Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism.
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ABSTRACT:
SUBMITTER: Das J
PROVIDER: S-EPMC4933920 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector.MethodsA series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino)-1-propylamine (DMAPA-Glyp) and 1-(2-aminoethyl) piperazine (AEPZ-Glyp) residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid-base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA) condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney) and the CNE2 (human nasopharyngeal carcinoma) cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley rats) was evaluated to identify the safety and transfection efficiency.ResultsThe hyperbranched cationic glycogen derivatives conjugated with DMAPA and AEPZ residues were synthesized. They exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI). They were able to bind and condense pDNA to form the complexes of 100-250 nm in size. The transfection efficiency of the DMAPA-Glyp/pDNA complexes was higher than those of the AEPZ-Glyp/pDNA complexes in both the 293T and CNE2 cells, and almost equal to those of bPEI. Furthermore, pDNA could be more safely delivered to the blood vessels in brain tissue of Sprague Dawley rats by the DMAPA-Glyp derivatives, and then expressed as green fluorescence protein, compared with the control group.ConclusionThe hyperbranched cationic glycogen derivatives, especially the DMAPA-Glyp derivatives, showed high gene-transfection efficiency, good blood compatibility, and low cyto toxicity when transfected in vitro and in vivo, which are novel potential nonviral gene vectors.
| S-EPMC3917921 | biostudies-literature
Project description:Polyethylenimine (PEI) is one of the most broadly used polycations for gene delivery due to its high transfection efficiency and commercial availability but materials are cytotoxic and often polydisperse. The goal of current work is to develop an alternative family of polycations based on controlled living radical polymerization (CLRP) and to optimize the polymer structure for efficient gene delivery. In this study, well-defined poly(glycidyl methacrylate)(P(GMA)) homopolymers were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization followed by decoration using three different types of oligoamines, i.e., tetraethylenepentamine (TEPA), pentaethylenehexamine (PEHA), and tris(2-aminoethyl)amine (TREN), respectively, to generate various P(GMA-oligoamine) homopolycations. The effect of P(GMA) backbone length and structure of oligoamine on gene transfer efficiency was then determined. The optimal polymer, P(GMA-TEPA)(50), provided comparable transfection efficiency but lower cytotoxicity than PEI. P(GMA-TEPA)(50) was then used as the cationic block in diblock copolymers containing hydrophilic N-(2-hydroxypropyl) methacrylamide (HPMA) and oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA). Polyplexes of block copolymers were stable against aggregation in physiological salt condition and in Opti-MEM due to the shielding effect of P(HPMA) and P(OEGMA). However, the presence of the HPMA/OEGMA block significantly decreased the transfection efficacy of P(GMA-TEPA)(50) homopolycation. To compensate for reduced cell uptake caused by the hydrophilic shell of polyplex, the integrin-binding peptide, RGD, was conjugated to the hydrophilic chain end of P(OEGMA)(15)-b-P(GMA-TEPA)(50) copolymer by Michael-type addition reaction. At low polymer to DNA ratios, the RGD-functionalized polymer showed increased gene delivery efficiency to HeLa cells compared to analogous polymers lacking RGD.
| S-EPMC3544971 | biostudies-literature
Project description:Cyclodextrin polymers have high applicability in pharmaceutical formulations due to better biocompatibility, solubility enhancement, loading capacity and controlled drug release than their parent, cyclodextrins. The cytotoxicity and cell uptake of new cationic beta-cyclodextrin monomers and polymers were evaluated as suitable materials for nasal formulations and their protective effects on cells exposed to hydrogen peroxide were studied. PC12 and CACO-2 cells were selected as the neuronal- and epithelial-type cells, respectively, to mimic the structure of respiratory and olfactory epithelia of the nasal cavity. All cationic beta-cyclodextrin polymers tested showed dose- and time-dependent toxicity; nevertheless, at 5 µM concentration and 60 min of exposure, the quaternary-ammonium-beta-cyclodextrin soluble polymer could be recognized as nontoxic. Based on these results, a fluorescently labelled quaternary-ammonium-beta-cyclodextrin monomer and polymer were selected for uptake studies in CACO-2 cells. The monomeric and polymeric beta-cyclodextrins were internalized in the cytoplasm of CACO-2 cells; the cationic monomer showed higher permeability than the hydroxypropyl-beta-cyclodextrin, employed as comparison. Therefore, these cationic beta-cyclodextrins showed potential as excipients able to improve the nasal absorption of drugs. Furthermore, amino-beta-cyclodextrin and beta-cyclodextrin soluble polymers were able to reduce oxidative damage in PC12 and CACO-2 cells and thus could be studied as bioactive carriers or potential drugs for cell protection against oxidative stress.
| S-EPMC7407921 | biostudies-literature
Project description:The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR.
| S-EPMC4395777 | biostudies-literature
Project description:The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA-), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.
| S-EPMC3867963 | biostudies-literature
Project description:Two cationic, amphipathic peptoids (poly-N-substituted glycines) were developed as new molecular transporters, which have extensive cellullar uptake and utilize different internalization mechanisms from purely cationic polyguanidine comparators.
