ABSTRACT Cationic Solid Lipid Nanoparticles (cSLNs) are one of the most promising nonviral vectors for gene therapy and DNA / RNA delivery. The aim of this thesis is the development and research of new formulations of SLNs that will improve the formulation already existing in the research group. Thus, after different experiments, SLNs with cholesteryl oleate as matrix lipid have been developed, improving the transfection efficiency of the nanoparticles without affecting cell viability.
For this development, 5 different lyophilized formulations with different proportions of cholesteryl oleate were manufactured, to study and understand its influence depending on the amount to be incorporated in morphology, stability, physicochemical characteristics and biological characteristics (such as efficiency of transfection and cytotoxicity). Thus, this study would also allow identifying the most appropriate formulation to continue the investigation. The Reference 14 was identified as the formulation with the best physicochemical characteristics and efficiency of transfection in assays in vitro. The composition of Reference 14 is 600 mg of octadecylamine, 300 mg of cholesteryl oleate, 200 mg of stearic acid and 100 mg of poloxamer 188.
Once a definitive formulation was identified, the next step was to study more thorougly its physical and chemical characteristics once the lyophilized was reconstituted, applying different techniques such as DSC, DRX or TEM microscopy. The objective of this complete characterization was to know the formulation and its key process to be able to reproduce exactly the formulation. In addition, stability studies were conducted, and it was observed that lyophilized SLNs were stable for at least 1 year. This improved the stability of the SLNs in suspension, which have a maximum stability of 15 days at 4 ° C.
Subsequently, different tests were performed in the Biology laboratory to test the efficiency and safety of the SLNs in different cell lines, such as HeLa, HEK293T, Jurkat or A549 cells. Thus, it was found that Reference 14 was capable of transfecting about 45% of HEK293T cells in vitro, and was capable of transfecting siRNA giving about 35% silencing in HeLa cells. These results were also confirmed by confocal microscopy. Finally, in vitro experiments were performed that indicated a possible therapeutic application of the formulation: the treatment of flavivirus infections such as Dengue virus, giving a decrease of about 85% of the infection in A549 cells treated with siRNA by silencing RPLP1/2 transfected by reference 14. This promising result opens avenues to deepen in such a line, as well as to find other therapeutic applications of this formulation and make the transition to the design of future in vivo experiments.
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