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Design of multifunctional polymeric nanoparticles for biomedical applications

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Universitat de Barcelona. Facultat de Química

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[spa] El objetivo general de esta investigación fue el diseño de nanopartículas poliméricas catiónicas, a partir de nano-emulsiones obtenidas por métodos de baja energía, para aplicaciones biomédicas. Este objetivo incluye la preparación de nano-emulsiones catiónicas de tipo aceite en agua (O/W) con métodos de baja energía y su caracterización, la preparación y caracterización de nanopartículas poliméricas catiónicas utilizando los O/W nano-emulsiones catiónicas como plantilla, la funcionalización de las nanopartículas poliméricas y estudios in vitro de las nanopartículas funcionalizadas. Se han obtenido nanopartículas catiónicas poliméricas a partir de nano-emulsiones, utilizando un polímero preformado que era incorporado en la fase oleosa. Se confirmó la posibilidad de formar complejos entre nanopartículas seleccionadas y compuestos bioactivos, tales como ácido fólico y oligonucleótido antisentido (ASO) fosforotioato. Las características (tamaño y carga superficial) de las nanopartículas y de los complejos formados con ácido fólico o ASO se hallan en un rango que permitiría su aplicación como agentes terapéuticos. Los estudios in vitro de nanopartículas y de los complejos de nanopartículas: oligonucleótido revelaron que las nanopartículas, a concentraciones iguales o inferiores a 3 mM no son tóxicas para las células HeLa, que la concentración de especies catiónicas de las nanoparticluas debe ser baja para obtener valores bajos de hemólisis y que la presencia de ácido fólico unido a las nanopartículas contribuye a la obtención de valores bajos de hemólisis. Además, los complejos de nanopartícula: oligonucleótido mostraron una inhibición de genes en ausencia de suero bovino fetal (FBS), por lo que son candidatos prometedores para la inhibición de genes in vitro. Sin embargo, en presencia de proteínas del suero, no se detectó silenciamiento génico, lo que limita el uso de estas nanopartículas para experimentos in vivo. No obstante, con una funcionalización adecuada (por ej. con CPP) posiblemente se podría favorecer la penetración celular y por tanto conseguir transfección.

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[eng] The general objective of this research was the design of polymeric cationic nanoparticles from nano-emulsions obtained by low-energy methods for biomedical applications. This general objective includes the preparation of oil inwater (O/W) cationic nano-emulsions by low-energy methods and their characterization, the preparation and characterization of cationic polymeric nanoparticles using the O/W cationic nano-emulsions as templates, the functionalization of the polymeric nanoparticles and in vitro studies of functionalized nanoparticles. Nano-emulsions with droplet sizes < 300 nm, positive surface charges and acceptable stability were successfully obtained in the Aqueous solution / [CatA: nonionic surfactant] / [Polymer in ethyl acetate] system. Nano-emulsion properties were controlled by the type of the nonionic surfactant, the CatA: nonionic surfactant ratio, composition of the aqueous solution, polymer concentration and polymer molecular weight. Nano-emulsion properties using a castor oil surfactant derivative, i.e. Cremophor® WO7 or Cremophor® EL, are more suitable for the intended application than those using a sorbitan ester surfactant which suggests that the surfactant structure plays a significant role. The CatA : nonionic surfactant mixing ratio of 1:1 was found to be optimum for nano-emulsion formation with small droplet size, appropriate surface charge and stability. The properties of nano-emulsions prepared with MilliQ® water or HEPES solution did not differ significantly while nano-emulsions prepared with a phosphate buffer were highly unstable. The incorporation of glucose (to adapt the solution to the osmolarity required for in vitro studies) had no influence on droplet size and surface charge. Increasing the polymer concentration (from 6 wt% to 10wt%) did not improve nano-emulsion properties. Decreasing the polymer molecular weight (from EC10 to EC4) did not significantly change nano-emulsion properties. Cationic nanoparticles with small particle sizes (<250 nm), positive surface charges and acceptable stability were obtained from nano-emulsion templating by the solvent evaporation method. Nanoparticle surface charge and stability were similar to those of the template nano-emulsions while particle size was smaller. The nature of the nano-emulsion aqueous component did not influence significantly nanoparticle size and stability. Surface charge values, however, were lower in HEPES solution than in water. Decreasing the polymer molecular weight (from EC10 to EC4) did not significantly change nanoparticle size and surface charge, independently on the aqueous solution used. Complex formation between nanoparticles and folic acid at different charge ratios was achieved. Complex formation was not influenced by the nature of the aqueous component. The size of the complexes formed with different cationic/anionic (c+/a) charge ratios was not significantly changed compared to that of uncomplexed nanoparticles. However, surface charge values were significantly decreased. Complexation between nanoparticles and an antisense oligonucleotide (ASO) at different nanoparticle:antisense oligonucleotide (N/P) ratios was achieved. The complexes showed the same trends regarding surface charge and size as those of nanoparticle:folic acid complexes and are hence suitable for therapeutic applications. In the presence of Fetal Bovine Serum (FBS), surface charge values of the nanoparticle:ASO complexes remained in the negative range suggesting that serum components were absorbed onto the nanoparticle surface. In vitro studies of nanoparticle dispersions revealed that nanoparticles are nontoxic to HeLa cells up to a concentration of 3 mM. A low concentration of cationic species is required for obtaining low hemolytic activity. Moreover, low hemolysis values are achieved with folic acid attached to the nanoparticles. In the absence of Fetal Bovine Serum, Renilla luciferase gene inhibition showed an optimum for the N/P ratio 28 (40%) which makes them promising candidates for gene inhibition in cell cultures. However, in the presence of serum proteins, no gene silencing was detected, precluding the use of these nanoparticles for in vivo experiments. However, with appropriate functionalization (e.g. with CPP), cell uptake and consequently transfecction could be favored.

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Design of multifunctional polymeric nanoparticles for biomedical applications