Theranostic applications of fluorescent liquid crystalline nanoparticles

Lipid liquid crystalline nanoparticles can find application as nanocarriers in several fields of the daily life but, very likely, the pharmaceutical arena is the most relevant. Indeed, several problems encountered in drugs administration (e.g. critical sideeffects from antitumor drugs) require alternative, less invasive, but simultaneously efficient therapeutic routes to be explored. Novel fields of personalized nanomedicine are developing in this direction. One of the most interesting is theranostic, which calls for the design of platforms capable of combining therapeutic and diagnostic functionalities. In this optic, we explored the potential of monoolein-based cubosomes and hexosomes as nanocarriers for theranostic purposes. Our work focussed on the design of lipid nanoparticles able to deliver antineoplastic drugs and imaging probes for fluorescent optical in vitro and in vivo imaging. We developed cubosome formulations loaded with antineoplastic drugs and useful for the fluorescence imaging of cells. Such formulations were also actively targeted to cancer cells and coupled with a NIR-emitting fluorophore, which was the promise for in vivo applications. We also investigated hexosomes with encouraging results encapsulating in their lipid matrix a BODIPY derivative with solvatochromic properties, helpful for the understanding of the dye localization. Importantly, we reported (manuscript submitted) the first proof-of-principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. Finally, since relatively little is known about the interaction of cubosomes with biological systems, their effects on lipid droplets, mitochondria and lipid profile of HeLa cells were deeply studied. This thesis is divided in two main parts. The introduction section reports on the essential background of the research field, and it is followed by the publications (published or submitted) resulting from these three years of work

Experimental Modelling of Flavonoid Membrane Interactions

Flavonoids, a class of polyphenols, are commonly found in fruits, vegetables, nuts, and grains. Increasing evidence from epidemiological and clinical studies show a relationship between high flavonoid consumption in diet and reduced risk of several chronic diseases. Several mechanisms, including specific binding of flavonoids to proteins, have been proposed for flavonoids to exert their biological activities. However, it has also been reported that nonspecific interactions of flavonoids with phospholipids can induce structural changes in the membrane’s features (e.g., thickness and fluctuations) and indirectly modulate membrane proteins, as well as influence their pharmacological potentials. This thesis investigates the interactions between flavonoids and model biomembranes through three distinctively different but complementary approaches, with a special emphasis on employing monolayer membrane model in proof of concept experiments using one lipid type; 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC). Hence, a well characterised electrochemical sensor system; phospholipid monolayer coated mercury (Hg) film electrode was established by rapid cyclic voltammetry (RCV) to screen structure-dependent interactions of a variety of flavonoids. The data revealed that flavonoids adopting a planar configuration altered the membrane properties more significantly than nonplanar flavonoids. The extent of interactions can be ranked in the order of quercetin > kaempferol > naringenin > hesperetin > catechin for flavonoid aglycones and tiliroside > rutin > naringin for flavonoid glycosides. Quercetin, rutin, and tiliroside were selected for follow-up experiments with Langmuir monolayers, Brewster angle microscopy (BAM), and small-angle X-ray scattering (SAXS). Relaxation phenomena in DOPC monolayers and visualisation of the surface with BAM revealed a pronounced monolayer stabilisation effect with both quercetin and tiliroside, whereas rutin disrupted the monolayer structure rendering the surface entirely smooth. The following ranking of the interactions: quercetin>tiliroside>rutin, yielded comparable results to those obtained from the previous technique. SAXS showed a monotonous membrane thinning for all flavonoids studied associated with an increase in the mean fluctuations of the membrane. The extent of interactions was concentration and temperature dependent with an order of quercetin>tiliroside>rutin except for tiliroside where a high concentration of tiliroside (>2 mol%) revealed the most pronounced response. In addition to the novelty of employing phospholipid monolayers for the systematic characterisation of a variety of flavonoids, this is the first report investigating the effect of tiliroside with biomimetic membrane models. All the flavonoids studied are believed to be localised in the lipid/water interface region. Both this location and the membrane perturbations might have implications for the therapeutic features of flavonoids

