Investigation of in vitro and in vivo skin permeability using laser scanning microscopy

Understanding the skin permeability, especially, live human skin permeability is important for several reasons. Firstly, transdermal (through skin) drug delivery is proclaimed as a key delivery mechanism of the future. It offers a targeted drug delivery to the ailment (e.g. muscular pain), with the potential for lower dosages and increased patient compliance. Secondly, skin is the largest organ of an organism serving to protect it from environmental assault. Therefore, thorough understanding of the skin permeability to molecules, macromolecules, and nanoparticles will serve to optimise targeted drug delivery strategies, and minimise potentially hazardous environmental impact. -- In this thesis, these important tasks were addressed by applying emerging optical imaging techniques, e.g. Fluorescence Confocal Scanning Microscopy (FCSM) and Fluorescence Multiphoton Microscopy (FMM), to study of the skin permeability, its kinetics and penetration pathways, with a particular focus on in vivo imaging. These microscopes are especially suitable for imaging of a thick and heterogeneous tissue such as skin. They provide real-time imaging of distribution of endogenous (intrinsic to tissue) and exogenous (externally introduced) fluorophores, as well as luminescent nanoparticles within superficial skin layers in vivo and in vitro on the subcellular scale. Thus, both three-dimensional skin architecture and exogenous fluorophores/nanoparticles can be simultaneously visualised. Of critical importance is to answer a question, whether molecules and nanoparticles under study are absorbed sub-dermally, passing stratum corneum, the topmost layer of the skin? -- As a result of multidisciplinary collaborative team investigations, particularly useful nanoparticle transdermal models have been identified and employed: surface-modified quantum dots (CdSe/ZnS) and metal oxide wide bandgap nanostructures, such as zinc oxide (ZnO-nano) and titanium dioxide (TiO₂-nano) nanoparticles. It appeared that ZnO-nano exhibited remarkable in skin in FMM imaging context. It is explained by its enhanced optical non-linear and serendipitous spectral properties. -- Due to the widespread use of ZnO-nano in skin care products, this work may find useful applications in vivo non-invasive imaging of nanoparticle transdermal penetration. The overall outcome from the optical and electron microscopy imaging studies was that, in human in vivo, nanoparticles stayed in stratum corneum and accumulated into skin folds and/or hair follicle roots of human skin, with a remarkable exception of skin treated with chemical enhancers. The current body of evidence, including contributions of this study, suggests that the form of sunscreen-based nanoparticles studied here is unlikely to result in safety concerns. -- In order to demonstrate an approach for investigation of the permeation pathways and kinetics of low-molecular weight drugs and toxins, permeation of organic dye Rhodamine B (Rh:B) via intercellular (through lipid) and intracellular pathways (through corneocytes) was investigated, using FMM and its derivative, fluorescence recovery after photo-bleaching (FRAP) techniques. As a result of this study, the diffusion coefficient of this exogenous fluorophore has been determined, which turned out to be in a good agreement with the tabulated value.