated cell-penetrating peptide-graft-polyethylenimine as a gene delivery vector
Biomaterials, 2014
Polyethylenimine (PEI) is widely applied in non-viral gene delivery vectors. PEI with high molecular weight is highly effective in gene transfection but is high cytotoxic. Conversely, PEI with low molecular weight displays lower cytotoxicity but less delivering efficiency. To overcome this issue, a novel copolymer with mannosylated, a cell-penetrating peptide (CPP), grafting into PEI with molecular weight of 1800 (Man-PEI 1800 -CPP) were prepared in this study to target antigen-presenting cells (APCs) with mannose receptors and enhance transfection efficiency with grafting CPP. The copolymer was characterized by 1 H NMR and FTIR. Spherical nanoparticles were formed with diameters of about 80e250 nm by mixing the copolymer and DNA at various charge ratios of copolymer/DNA(N/P). Gel retardation assays indicated that Man-PEI 1800 -CPP polymers efficiently condensed DNA at low N/P ratios. Cytotoxicity studies showed that Man-PEI 1800 -CPP/DNA complexes maintained in a high percentage of cell viability compared to the PEI with molecular weight of 25 k (PEI 25k ). Laser scan confocal microscopy and flow cytometry confirmed that Man-PEI 1800 -CPP/DNA complexes resulted in higher cell uptake efficiency on DC2.4 cells than on Hela cells line. The transfection efficiency of Man-PEI 1800 -CPP was significantly higher than that of PEI 25k on DC2.4 cells. More importantly, the complexes were mainly distributed in the epidermis and dermis of skin and targeted on splenocytes after percutaneous coating based on microneedles in vivo. These results indicated that Man-PEI 1800 -CPP was a potential APCs targeted of non-virus vector for gene therapy.
Highly Effective Gene Transfection In Vivo by Alkylated Polyethylenimine
Journal of Drug Delivery, 2011
We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp.) while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.
European Journal of Pharmaceutics and Biopharmaceutics, 2011
Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siR-NA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.
Efficient gene delivery of primary human cells using peptide linked polyethylenimine polymer hybrid
Biomaterials, 2011
Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nanogels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly, when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of 'reactive oxygen species' (ROS) in the presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress.
Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotox-icity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotox-icity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5-to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex of LMW-PEI and CA as promising generic nonviral gene carriers.