Experimental Modelling of Flavonoid Membrane Interactions

Flavonoids, a class of polyphenols, are commonly found in fruits, vegetables, nuts, and grains. Increasing evidence from epidemiological and clinical studies show a relationship between high flavonoid consumption in diet and reduced risk of several chronic diseases. Several mechanisms, including specific binding of flavonoids to proteins, have been proposed for flavonoids to exert their biological activities. However, it has also been reported that nonspecific interactions of flavonoids with phospholipids can induce structural changes in the membrane’s features (e.g., thickness and fluctuations) and indirectly modulate membrane proteins, as well as influence their pharmacological potentials. This thesis investigates the interactions between flavonoids and model biomembranes through three distinctively different but complementary approaches, with a special emphasis on employing monolayer membrane model in proof of concept experiments using one lipid type; 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC). Hence, a well characterised electrochemical sensor system; phospholipid monolayer coated mercury (Hg) film electrode was established by rapid cyclic voltammetry (RCV) to screen structure-dependent interactions of a variety of flavonoids. The data revealed that flavonoids adopting a planar configuration altered the membrane properties more significantly than nonplanar flavonoids. The extent of interactions can be ranked in the order of quercetin > kaempferol > naringenin > hesperetin > catechin for flavonoid aglycones and tiliroside > rutin > naringin for flavonoid glycosides. Quercetin, rutin, and tiliroside were selected for follow-up experiments with Langmuir monolayers, Brewster angle microscopy (BAM), and small-angle X-ray scattering (SAXS). Relaxation phenomena in DOPC monolayers and visualisation of the surface with BAM revealed a pronounced monolayer stabilisation effect with both quercetin and tiliroside, whereas rutin disrupted the monolayer structure rendering the surface entirely smooth. The following ranking of the interactions: quercetin>tiliroside>rutin, yielded comparable results to those obtained from the previous technique. SAXS showed a monotonous membrane thinning for all flavonoids studied associated with an increase in the mean fluctuations of the membrane. The extent of interactions was concentration and temperature dependent with an order of quercetin>tiliroside>rutin except for tiliroside where a high concentration of tiliroside (>2 mol%) revealed the most pronounced response. In addition to the novelty of employing phospholipid monolayers for the systematic characterisation of a variety of flavonoids, this is the first report investigating the effect of tiliroside with biomimetic membrane models. All the flavonoids studied are believed to be localised in the lipid/water interface region. Both this location and the membrane perturbations might have implications for the therapeutic features of flavonoids

Delivery of small nucleic acids by conjugation to carbohydrates and lipids as novel research and therapeutic tools

399 p. : il., graf.Small nucleic acids present a great potential to silence specific genes or inhibit the biological activity of specific proteins. Unfortunately, their poor cellular accessibility presents a big challenge for developing an efficient delivery system. In this regard, conjugation of appropriate molecules to small nucleic improves their pharmacokinetic behaviours and cellular uptake efficiencies, endowing them with entirely new properties. In the present work different carbohydrate- and lipid-oligonucleotide conjugates have been studied as novel tools for targeted delivery and enhancement of cellular permeability. The results obtained in this work indicate that keeping a certain distance between DNA and sugar modification could be important for a better incorporation of this type of conjugates into the target cell. Also long or double-tailed lipid modifications are preferred for an enhanced incorporation of lipid-oligonucleotide conjugates into membrane-model and cell systems.The present thesis has been performed Departamento de Bioquímica y Biología Molecular de la Universidad del País Vasco and Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU). This work has been supported mainly by CSIC (CARBINH, PIF06-045). The author received a research fellowship from the University of the Basque Country (PIFA01/2006/052, June 2007-May 2011) and a research contract supported by Fundación Biofísica Bizkaia (June 2011-May 2012)

Design of new hybrid nanomaterials with molecular gates as nanodevices for therapeutic applications

