Dispersão de agregados de C60-PVP : caracterização físico-química e estudos de absorção dérmica e status oxidativo sob a influência de radiação ultravioleta A

Objetivos: O objetivo principal da presente tese foi avaliar as interações entre a pele e agregados de fulerenos C60 estabilizados com polivinilpirrolidona (C60-PVP), considerando sua incorporação recente em cosméticos, comparando-se a absorção dérmica in vitro entre pele suína e humana e avaliando-se o status oxidativo ex vivo de lipídios e proteínas de pele humana, sob radiação ultravioleta A, para verificar a influência deste fator externo nos estudos. Métodos: As dispersões de agregados de C60 preparadas foram caracterizadas quanto ao pH, tamanho de partícula, potencial zeta e morfologia. Os estudos de absorção dérmica foram realizados com pele suína e humana, sob uma fonte de radiação UVA, em equipamento automatizado de células do tipo Franz. Estudos in vitro foram realizados com homogenato de pele humana, na presença ou não de um sistema pró-oxidante, variando-se o tempo de incubação e a concentração de C60-PVP, avaliando-se os níveis de espécies reativas ao ácido tiobarbitúrico (TBARS) e proteína carbonila. Utilizou-se pele humana recém excisada para estudos ex vivo de difusão de C60- PVP na presença de radiação UVA e avaliação da peroxidação lipídica e oxidação de proteínas. Resultados: O tamanho dos agregados de C60 obtido por análise de rastreamento de nanopartículas (NTA) foi 129 ± 54 nm e o potencial zeta foi -4,93 ± 1,72 mV. Os agregados apresentaram formato irregular e permearam através da pele humana e suína após 12 h de exposição. O C60-PVP não demonstrou atividade antioxidante, mas causou oxidação proteica, devido ao PVP. O C60 induziu oxidação de proteínas da epiderme humana ex vivo. Conclusões: A NTA foi considerada a técnica mais adequada para a caracterização do tamanho dos agregados de C60- PVP, devido à dinâmica de agregação não controlada do sistema. A pele humana mostrou-se menos permeável do que a pele suína e a irradiação UVA aumentou o teor de C60 até a derme. O C60 não demonstrou capacidade antioxidante, mas próoxidante, e induziu a carbonilação de proteínas da epiderme humana.Objectives: The main objective of the present dissertation was to evaluate the interactions between the skin and fullerene C60 aggregates stabilised with poly(vinylpyrrolidone) (C60-PVP), considering its recent incorporation into cosmetics, by comparing in vitro dermal absorption with porcine and human skin, and investigating the ex vivo oxidative effects to lipids and proteins of the human skin, under UVA irradiation to verify the influence of this external factor to the studies. Methods: Aggregate dispersions were characterised for pH, particle size, zeta potential, and morphology. Skin absorption studies were performed using porcine or human skin under UVA or sham irradiation, in automated Franz diffusion cells equipment. In vitro studies were performed with human skin homogenates, with or without a pro-oxidant system, varying the time of incubation and the C60-PVP concentration, to evaluate thiobarbituric acid reactive species (TBARS) and protein carbonyl levels. Freshly excised human skin was used for ex vivo studies of C60-PVP diffusion with UVA radiation and subsequent determination of lipid peroxidation and protein oxidation content. Results: C60 aggregate size through nanoparticle tracking analysis (NTA) was 129 ± 54 nm and zeta potential was -4,93 ± 1,72 mV. C60 aggregates presented irregular shape and permeated through human and porcine skin after 12 h exposure. C60-PVP did no demonstrate antioxidant activity, but caused protein oxidation due to PVP. C60 induced protein oxidation of human epidermis ex vivo. Conclusions: NTA was considered the most suitable method for aggregate size characterisation of C60-PVP, due to the non-controllable aggregation dynamics of the system. Human skin was less permeable than porcine skin and the presence of UVA-R increased C60 content up to the dermis. C60 demonstrated no antioxidant capacity, but pro-oxidant activity, and induced protein carbonylation in human epidermis

Evaluation of cytotoxicity profile and intracellular localisation of doxorubicin-loaded chitosan nanoparticles

In the emerging field of nanomedicine, targeted delivery of nanoparticle encapsulated active pharmaceutical ingredients (API) is seen as a potential significant development, promising improved pharmacokinetics and reduced side effects. In this context, understanding the cellular uptake of the nanoparticles and subsequent subcellular distribution of the API is of critical importance. Doxorubicin (DOX) was encapsulated within chitosan nanoparticles to investigate its intracellular delivery in A549 cells in vitro. Unloaded (CS-TPP) and doxorubicin-loaded (DOX-CS-TPP) chitosan nanoparticles were characterised for size (473±41 nm), polydispersity index (0.3±0.2), zeta potential (34±4 mV), drug content (76±7 µM) and encapsulation efficiency (95±1%). The cytotoxic response to DOX-CS-TPP was substantially stronger than to CS-TPP, although weaker than that of the equivalent free DOX. Fluorescence microscopy showed a dissimilar pattern of distribution of DOX within the cell, being predominantly localised in the nucleus for free form and in cytoplasm for DOX-CS-TPP. Confocal microscopy demonstrated endosomal localisation of DOX-CS-TPP. Numerical simulations, based on a rate equation model to describe the uptake and distribution of the free DOX, nanoparticles and DOX loaded nanoparticles within the cells, and the subsequent dose and time dependent cytotoxic responses, were used to further elucidate the API distribution processes. The study demonstrates that encapsulation of the API in nanoparticles results in a delayed release of the drug to the cell, resulting in a delayed cellular response. This work further demonstrates the potential of mathematical modelling in combination with intracellular imaging techniques to visualise and further understand the intracellular mechanisms of action of external agents, both APIs and nanoparticles in cells

Chitosan-Coated Nanoparticles: Effect of Chitosan Molecular Weight on Nasal Transmucosal Delivery

Drug delivery to the brain represents a challenge, especially in the therapy of central nervous system malignancies. Simvastatin (SVT), as with other statins, has shown potential anticancer properties that are difficult to exploit in the central nervous system (CNS). In the present work the physico⁻chemical, mucoadhesive, and permeability-enhancing properties of simvastatin-loaded poly-ε-caprolactone nanocapsules coated with chitosan for nose-to-brain administration were investigated. Lipid-core nanocapsules coated with chitosan (LNCchit) of different molecular weight (MW) were prepared by a novel one-pot technique, and characterized for particle size, surface charge, particle number density, morphology, drug encapsulation efficiency, interaction between surface nanocapsules with mucin, drug release, and permeability across two nasal mucosa models. Results show that all formulations presented adequate particle sizes (below 220 nm), positive surface charge, narrow droplet size distribution (PDI < 0.2), and high encapsulation efficiency. Nanocapsules presented controlled drug release and mucoadhesive properties that are dependent on the MW of the coating chitosan. The results of permeation across the RPMI 2650 human nasal cell line evidenced that LNCchit increased the permeation of SVT. In particular, the amount of SVT that permeated after 4 hr for nanocapsules coated with low-MW chitosan, high-MW chitosan, and control SVT was 13.9 ± 0.8 μg, 9.2 ± 1.2 µg, and 1.4 ± 0.2 µg, respectively. These results were confirmed by SVT ex vivo permeation across rabbit nasal mucosa. This study highlighted the suitability of LNCchit as a promising strategy for the administration of simvastatin for a nose-to-brain approach for the therapy of brain tumors