RESUMO
The light-driven hybrid P450 enzyme approach utilizing the photochemical properties of a covalently attached Ru(II)-diimine photosensitizer was extended to the archaeal Sulfolobus acidocaldarius CYP119 enzyme leading to high photocatalytic activity in the hydroxylation of the chromogenic substrate, 11-nitrophenoxyundecanoic acid. The determined kcat was greater than those reported with various natural redox partners. In addition, the sacrificial electron donor, diethyldithiocarbamate, used in the photocatalytic reaction is shown to play a dual role. It acts as an efficient quencher of the Ru(II) excited state leading to a highly reducing species necessary to inject electrons into the heme. It is also known for its antioxidant properties and is shown herein to be a useful probe to determine coupling efficiency in the light-driven hybrid enzymes.
Assuntos
Proteínas Arqueais/química , Sistema Enzimático do Citocromo P-450/química , Proteínas Arqueais/genética , Proteínas Arqueais/efeitos da radiação , Biocatálise/efeitos da radiação , Complexos de Coordenação/química , Complexos de Coordenação/efeitos da radiação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/efeitos da radiação , Ditiocarb/química , Heme/química , Cinética , Luz , Mutação , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/efeitos da radiação , Rutênio/química , Sulfolobus acidocaldarius/enzimologiaRESUMO
In this study, we evaluated the potential of lipid nanocapsules (LNC) of 120 nm as drug nanocarriers to treat skin diseases. As a model molecule, we encapsulated the fluorescent dye curcumin, which also is an antioxidant. Curcumin-loaded LNC showed interesting antioxidant properties and a low toxicity on human skin cells. The penetration of curcumin in the skin was determined by 2 complementary methods: high performance liquid chromatography was used to measure total curcumin accumulation in the skin, whereas fluorescence confocal spectral imaging of skin sections showed that curcumin preferentially accumulates in the stratum corneum and the viable epidermis. These results confirm that LNC of a size above 100 nm can vectorize hydrophobic compounds to the keratinocytes without transdermal delivery. They also demonstrate the interest of combining 2 analytical methods when studying the skin penetration of nanovectorized molecules.
Assuntos
Curcumina/administração & dosagem , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Nanocápsulas/administração & dosagem , Absorção Cutânea/efeitos dos fármacos , Administração Tópica , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Curcumina/química , Curcumina/metabolismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Humanos , Lipídeos/administração & dosagem , Lipídeos/química , Nanocápsulas/química , Técnicas de Cultura de Órgãos , Absorção Cutânea/fisiologia , SuínosRESUMO
Calcium alginate nanocarriers (CaANCs) were developed as a potential tool for delivery of hydrophobic active molecules such as pharmaceutical and cosmetic active ingredients. In this study, we focused on interactions between CaANCs and keratinocytes in culture and examined toxicity, internalization and drug release. Prior to cellular interactions, cryogenic transmission electron microscopy images showed that CaANCs appear as regular, spherical and dense particles, giving evidence of the surface gelation of CaANCs. Their size, around 200nm, was stable under tested conditions (temperature, culture media, presence of serum and presence of encapsulated dye), and their toxicity on keratinocytes was very low. Flow cytometry assays showed that CaANCs are internalized into keratinocytes by endocytosis with a predominant implication of the caveolae-mediated route. Förster resonance energy transfer (FRET) demonstrated that after a 2h contact, the release of CaANC contents in the cytoplasm of keratinocytes was almost complete. The endocytosis of CaANCs by a lysosome-free pathway, and the rapid release of their contents inside keratinocytes, will allow vectorized molecules to fully exhibit their pharmacological or cosmetic activity.