Tesis por compendioLa presente tesis doctoral, que lleva por título “Diseño de nuevos nanomateriales híbridos con puertas moleculares como nanodispositivos para aplicaciones terapéuticas” está centrada en el desarrollo de nuevos materiales funcionales híbridos orgánico-inorgánicos para aplicaciones de liberación controlada. Los dos capítulos de la presente tesis en los que se describen los resultados obtenidos (el segundo y el tercer capítulos) están directamente relacionados con el uso de las nanopartículas mesoporosas de sílice como matriz inorgánica en el desarrollo de nuevos materiales híbridos orgánico-inorgánicos para aplicaciones en liberación controlada. Aun así, los resultados se han dividido en dos capítulos, dependiendo del estímulo aplicado para la liberación de la molécula encapsulada. En uno de los capítulos, los diferentes materiales desarrollados se basan en nanodispositivos controlados enzimáticamente, mientras que en el otro capítulo es un cambio de pH o de fuerza electroestática (en los dos casos debido a la presencia de un microorganismo patógeno) el que causa la consecuente liberación de la carga. En el caso de los nanodispositivos controlados enzimáticamente, los cuales se describen en el Capítulo 2, se desarrollaron tres sólidos diferentes. El primer ejemplo se basó en el diseño, síntesis y caracterización de nanopartículas mesoporosas de sílice recubiertas con sales de azopiridinio, que se hidrolizan en presencia de esterasas y reductasas, las cuales se encuentran en la microflora del colon. Estas sales, que contienen un enlace azoico, se seleccionaron para una posible liberación selectiva en el colon. Los estudios de viabilidad celular e internalización se llevaron a cabo con células HeLa, así como los estudios de liberación del agente quimioterapéutico camptotecina. Un segundo ejemplo se centró en el diseño, síntesis, caracterización y aplicaciones de un nuevo nanodispositivo que responde a la presencia de proteasas para liberación controlada, empleando nanopartículas de sílice cubiertas con el polímero -poli-L-lisina. En este caso, se pretendía evaluar dos mecanismos diferentes de anclaje del polímero y los dos dieron resultados positivos, aunque presentaron diferentes perfiles de liberación en cada caso. También se realizaron estudios de viabilidad e internalización celular con este nuevo nanodispositivo, así como la liberación de camptotecina en células HeLa. Finalmente, el último nanodispositivo que responde a la acción de un enzima; incluye el diseño y aplicación de un “scaffold” 3D inteligente con puertas moleculares, el cual consiste en la combinación de nanopartículas mesoporosas de sílice con puertas y biomateriales porosos clásicos. En este caso, las nanopartículas mesoporosas de sílice se cubrieron con poliaminas y ATP. Estas nanopartículas se incorporaron durante la síntesis de un “scaffold” de gelatina, el cual se preparó mediante técnicas de prototipado rápido (RP). En presencia de fosfatasa ácida se induce la liberación del colorante encapsulado en los poros de las nanopartículas. La fosfatasa ácida se seleccionó como estímulo activador de este material diseñado, ya que es un enzima cuya concentración se emplea para evaluar la actividad de los osteoclastos en procesos de remodelación ósea y como marcador en metástasis de huesos. Estas propiedades abren posibilidades de uso de esta combinación en el diseño de materiales funcionales para la preparación de numerosos “scaffolds” avanzados con puertas moleculares, que puedan ayudar en aplicaciones de medicina regenerativa y terapias de cáncer de huesos. Con respecto al otro tipo de nanodispositivos, que se muestra en el Capítulo 3, se ha evaluado el posible uso de las nanopartículas mesoporosas de sílice con puertas moleculares como posibles vehículos para la liberación controlada de fármacos cuando un microorganismo patógeno está presente. En este caso, el diseño y desarrollo de nuevos materiales híbridos orgánico-inorgánicos se ha basado en el uso de nanopartículas mesoporosas de sílice como matriz inorgánica, cubiertas con entidades moleculares orgánicas que podrían responder a un cambio en el pH del ambiente o a un cambio en la fuerza electroestática, debido a la presencia de un microorganismo patógeno, tales como hongos o bacterias. Uno de estos nanodispositivos desarrollados demuestra las aplicaciones y propiedades antifúngicas de un soporte cargado con tebuconazol y cubierto con moléculas que actúan de puerta molecular dirigida mediante un cambio de pH. El otro material presenta aplicaciones antibacterianas contra bacterias gram-positivas y gram-negativas, ya que se utiliza un nanodispositivo cargado con vancomicina y funcionalizado con -poli-L-lisina. En los dos casos, se ha demostrado que el uso de la nanoformulación puede mejorar la efectividad del fármaco encapsulado, mejorando y ampliando el espectro de acción del mismo, lo cual abre un gran abanico de posibilidades en aplicaciones de estos nanodispositivos en el tratamiento de infecciones. En resumen, se puede concluir que en la presente tesis se han desarrollado nuevos sólidos híbridos orgánico-inorgánicos, así como se han descrito las aplicaciones de estos nanodisposotivos como sistemas de liberación controlada. Los resultados obtenidos podrían ser útiles en futuros diseños de materiales híbridos avanzados en biotecnología, biomedicina y, concretamente, en aplicaciones terapéuticas (como terapias contra el cáncer, tratamiento de infecciones, medicina regenerativa, etc.)Mas Font, N. (2014). Design of new hybrid nanomaterials with molecular gates as nanodevices for therapeutic applications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48491TESISCompendi

