Light-sensitive nanocarriers for drug delivery in photodynamic therapy

Aquesta tesi aprofundeix en l’estudi de nanotransportadors com a sistemes de vehiculització i en alguns casos, alliberació de fotosensibilitzadors emprats en teràpia fotodinàmica. S’han fet servir dos nanotransportadors de naturalesa diferent: proteïnes i liposomes. En primer lloc s’ha investigat els complexos formats entre la hipericina i les proteïnes apomioglobina i β-lactoglobulina. S’han estudiat les característiques fisicoquímiques i fotofísiques, avaluant l’activitat antimicrobiana en front a bacteris gram-positius i gram-negatius. En ambdues matrius proteiques el fotosensibilitzador es troba majoritàriament en forma monomèrica, preservant les seves propietats fotofísiques i formant un complex estable. En el cas de la β-lactoglobulina s’estudia a més, la formació del complex amb l’adició d’un 20% de DMSO com a co-solvent, fet que millora les propietats fotofísiques en detriment de la capacitat antimicrobiana. Ambdós complexos proteics son efectius contra bacteris gram-positius però no contra gram-negatius. Per altra banda, es demostra que la hipericina incorporada a la cavitat de l’apomioglobina pot ser utilitzada en microscòpia de super-resolució STED. Amb aquesta tècnica es pot monitoritzar els llocs d’unió del fotosensibilitzador a la membrana dels bacteris. Així mateix, s’estudia l’ús de la β-lactoglobulina com a portador dual d’hipericina i àcid retinoic. En aquest últim sistema multi-component s’avaluen les propietats fotofísiques per a verificar la formació i estabilitat del complex. En segon lloc, es desenvolupa un nanovehicle per la seva aplicació en teràpia combinada en el qual s’incorporen fàrmacs quimioterapèutics convencionals amb agents fotosensibilitzants, per superar resistències i millorar l’eficàcia dels tractaments individuals. Amb aquest objectiu, s’han dissenyat i estudiat dues formulacions liposomals diferents, ambdues amb el mateix fotosensibilitzador però encapsulant diferents agents quimioterapèutics. Es preparen formulacions bimodals on s’incorporen els dos agents al mateix vehicle i els seus homòlegs unimodals, amb la incorporació única d’un dels dos agents. S’han avaluat les característiques fisicoquímiques, fotofísiques i fotobiològiques de les suspensions bimodals i unimodals. La lozalització subcel·lular demostra que cada principi actiu es localitza a orgànuls diferents desencadenant rutes de senyalització cel·lular diferents, eludint els possibles mecanismes de resistència. El tractament in vitro en cèl·lules cancerígenes amb aquests sistemes tenen un efecte prometedor, ja que com a mínim presenten un comportament additiu respecte els tractaments individuals. Finalment, s’ha avaluat el potencial de la vehiculització activa mitjançant la unió covalent d’un anticòs monoclonal a la superfície, el que millora lleugerament els resultats per una de les dues formulacions.Esta tesis profundiza en el estudio de nanotransportadores como sistema de vehiculización y en algunos casos, liberación de fotosensibilizadores empleados en terapia fotodinámica. Se emplean dos nanotransportadores de naturaleza distinta: proteínas y liposomas. En primer lugar se han investigado los complejos formados entre hipericina y las proteínas apomioglobina y β-lactoglobulina. Se han estudiado las características fisicoquímicas y fotofísicas, evaluando la actividad antimicrobiana frente bacterias gram-positivas y gram-negativas. En ambas matrices proteicas el fotosensibilizador se encuentra mayoritariamente en forma monomérica, preservando sus propiedades fotofísicas y formando un complejo estable. En el caso de la β-lactoglobulina se estudia además, la formación del complejo con la adición del 20% de DMSO como co-solvente, lo que mejora las propiedades físicas pero sorprendentemente, empeora la capacidad antimicrobiana. Ambos complejos proteicos son efectivos contra bacterias gram-positivas, pero no contra gram-negativas. Además, se demuestra que la hipericina en la cavidad de la apomioglobina es capaz de realizar microscopía de super-resolución STED, mediante la cual se puede monitorizar los sitios de unión a las bacterias. Asimismo, se ha estudiado la β-lactoglobulina