Diseño de nanoestructuras para vectorización selectiva de fármacos mediante el uso de nanopartículas de oro estabilizadas en soportes lipídicos. Aplicación en la terapia anticancerígena

En el presente proyecto de Tesis Doctoral, se ha hecho un estudio multidisciplinar concerniente al diseño de nanoplataformas lipídicas estables de fármacos citotóxicos que sirven como sistemas transportadores para vectorización de dichos fármacos a nivel celular para tratar patologías de origen celular, como es el cáncer. Para ello, hemos iniciado los estudios con unos planteamientos preliminares sobre el diseño, caracterización y evaluación de la liberación in vitro de formulaciones estables de liposomas de calceína, con características adecuadas de tamaño, carga superficial y eficacia de encapsulación. Asimismo, hemos realizado los estudios pertinentes mediante distintas técnicas de elaboración y evaluando distintas mezclas de fosfolípidos con el fin de seleccionar aquella formulación que aporte cargas netas superficiales favorables para anclar nanopartículas de oro (AuNPs). A continuación hemos llevado a cabo la preparación y caracterización de películas lipídicas mediante técnicas fisicoquímica basadas en la preparación de monocapas en las interfases aire/agua de la balanza de Langmuir, con el fin de determinar la composición lipídica termodinámicamente óptima para elaborar los liposomas, y su posterior transferencia a electrodos monocristalinos de oro (111), para analizar su estabilidad en presencia de campos eléctricos de intensidades similares a los presentes en las interfases biológicas, mediante espectroscopía de impedancias. Posteriormente se ha empleado la plataforma optimizada en el anterior capítulo para encapsular doxorrubicina (DXR) y anclar AuNPs. Tras llevar a cabo los estudios de encapsulación por carga remota en gradiente de pH, y posterior caracterización, luego se ha procedido a analizar el proceso de liberación in vitro, con el fin de desarrollar una posible nanoplataforma para el tratamiento de cáncer. Finalmente, se ha llevado a cabo el estudio de las interacciones del fármaco anticancerígeno DXR con los componentes de las nanoplataformas complejas diseñadas para su liberación. Para ello, se ha estudiado la reducción electroquímica de la DXR sobre electrodos monocristalinos de oro (111) modificados con monocapas lipídicas, de composiciones previamente optimizadas. Analizando la influencia que un componente catiónico, bromuro de didodecil dimetil amonio (DDAB), empleado para el anclaje de las NPs, ejerce sobre la inclusión de la DXR en las monocapas

Didodecyldimethylammonium Bromide Role in Anchoring Gold Nanoparticles onto Liposome Surface for Triggering the Drug Release

Liposomes with their capacity to anchor gold nanoparticles (AuNPs) onto their surface are used in the treatment of several pathologies such as cancer. The objective of this work was the optimization of the vesicle composition by using cationic agents in order to reinforce the anchoring process of AuNPs, and for the study of the influence of local temperature and vesicle size on drug release. A Plackett–Burman design was conducted to determine the optimal composition for the anchoring of AuNPs. A comprehensive study of the influence of lipid bilayer composition on the surface charge, size, and polydispersity index (PdI) of liposomes was carried out. Afterwards, in vitro release studies by dialysis were performed and several release parameters were evaluated as a function of temperature. Cholesterol was fixed as the rigid agent and Didodecyldimethylammonium bromide (DDAB) was selected as the cationic lipid into the liposome bilayer. Photomicrographs revealed that DDAB facilitated the anchoring of AuNPs onto the liposomal surface. The anchoring of AuNPs also enhanced the amount and rate of calcein released, especially in extruded samples, at several incubating temperatures. In addition, it was observed that both the anchoring of AuNPs and the calcein release were improved by increasing the surface of the vesicles. The contributions of liposome composition (DDAB inclusion, incubation temperature, anchoring of AuNPs) and size and surface availability of the vesicles on calcein release could be used to design improved lipid nanostructures for the controlled release of anticancer drugs.Ministerio de Economía y Competitividad CTQ2014-57515-C2-1-

Electrochemical characterization of a mixed lipid monolayer supported on Au(111) electrodes with implications for doxorubicin delivery

The cationic lipid didodecyldimethylammonium bromide (DDAB) is one of the agent that is included in formulations for liposomes with anchored gold nanoparticles as drugs carriers because its positive charge facilitates the anchoring of the negatively charged stabilized gold nanoparticles to the lipid components of the liposomes. In this paper a thermodynamic analysis of Langmuir isotherms was performed, as a first step in the preparation of liposomes including DDAB, the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and cholesterol(Ch),to decide about the most stable combination. Monolayers of DMPC:Ch:DDAB with the most energetically favourable composition, were transferred to Au(111) electrodes by the Langmuir-Schaefer technique in the electrochemical cell and characterized by impedance spectroscopy. The results were compared with those obtained with electrodes coated with DMPC:Ch films that did not contain DDAB. In both cases the frequency dispersion of impedance data indicates high homogeneity of the films in a wide potential range around the capacitance minimum. The inclusion of the anticancer drug doxorubicin (DOX) into the mixed lipid monolayers and its electrochemical reduction at pH 4.5 were studied by voltammetry and by impedance spectroscopy. At potentials out of the faradaic region the inclusion of DOX does not affect significantly the frequency dispersion of the impedance but decreases the capacitance. However, at negative potentials the analysis of the high frequency dispersion of the impedance and the influence of the scan rate on the voltammograms indicate a reduction process with contribution of adsorption and diffusion of DOX. Diffusion was avoided by transferring the electrodes coated in the Langmuir trough to the electrochemical cells that do not contain the drug. Under these conditions the reduction of the adsorbed DOX was found to obey the model of a surface confined electrode and the charge transfer resistance, Ra, and adsorption capacitance, Ca, were obtained at potentials of the faradaic region. The combination of both parameters provides the rate constant for the reduction in a wide potential range that allows us to propose a sequential mechanism with two determining steps

Cholesterol levels affect the performance of aunps-decorated thermo-sensitive liposomes as nanocarriers for controlled doxorubicin delivery

Stimulus-responsive liposomes (L) for triggering drug release to the target site are particularly useful in cancer therapy. This research was focused on the evaluation of the effects of cholesterol levels in the performance of gold nanoparticles (AuNPs)-functionalized L for controlled doxorubicin (D) delivery. Their interfacial and morphological properties, drug release behavior against temperature changes and cytotoxic activity against breast and ovarian cancer cells were studied. Langmuir isotherms were performed to identify the most stable combination of lipid components. Two mole fractions of cholesterol (3.35 mol% and 40 mol%, L1 and L2 series, respectively) were evaluated. Thin-film hydration and transmembrane pH-gradient methods were used for preparing the L and for D loading, respectively. The cationic surface of L allowed the anchoring of negatively charged AuNPs by electrostatic interactions, even inducing a shift in the zeta potential of the L2 series. L exhibited nanometric sizes and spherical shape. The higher the proportion of cholesterol, the higher the drug loading. D was released in a controlled manner by diffusion-controlled mechanisms, and the proportions of cholesterol and temperature of release media influenced its release profiles. D-encapsulated L preserved its antiproliferative activity against cancer cells. The developed liposomal formulations exhibit promising properties for cancer treatment and potential for hyperthermia therapy.Ministerio de Ciencia e Innovación CTQ2014- 57515-C2-