Suchergebnisse

We designed niosomes based on three lipids that differed only in the polar-head group to analyze their influence on the transfection efficiency. These lipids were characterized by small-angle X-ray scattering before being incorporated into the niosomes which were characterized in terms of pKa, size, zeta potential, morphology and physical stability. Nioplexes were obtained upon the addition of a plasmid. Different ratios (w/w) were selected to analyze the influence of this parameter on size, charge and the ability to condense, release and protect the DNA. In vitro transfection experiments were performed in HEK-293, ARPE-19 and MSC-D1 cells. Our results show that the chemical composition of the cationic head-group clearly affects the physicochemical parameters of the niosomes and especially the transfection efficiency. Only niosomes based on cationic lipids with a dimethyl amino head group (lipid 3) showed a transfection capacity when compared with their counterparts amino (lipid 1) and tripeptide head-groups (lipid 2). Regarding cell viability, we clearly observed that nioplexes based on the cationic lipid 3 had a more deleterious effect than their counterparts, especially in ARPE-19 cells at 20/1 and 30/1 ratios. Similar studies could be extended to other series of cationic lipids in order to progress in the research on safe and efficient non-viral vectors for gene delivery purposes. ; This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32), the National Council of Science and Technology (CONAYT), Mexico, Reg. # 217101, the Spanish Ministry of Education (Grant CTQ2010-20541, CTQ2010-14897), the Basque Government (Department of Education, University and Research, predoctoral BFI-2011-2226 grant), the Generalitat de Catalunya (2009SGR208, 2009SGR1331) and the Instituto de Salud Carlos III. Technical and human support provided by SGIker (UPV/EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the platform for Drug Formulation (NANBIOSIS) CIBER-BBN. ; Peer reviewed

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. ; This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32 and UFI11/35), the Basque Government (Department of Education, GIC-12/150 to J.S, University and Research, for the predoctoral BFI-2011-2226 fellowship, and Department of Industry SAIOTEK S-PE13UN193) and the National Council of Science and Technology (CONACYT, Mexico, Reg. 217101). Technical and human support provided by SGIker (UPV/EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the ICTS "NANBIOSIS", more specifically, by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU). ; Peer reviewed