Toxicity Of Polyethylenimine Research Articles

Polyethylenimine-associated cerium oxide nanoparticles: A novel promising gene delivery vector

AimsThe development of highly efficient and low toxic non-viral gene delivery vectors is the most challenging issues for successful application of gene therapy. A particular focus has been on understanding structure-activity relationships for transfection activity and toxicity of polyethylenimine (PEI). During the last decade, the use of cerium oxide nanoparticles (CeO2-NPs) in biomedicine has attracted much attention due to their pH-dependent antioxidant activity. CeO2-NPs provide protection normal cells from various forms of reactive oxygen species, but possess innate cytotoxicity and apoptosis to cancer cells. The purpose of this study was to design a new class of gene carriers by low molecular weight PEI (B-PEI 10 kDa) coordination onto CeO2-NPs. Main methodsB-PEI 10 kDa was conjugated to CeO2-NPs by Epichlorohydrin linker. Transfection efficiency, cytotoxic and apoptotic effects of pDNA-PEI-CeO2 NPs were evaluated on WEHI 164 cancer cells and normal L929 cells lines. Key findingsPEI-CeO2 NPs was able to condense the pDNA at carrier/plasmid (C/P) weight ratios of 0.5. The size and zeta potential of pDNA-PEI-CeO2 NPs were 124 ± 7 nm and 22 ± 2 mV, respectively. The transfection efficacy of synthesized pDNA-PEI-CeO2 NPs improved and the cytotoxicity was decreased compared to pDNA-PEI. Moreover, pDNA-PEI-CeO2 NPs induced more apoptosis than unmodified PEI and CeO2-NPs control groups. pDNA-PEI-CeO2 NPs displayed more transfection, cytotoxicity, and apoptosis in WEHI 164 cancer cells than normal L929 cells. SignificanceIn conclusion, PEI-CeO2 nanocarriers could act as a potential candidate for gene and drug delivery to cancerous and tumor cells.

Improved biocompatibility of polyethylenimine (PEI) as a gene carrier by conjugating urocanic acid: In vitro and in vivo

Polyethylenimine (PEI) is a promising gene carrier among polymeric vectors because of its high transfection efficiency, buffering capacity, which is responsible for its escape from endosomes, facility of modification, reasonable price and wide range of molecular weight. However, toxicity of PEI is an important obstacle to overcome for using it as a gene carrier in gene therapy protocols. In our study, PEI (10 kDa) was modified with urocanic acid, which has an imidazole ring (PEIU). This modification of PEI played an important role by improving transfection efficiency and reducing toxicity. The PEIU was condensed with DNA in different nitrogen of polycation/phosphate of DNA (N/P) ratios starting from 1.25. The PEIU/DNA complexes formed smaller (60-80 versus 100-400 nm) and less dispersible particles than PEI/DNA complexes. Cell viability test showed that in all the transfection experiments PEIU was always found less toxic than the PEI on three different cell-lines, Cos-7, HeLa, and 293T. The PEIU demonstrated improved biocompatibility as compared to the PEI. The in vitro transfection experiments showed that the PEIU displayed similar or lower transfection efficiencies than the PEI in the absence of serum. In medium complemented with serum, the PEIU had higher transfection efficiencies than the PEI. In vivo transfection experiments were carried out with intravenous and subcutaneous injections into mice. Five different organs were taken and lysed for quantification of expressed GFP. The quantification of the fluorescence of these organs exhibited that the PEIU has better transfection efficiency in in vivo experiments compared to the PEI. This study generated a strong evidence indicating that PEIU can be considered as a promising versatile gene carrier because of its biocompatibility and good transfection efficiency.

Folate-targeted liposome encapsulating chitosan/oligonucleotide polyplexes for tumor targeting.

We previously reported that a liposome encapsulating polyethylenimine/oligonucleotides is suitable for in vivo delivery of nucleic acid therapeutics. However, toxicity of polyethylenimine is an obstacle in clinical application. To develop a liposome encapsulating polyplexes applicable to clinical use, we proposed to replace polyethylenimine with chitosan and thus constructed the liposome encapsulating low-molecular weight chitosan (LMWC)/oligonucleotide (ODN) polyplexes [LS(CO)]. ODN was completely complexed to LMWC at pH5.5 and an N/P ratio 10 with a positive zeta potential of 19.81 ± 1.11. The positively charged polyplexes were encapsulated into anionic liposome by membrane extrusion. Folate-targeted liposome encapsulating LMWC/ODN complex [FLS(CO)] was prepared by adding folate-conjugated phospholipid. The resulting LS(CO) and FLS(CO) were characterized with respect to size distribution, zeta potential, and colloidal stability. The LS(CO) and FLS(CO) were also evaluated for in vitro cellular uptake and cytotoxicity. The LS(CO) and FLS(CO) showed a narrow size distribution with a mean diameter of about 130nm and neutral zeta potentials and remained stable for 7days in 0.15-M NaCl at room temperature. FLS(CO) showed higher cellular uptake than LS(CO) in B16F10 murine melanoma cells. Furthermore, LS(CO) showed less toxicity as compared to liposome encapsulating polyethylenimine/oligonucleotides, representing a biocompatible nanocarrier of oligonucleotide therapeutics.