Water-dispersible conjugated polymer nanocomplexes with soft or hard nanostructures

Nanocomplexes are made of different components, benefiting from properties of each of the constituent material. As such, nanocomplexes present unique properties including chemical activity due to high surface area, tuneable size and morphology, variable surface charges and efficient uptake by cells. They have been used in different therapeutic approaches such as drug delivery systems, biosensors and tissue engineering. Introducing an electrically conductive material, such as metal or conjugated polymer, as one component of nano-complexes adds electronic and optical properties to the system, broadening their applications. While metals are excellent conductors, their hard nature does not match the soft tissue. Conjugated polymers on the other hand, as organic materials, are soft and flexible; thus, they are the choice material in this thesis for the development of soft and hard conjugated polymer nanocomplexes. Liposome and anionic polymeric nanoparticles with a core morphology were used as soft and hard templates, respectively, around which conjugated polymers, polyaniline and polypyrrole, were synthesised. Their chemical, physical, electrochemical and biomedical properties were optimised and fully characterised. A single-step synthesis was developed for fabricating the soft polyaniline-liposome nano-complexes in which polyaniline, doped with phytic acid, was either embedded in the liposome bilayer or bound on the liposome outer surface. Hard polypyrrole nanocomplexes were based on the anionic poly(potassium 3- sulfopropyl methacrylate)-block- poly (benzyl methacrylate) nanoparticle. In a novel step, the nanoparticle served as the dopant for the conjugated polymer. The conjugated polymer nanocomplexes were water-dispersible, conductive and exhibited low impedance. In-vitro cell studies demonstrated the complexes are not cytotoxic with capability of targeted cell uptakes. This research presents novel and promising routes for xiv fabrication of water-dispersible conductive nanocomplexes with potential biomedical applications

Design, Engineering and Biological Performance of Responsive Lipid Vesicles for Enhanced Drug Delivery by Mild Hyperthermia

The design of a delivery system that specifically delivers anticancer drug to the tumour site avoiding normal tissues damage has always been a challenge. In this thesis we describe the engineering and biological performance of novel temperature-sensitive liposomes (TSL) that have both a substantial in vivo stability and an efficient content-release by mild hyperthermia (HT). First, we explain the development of novel lipid-peptide hybrids (Lp-Peptide) by anchoring leucine zipper temperature-sensitive peptide within the liposomal lipid bilayer. We characterized this system by studying its physicochemical properties and the interaction of the peptide with the lipid bilayer. Then we examined its potential to retain and trigger the release of the anticancer drug, doxorubicin, in vitro at physiological temperatures and after exposure to mild HT. In addition, the blood kinetics, tumour and other tissues accumulation were explored when we studied the system in vivo. Our data suggested that Lp-Peptide hybrids can increase both immediate and long-term drug accumulation in the tumour. Therefore, we studied their therapeutic activity comparing two different heating protocols to mimic intravascular and interstitial drug release. The last chapter of this thesis explored the opportunities of increasing the therapeutic specificity of TSL by designing anti-MUC-1 targeted vesicles based on the traditional TSL (TTSL) to trigger drug release after specific uptake into cancer cells. The system was evaluated by studying the in vitro cellular binding, uptake and therapeutic efficacy. Taking this system a step further, its biodistribution and therapeutic potential were also examined. Different protocols were applied to explore the effect of HT on the accumulation of targeted TTSL into the tumour and their therapeutic efficacy. In summary, our studies demonstrate the critical factors to consider in the design of clinically relevant TSL and the importance of matching the heating protocol to their physicochemical and pharmacokinetic parameters to maximise therapeutic benefits