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RNA interference (RNAi) is a promising strategy to treat the chronic infection by hepatitis C virus (HCV). The objective of this work was to develop a non-viral vector based on solid lipid nanoparticles (SLN) and RNAi to inhibit the internal ribosome entry site (IRES) mechanism of the HCV. The vectors were prepared with SLN, protamine, hylauronic acid (HA) or dextran (DX), and a short-hairpin RNA expression plasmid targeted to the stem loop II of the 5' UTR (shRNA74). The particle size, surface charge, and capacity to bind, release and protect the shRNA74 against nucleases were evaluated. Cell uptake, silencing capacity and cell viability were evaluated in HepG2 cells. All the vectors presented particle size in the range of nanometers and positive surface charge, and they were able to protect the shRNA74 against DNase. An effective and rapid uptake into the cells was observed. Silencing capacity ranged from 3% to 67% depending on the presence of DX or HA in the vector, the shRNA74 to SLN ratio, and the shRNA74 dose. Vectors prepared with HA showed to be twice more effective than those prepared with DX. Differences in the intracellular trafficking may justify the higher efficacy of the HA-prepared vectors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The introduction of therapeutic genes in target tissues is considered as a novel tool for the treatment of several diseases. We have developed nanoparticles consisting on SLNs, protamine (P) and hyaluronic acid (HA) as carrier for gene therapy. Stable complexes positively charged and with a particle size ranging from 240 nm to 340 nm were obtained. Transfection studies in ARPE-19 and HEK-293 cells showed the versatility of vectors to efficiently transfect cells with different division rate, widening the potential applications of SLN-based vectors. In ARPE 19cells, the incorporation of P and HA induced almost a 7-fold increase in the transfection capacity of SLNs. The CD44 inhibition studies suggested the participation of this receptor in the internalization of the vectors in this cell line. The intracellular disposition of DNA showed that the HA is able to modulate the high degree of condensation of DNA due to the protamine inside the cells; an important fact, if the vector is uptaken via non-degradative endocytosis. Besides, the therapeutic plasmid which encodes the protein retinoschisin was employed achieving a positive transfection in ARPE-19 cells, showing a promising application of this new non-viral system for the treatment of X-linked juvenile retinoschisis by gene therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of present work is to evaluate the transfection capacity of a new multicomponent system based on dextran (Dex), protamine (Prot), and solid lipid nanoparticles (SLN) after intravenous administration to mice. The vectors containing the pCMS-EGFP plasmid were characterized in terms of particle size and surface charge. In vitro transfection capacity and cell viability were studied in four cell lines, and compared with the transfection capacity of SLN without dextran and protamine. Transfection capacity was related to the endocytosis mechanism: caveolae or clathrin. The Dex-Prot-DNA-SLN vector showed a higher transfection capacity in those cells with a high ratio of activity of clathrin/caveolae-mediated endocytosis. However, the complex prepared without dextran and protamine (DNA-SLN) was more effective in those cells with a high ratio of activity of caveolae/clathrin-mediated endocytosis. The interaction with erythrocytes and the potential hemolytic effect were also checked. The Dex-Prot-DNA-SLN vector showed no agglutination of erythrocytes, probably due to the presence of dextran. After intravenous administration to BALB/c mice, the vector was able to induce the expression of the green fluorescent protein in liver, spleen and lungs, and the protein expression was maintained for at least 7 days. Although additional studies are necessary, this work reveals the promising potential of this new gene delivery system for the treatment of genetic and non-genetic diseases through gene therapy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Naked plasmid DNA is a powerful tool for gene therapy, but it is rapidly eliminated from the circulation after intravenous administration. Therefore, the development of optimized DNA delivery systems is necessary for its successful clinical use. Solid lipid nanoparticles (SLNs) have demonstrated transfection capacity in vitro, but their application for gene delivery has not been conveniently investigated in vivo. We aimed to evaluate the capacity of SLN-DNA vectors to transfect in vivo after intravenous administration to mice. The SLNs, composed of Precirol ATO 5, DOTAP and Tween 80 were complexed with the plasmid pCMS-EGFP which encodes the enhanced green fluorescent protein (EGFP). The resulting systems were characterized in vitro showing a mean particle size of 276 nm, superficial charge of +28 mV, the ability to protect the plasmid and transfection capacity in culture cells. The intravenous administration in mice led to transfection in hepatic tissue and spleen. Protein expression was detected from the third day after administration, and it was maintained for at least 1 week. This work shows for the first time the capacity of SLN-DNA vectors to induce the expression of a foreign protein after intravenous administration, supporting the potential of SLNs for gene therapy., (2009 Elsevier B.V. All rights reserved.)