Nanoparticles Composed by Oligochitosan and Polyethylenimine for Gene Delivery

Cationic carriers including polyethylenimine, liposomes, and chitosan have been used to transfer plasmid DNA in vitro by condensing anionic DNA. Here, oligochitosan (OC) was found to have capacity for in-vitro gene delivery in four cell lines. Plasmid containing green fluorescent protein (GFP) gene was used as a reporter gene. The transfection efficacy and cell viability of the transfection vehicles were analyzed by using a high-throughput image analyzer. We found that DNA polyplexes formed by high dosage of OC could be efficiently delivered into the cells. The combination of OC and polyethylenimine (PEI) were found to significantly enhance the fluorescence protein expression. The introduction of oligochitosan in PEI-mediated transfection could increase the transfection efficacy and could reduce the toxicity of PEI. Additionally, the synergistic effects of PEI and OC were confirmed in CHO, Caco2, Hep-SK, and 3T3 cell lines. The detailed mechanism of PEI and oligochitosan on transfection was investigated by using gel retardation and DNase degradation experiments. A facile and inexpensive construction of gene delivering vehicles was developed herein by using oligochitosan and PEI.

Pharmacokinetics and bone formation by BMP-2 entrapped in polyethylenimine-coated albumin nanoparticles

The osteoinductive growth factor, bone morphogenetic protein-2 (BMP-2), is capable of inducing de novo bone formation after implantation. A nanoparticulate (NP) system was developed for BMP-2 delivery based on NPs fabricated from bovine serum albumin (BSA) and stabilized by polyethylenimine (PEI) coating. In this study, the pharmacokinetics and osteoinductivity of BMP-2 delivered with different BSA NP formulations were determined by subcutaneous implantation in rats. A 7-day pharmacokinetics study showed that PEI coating on NPs effectively reduced the initial burst release of BMP-2 and prolonged the BMP-2 retention at implantation site. However, the uncoated BMP-2 NPs (BMP-2 loading of 1.44% w/w) were able to induce a robust ectopic bone formation, while no bone formation was found by the BMP-2 NPs coated with PEI. The toxicity of the PEI used for NP coating was determined to be the reason for lack of osteoinduction. Increasing BMP-2 loading (up to 5.76% w/w) was then employed to formulate NPs with lower PEI content; the higher BMP-2 loading was found to better promote induction of de novo bone. Our findings indicated that PEI coating on BSA NPs was effective for controlling BMP-2 release from NPs, but the toxicity of cationic PEI was a concern for the osteoinductive activity, which should be alleviated by further optimization of NP formulations.

Effect of acyl chain length on transfection efficiency and toxicity of polyethylenimine

Polyethylenimine (PEI) is an efficient nonviral gene delivery vector because of its high buffering capacity and DNA condensation ability. In our study, the amino groups on the polymeric backbone were acylated using acetic or propionic anhydride to alter the protonation behaviour and the hydrophilic/hydrophobic balance of the polymer. The concentration of acylated primary amines was determined using trinitrobenzene sulphonic acid assay. Results showed that our modified polymers had lower buffering capacities in solutions compared to PEI. The polymers were complexed with plasmid encoding enhanced green fluorescent protein at three different ratios (1:1, 1:2 and 1:10 w/w DNA to polymer) to form polyplexes and their toxicities and transfection efficiencies were evaluated in HEK 293 cells. Acylation reduced the number of primary amines on the polymer and the surface charge, improving haemocompatibility and reducing cytotoxicity. The reduction in the concentration of amino groups helped to optimise DNA compaction and facilitated polyplex dissociation in the cell, which increased transfection efficiency of the modified polymers compared to the parent polymer. Polymers with buffering capacities greater than 50% and less than 80% relative to PEI, showed higher transfection efficiencies than PEI. The propionic anhydride modified polymers had appropriate interactions with DNA which provided both DNA compaction and polyplex dissociation. These systems interacted better with the cell membrane because of their slightly higher lipophilicity and formed polyplexes which were less cytotoxic than polyplexes of acetic anhydride modified polymers. Among the vectors tested, 1:0.3 mol/mol PEI:propionic anhydride in a 1:2 w/w DNA:polymer composition provided the best transfection system with improved transfection efficiency and reduced cytotoxicity.

Subcutaneous Peripheral Injection of Cationized Gelatin/DNA Polyplexes As a Platform for Non-viral Gene Transfer to Sensory Neurons

Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.

Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures.

In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3). Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used. Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery. Transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found. In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines. However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF).