ABSTRACT
Nanotechnology based non-viral vectors hold great promise to deliver therapeutic genes into the central nervous system (CNS) in a safe and controlled way. Vascular endothelial growth factor (VEGF) is a potential therapeutic gene candidate for CNS disorders due to its specific roles in brain angiogenesis and neuroprotection. In this work, we elaborated three different non-viral vectors based on magnetic, cationic lipid and polymeric nanoparticles complexed to the phVEGF165aIRESGFP plasmid, which codifies the VEGF protein -extracellular- and the green fluorescent protein (GFP) -intracellular-. Nanoparticles and corresponding nanoplexes -magnetoplexes, lipoplexes and polyplexes- were characterized in terms of size, zeta potential, polydispersity index, morphology and ability to bind, release and protect DNA. Transfection efficiencies of nanoplexes were measured in terms of percentage of GFP expressing cells, mean fluorescent intensity (MFI) and VEGF (ng/ml) production in HEK293, C6 and primary neuronal culture cells. Magnetoplexes showed the highest transfection efficiencies in C6, followed by lipoplexes, and in primary neuronal culture cells, followed by polyplexes. Lipoplexes were the most efficient in HEK293 cells, followed by magnetoplexes. The biological activity of VEGF was confirmed by its proliferative effect in HUVEC cells. Overall, these results provide new insights for VEGF gene delivery into CNS cells using non-viral vectors.
Subject(s)
Gene Transfer Techniques , Genetic Vectors/metabolism , Lipids/pharmacokinetics , Magnetite Nanoparticles , Neurons/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cells, Cultured , Drug Compounding , Genetic Vectors/administration & dosage , Genetic Vectors/chemistry , HEK293 Cells , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Lipids/administration & dosage , Lipids/chemistry , Magnetite Nanoparticles/administration & dosage , Magnetite Nanoparticles/chemistry , Neurons/drug effects , Rats , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor A/administration & dosage , Vascular Endothelial Growth Factor A/chemistryABSTRACT
Niosome formulations for gene delivery purposes are based on nonionic surfactants, helper lipids, and cationic lipids that interact electrostatically with negatively charged DNA molecules to form the so-called nioplexes. Niosomes are elaborated by different techniques, such as solvent emulsion-evaporation, thin film hydration, hand-shaking, dissolvent injection, and microfluidization method, among many others. In this chapter, we have described some protocols for the elaboration of niosomes and nioplexes and their physicochemical characterization that guarantees the quality criteria of the formulation in terms of size, morphology, ζ-potential, and stability.
Subject(s)
Cations/chemistry , Chemistry, Pharmaceutical/methods , Liposomes/chemical synthesis , DNA/chemistry , DNA/genetics , Gene Transfer Techniques , Liposomes/chemistry , Particle Size , Surface-Active Agents/chemistry , TransfectionABSTRACT
In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.
Subject(s)
Epithelium/metabolism , Gene Transfer Techniques , Lipids/chemistry , Retina/metabolism , Cell Line , Cell Survival , DNA/administration & dosage , Endocytosis , Humans , Liposomes , PlasmidsABSTRACT
The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies requires a profound knowledge of the whole transfection process. In this work, we elaborated and characterized in terms of size and zeta potential three different nonviral vectors: lipoplexes (144 nm; -29.13 mV), nioplexes (142.5 nm; +35.4 mV), and polyplexes (294.8 nm; +15.1 mV). We compared the transfection efficiency, cellular uptake, and intracellular trafficking of the three vectors in NT2 cell line. Lipoplexes exhibited the highest percentages of EGFP positive cells. The values obtained with polyplexes were lower compared to lipoplexes but higher than the percentages obtained with nioplexes. Cellular uptake results had a clear correlation with respect to the corresponding transfection efficiencies. Regarding the endocytosis mechanism, lipoplexes enter in the cell, mainly, via clathrin-mediated endocytosis (CME) while polyplexes via caveolae-mediated endocytosis (CvME). Nioplexes were discarded for this experiment due to their low cellular uptake. By simulating an artificial endosome, we demonstrated that the vectors were able to release the DNA cargo once inside the late endosome. The data collected from this assay showed that at 6 h the genetic material carried by polyplexes was still located in the late endosome, while DNA carried by lipoplexes was already in the nucleus. This result indicates a faster intracellular traffic of the lipid-based vectors. Overall, our work gives new insights into the transfection process of NT2 cells by different nonviral vectors as a first step in the development of ex vivo gene therapy platform.
Subject(s)
Embryonal Carcinoma Stem Cells/metabolism , Gene Transfer Techniques , Genetic Therapy/methods , Lipids/chemistry , Liposomes/chemistry , Neurons/metabolism , Cell Survival , Embryonal Carcinoma Stem Cells/pathology , Endocytosis/physiology , Green Fluorescent Proteins/metabolism , Humans , Neurons/pathology , Plasmids/administration & dosage , Polymers/chemistry , TransfectionABSTRACT
Ultrapure oligochitosans have been recently reported as efficient non-viral vectors for the delivery of pCMS-EGFP plasmid (5.5kbp) to the cornea and retina. However, the delivery of oncolytic adenoviral plasmids (40kbp) represents a unique challenge. In this work, we elaborated self assembled O15 and O25 UOC/pAdTLRGD polyplexes, and we studied the influence of the N/P ratio, the pH of the transfection medium and the salt concentration on the particle size and zeta potential by an orthogonal experimental design. All polyplexes showed a particle size lower than 200nm and a positive zeta potential. These parameters were influenced by the N/P ratio, salt concentration, and pH of the transfection medium. The selected polyplexes were able to bind, release, and protect the plasmid from DNase degradation. Transfection experiments in HEK293 and A549 cell lines demonstrated that UOC/pAdTLRGD polyplexes were able to deliver the plasmid and transfect both cell lines. These results suggest that O15 and O25 UOC based polyplexes are suitable for future in vivo applications.
Subject(s)
Adenoviridae/genetics , Chitin/analogs & derivatives , DNA/administration & dosage , Polymers/administration & dosage , Cell Line, Tumor , Chitin/administration & dosage , Chitosan , Genetic Vectors , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Oligosaccharides , Particle Size , Plasmids , Transfection/methodsABSTRACT
CONTEXT: Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications. OBJECTIVE: The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells. MATERIALS AND METHODS: We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay. RESULTS AND DISCUSSION: The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000™ showed decreased cell viability. CONCLUSION: This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.