como portador dual de hipericina y ácido retinoico. En este último sistema multi-componente se evalúan las propiedades fotofísicas para verificar la formación y estabilidad del complejo. En segundo lugar, se desarrolla un nanovehículo para su uso en terapia combinada en el que se incorpora fármacos quimioterapéuticos convencionales con agentes fotosensibilizantes, para superar las resistencias y mejorar la eficacia de los tratamientos individuales. Con este objetivo, se han diseñado y estudiado dos formulaciones liposomales diferentes, ambas con el mismo fotosensibilizador, pero con diferentes agentes quimioterapéuticos. Se preparan las formulaciones bimodales con ambos agentes en el mismo vehículo además de sus homólogos unimodales, con la incorporación única de uno de los dos agentes. Se han evaluado las características fisicoquímicas, fotofísicas y fotobiológicas de las suspensiones bimodales y unimodales. La localización subcelular demuestra que cada principio activo se localiza en orgánulos diferentes desencadenando rutas de señalización celular diferentes, eludiendo los posibles mecanismos de resistencia. El tratamiento in vitro en células cancerígenas de estos sistemas tiene un efecto prometedor siendo al menos aditivo en comparación con los tratamientos individuales. Finalmente, se ha evaluado el potencial de la vehiculización activa mediante la unión covalente de un anticuerpo monoclonal en la superficie, lo que lleva a resultados ligeramente superiores para una de las dos formulaciones.This thesis reports the study of nanocarriers as drug delivery systems for photosensitisers in photodynamic therapy. Proteins and liposomes are the two nanovehicles of different nature used for this purpose. Beginning with the proteins, the complexes formed between hypericin and the proteins apomyoglobin and β-lactoglobulin have been explored. The physicochemical and photophysical properties have been studied, as also assessing their photoantibacterial activity against Gram-positive and Gram-negative bacteria. In both protein scaffolds the photosensitiser is found mainly in monomeric form, preserving its fluorescence and singlet oxygen photosensitising properties and yielding a stable complex. In the case of β-lactoglobulin, the complex formation has also been tested with the addition of a 20% DMSO as a co-solvent, which improves the photophysical properties but surprisingly, worsens its antimicrobial activity. Both protein complexes are effective against Gram-positive but not against Gram-negative bacteria. Moreover, it has been proved that hypericin, inside the apomyoglobin cavity, can perform STED microscopy through which its localization in bacteria can be monitored. Additionally, the suitability of β-lactoglobulin as a dual carrier for hypericin and acid retinoic has also been exploited. In this last multi-component system, the photophysical properties have been evaluated to confirm the formation and complex stability. Secondly, a nanocarrier for its use in combined therapy has been developed, in which conventional chemotherapeutic drugs are combined with photosensitising agents to overcome resistance and improve the effectiveness of the individual treatments. For this purpose, two different liposome formulations have been designed and studied with a common photosensitiser but different anti tumour drugs. The bimodal formulations with both agents entrapped and their unimodal counterparts, having each drug loaded in separate liposomes, have been evaluated. The physicochemical, photophysical and photobiological properties of bimodal and unimodal suspensions have been studied. The subcellular localization shows different organelle accumulation by each agent, triggering different key signals transduction pathways, eluding the cellular resistance mechanisms. The treatment in vitro of these multi-component liposomes with cancer cells has a promising effect, since at least an additive outcome is observed when compared with the individual treatments. Finally, we have explored the potential of active targeting strategies by covalently linking a monoclonal antibody to the surface, leading to slightly greater outcomes for one of the liposomal formulations