| S-EPMC3431920 | biostudies-literature
Project description:Receptor-independent cellular uptake of small molecule therapeutics is limited by their physical interaction with the negatively charged surface of cellular membranes. Passive diffusion through the hydrophobic membrane bilayer follows this process. Unless specific carriers exist in the biological membrane, such interactions limit therapeutics to those that are hydrophobic with modest positive charge at physiological pH. Small negatively charged molecules are therefore not efficient as therapeutics. To enable delivery of such molecules into eukaryotic cells, cationic branched polymers with tetraalkylammonium pendant groups were synthesized by copolymerization of a functional monomer (glycidyl methacrylate) with degradable and non-degradable divinyl crosslinkers in the presence of an efficient chain transfer agent, CBr4, followed by reaction of the multiple pendant epoxide groups and most of the alkyl bromide chain ends with amines. Cationic branched polymers with covalently attached fluorescent labels entered human cancerous and non-cancerous cells. The non-labeled analogues were able to carry anionic cargo (carboxyfluorescein) into the cells, while no uptake was observed in the absence of the cationic carriers. Most of the polymers were not significantly toxic at the concentrations used. This pilot study showed that cellular uptake of anionic small molecules can be promoted even in the absence of natural uptake mechanisms.
| S-EPMC5830148 | biostudies-literature
Project description:This article reports on the application of organically modified silica (ORMOSIL) nanoparticles as a nonviral vector for efficient in vivo gene delivery. Highly monodispersed, stable aqueous suspension of nanoparticles, surface-functionalized with amino groups for binding of DNA, were prepared and characterized. Stereotaxic injections of nanoparticles, complexed with plasmid DNA encoding for EGFP, into the mouse ventral midbrain and into lateral ventricle, allowed us to fluorescently visualize the extensive transfection of neuronal-like cells in substantia nigra and areas surrounding the lateral ventricle. No ORMOSIL-based toxicity was observed 4 weeks after transfection. The efficiency of transfection equaled or exceeded that obtained in studies using a viral vector. An in vivo optical imaging technique (a fiber-based confocal fluorescent imaging system) provided an effective means to show the retention of viability of the transfected cells. The ORMOSIL-mediated transfections also were used to manipulate the biology of the neural stem/progenitor cells in vivo. Transfection of a plasmid expressing the nucleus-targeting fibroblast growth factor receptor type 1 resulted in significant inhibition of the in vivo incorporation of bromodeoxyuridine into the DNA of the cells in the subventricular zone and the adjacent rostral migratory stream. This in vivo approach shows that the nuclear receptor can control the proliferation of the stem/progenitor cells in this region of the brain. The results of this nanomedicine approach using ORMOSIL nanoparticles as a nonviral gene delivery platform have a promising future direction for effective therapeutic manipulation of the neural stem/progenitor cells as well as in vivo targeted brain therapy.
| S-EPMC1181239 | biostudies-literature
Project description:The design of intracellular drug delivery vehicles demands an in-depth understanding of their internalization and function upon entering the cell to tailor the physicochemical characteristics of these platforms and achieve efficacious treatments. Polymeric cationic systems have been broadly accepted to be membrane disruptive thus being beneficial for drug delivery inside the cell. However, if excessive destabilization takes place, it can lead to adverse effects. One of the strategies used to modulate the cationic charge is the incorporation of hydrophobic moieties, thus increasing the hydrophobic content. We have demonstrated the successful synthesis of nanogels based on diethylaminoethyl methacrylate and poly(ethylene glycol) methyl ether methacrylate. Addition of the hydrophobic monomers tert-butyl methacrylate or 2-(tert-butylamino)ethyl methacrylate shows improved polymer hydrophobicity and modulation of the critical swelling pH. Here, we evaluate the cytocompatibility, uptake, and function of these membrane-destabilizing cationic methacrylated nanogels using in vitro models. The obtained results suggest that the incorporation of hydrophobic monomers decreases the cytotoxicity of the nanogels to epithelial colorectal adenocarcinoma cells. Furthermore, analysis of the internalization pathways of these vehicles using inhibitors and imaging flow cytometry showed a significant decrease in uptake when macropinocytosis/phagocytosis inhibitors were present. The membrane-disruptive abilities of the cationic polymeric nanogels were confirmed using three different models. They demonstrated to cause hemolysis in sheep erythrocytes, lactate dehydrogenase leakage from a model cell line, and disrupt giant unilamellar vesicles. These findings provide new insights of the potential of polymeric nanoformulations for intracellular delivery.
| S-EPMC6336667 | biostudies-literature
Project description:PurposeTo formulate f-MWNTs-cationic liposome hybrids for the simultaneous delivery of siPLK1 and doxorubicin to cancer cells.Methodf-MWNTs-cationic liposome hybrids were prepared by the thin film hydration method where the lipid film was hydrated with 100 μg/ml or 1 mg/ml of ox-MWNTs-NH3 (+) or MWNTs-NH3 (+) in 5% dextrose. siRNA complexation and protection ability was determined by agarose gel electrophoresis. f-MWNTs and liposome interaction was evaluated using Nile Red (NR) fluorescence spectroscopy. Cellular uptake in A549 cells was assessed by flow cytometry. Silencing of target proteins was determined by Luciferase and MTT assays. Sub-G1 analysis was performed to evaluate apoptosis following co-delivery of siPLK1 and Doxorubicin (Dox).ResultsZeta potential and siRNA complexation profile obtained for all hybrids were comparable to those achieved with cationic liposomes. ox-MWNTs-NH3 (+) showed greater extent of interaction with cationic liposomes compared to MWNTs-NH3 (+). ox-MWNTs-NH3 (+) was able to protect siRNA from nuclease-mediated degradation. Enhanced cellular uptake of both the carrier and loaded siRNA in A549 cell, were observed for this hybrid compared to the liposomal carrier. A synergistic pro-apoptotic effect was obtained when siPLK1 silencing was combined with doxorubicin treatment for the hybrid:siRNA complexes compared to the lipoplexes, in A549 cells in vitro.Conclusionsf-MWNTs-cationic liposome hybrid designed in this study can serve as a potential vehicle for the co-delivery of siRNA and cytotoxic drugs to cancer cells in vitro.
| S-EPMC4577551 | biostudies-literature