The Photochemistry of Functionalised Rhenium (I) Tricarbonyl Complexes

This research explores the fundamental photochemistry of neutral and cationic rhenium(I)-NHC complexes with formula [Re(N^C)(CO)3(L)]+/0, where N^C represents a bidentate N-heterocyclic carbene ligand and L resembles various pyridine substituents. In addition, rhenium(I) phen tetrazolato complexes with formula [Re(phen)(CO)3(L)], where phen is 1,10-phenanothroline and L represents a tetrazolato moiety coordinated to various amino acids are studied. The basis of this research is to study the potential effect of electron transfer on the photophysics of rhenium(I) complexes

Picosecond studies of excited states in conjugated polymers

This thesis reports on the interplay between molecular structure and photophysics in light emitting conjugated polymers revealed by steady state and picosecond time-resolved fluorescence spectroscopy. The fundamental excited state relaxation of a polyfluorene derivative is compared to that of two oligofluorenes using isolated molecules in dilute solution. Their long-time time-dependent spectral dynamics are monitored by means of picosecond streak camera and single photon counting techniques. Excited state relaxation in oligofluorenes is entirely conformational and depends on solvent viscosity. The intrachain photophysics of the polyfluorene is dominated by fast excitation migration with a slow conformational component. A rigid ladder-type polymer exhibits only migrational relaxation. In analogy, the spectral dynamics of an alkoxy-substituted polyspirobifluorene are studied in dilute solution. Their qualitative dependence on solvent viscosity is elucidated by further femtosecond photobleaching measurements. Two excited states are proposed with the lower energy state involving strong spiroconjugation as confirmed by electronic structure calculations. Conversion between them occurs via conformational relaxation of the fluorene side groups. The sensitive reaction of these photophysics to the substitution pattern of the polymer suggests an easy chemical tunability of polyspirobifluorenes towards optimised charge carrier transport properties. Finally, the formation of the beta phase in amorphous polydioctylfluorene is investigated as a function of spin coating fabrication in the solid state. It is suggested that it forms by condensation at colloidal sites, which arise from incomplete solvation in the master solution. A further room temperature phase exists in the absence of these nuclei. The excited state relaxation after energy transfer from amorphous to beta phase is monitored via time-resolved spectroscopy. Within the beta phase, exciton migration is restricted as confirmed by steady state anisotropy data. This evidence for exciton confinement is an important step towards the application of the beta phase as a polymer laser

Sedimentology and Stratigraphy of the Upper Cretaceous Puskwaskau Formation in North-Central Alberta, Western Canada Foreland Basin

The Santonian to early Campanian mudstone-dominated Puskwaskau Formation was correlated throughout a study area of 50,000 km2 in north-central Alberta using 988 well logs. Fourteen informal allomembers, established by previous studies, are bounded by regionally-mappable marine flooding and/or transgressive surfaces that are traceable for hundreds of kilometres within the study area. These laterally continuous bounding surfaces are parallel to very gently converging, and mostly terminate by onlap onto underlying surfaces. Observations in thin section and in SEM revealed ten mudstone microfacies, grouped into five microfacies associations. The facies preserve evidence for repeated storm-generated reworking of the seafloor. The Puskwaskau Fm. exhibits an assemblage of authigenic cements that are mainly intergranular pore-filling phases indicative of early diagenesis. The geometric style of allomember bounding surfaces, combined with microfacies analysis, suggests that the seafloor was a low-gradient ramp that was repeatedly reworked by storms. The physiography of the ramp was maintained by a near equilibrium between the rates of accommodation and sediment supply. Allomembers are grouped into three tectono-stratigraphic ‘units’, each of which forms a broadly arcuate, wedge-shaped package of rock with a strike length of \u3e 800 km. The thickest part of successive units is laterally offset from the underlying unit, suggesting that the locus of tectonic loading underwent a corresponding shift. The flexural forebulge surrounding unit 2 is exposed in the southern Alberta foothills where 2 m of sandy, bioclast-rich sediment is equivalent to 100 m of mudstone in the unit depocentre. The same forebulge exposed \u3e 250 km distal to the orogen is instead mantled by clay- and organic-rich sediment. Isopach maps of individual allomembers show little evidence of thickening toward the orogen as predicted by flexural models of subsidence. Subsidence patterns are, instead, interpreted to have been governed by episodic movement along four inferred deep-seated faults, that are interpreted to have generated localised horst and graben structures that resulted in localised regions of accommodation. Movement of these faults is interpreted to have been controlled by changes in the magnitude of in-plane stress within the plate, associated with orogen-related flexure

HIGHLY EMISSIVE CHIRAL LANTHANIDE(III) COMPLEXES FOR LABELLING AND IMAGING

Sensitised lanthanide complexes based on macrocyclic chelating ligands have been used extensively to study biological function at a cellular level, due to their very bright and long-lived emission, sharp emission bands and high stability to photo-degradation. These photophysical properties, in addition to their excellent chiroptical behaviour, also make these complexes promising candidates for security labelling and as anti-counterfeiting tools. Novel highly emissive chiral europium(III) complexes based on macrocyclic ligands have been synthesised and their photophysical properties studied. They possess high molar extinction coefficients and are amenable to excitation using commonly available light sources. These complexes have been resolved by chiral HPLC and the circularly polarised luminescence (CPL) spectra of their enantiomers recorded. They exhibit strong circularly polarised emission in response to excitation using near ultra-violet light, and are stable to thermally-activated racemisation. A simple off-the-shelf camera set up has been developed which is capable of discriminating ‘real’ europium(III) emission from emission from a ‘fake’ marking, based on emission lifetime and wavelength. Additionally, chiroptical discrimination has been achieved using a custom built microscope incorporating a quarter-wave plate and linear polariser. The solvent dependent emission behaviour of a series of C3-symmetric lanthanide(III) complexes has been studied, demonstrating that the form of the total emission and CPL is extremely sensitive to minor changes in the outer solvation sphere of the complex. Finally, macropinocytosis has been identified as the mechanism of cell uptake of this family of complexes in NIH-3T3 cells, and the internalisation and subsequent sub-cellular localisation has been shown to be dependent on complex chirality