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Skin is a complex structured system primarily involved in Transdermal Drug Delivery (TDD). The outer stratum corneum represents the main barrier to the entrance of external molecules. Nowadays, none of the recognized methods, used for the investigation of penetration processes, is able to give a complete overview of the transport mechanisms involved. Standard protocols are based on the use of human or animal skin samples, which are difficult and expensive to obtain. Here, we present a novel experimental setup to investigate TDD by using Confocal Laser Scanning Microscopy and image analysis. The methodology is based on diffusion experiments of fluorescent-labelled fluids (water, oil, and oil-in-water emulsions), in a model matrix. Using an agarose gel as model for a proof of concept, different penetration efficiencies were observed, suggesting an important role of both chemical composition and fluid microstructure on the transport mechanism. An adequate model system should be used to better mimic the stratum corneum morphology and properties. To this aim, several bicontinuous emulsion gels, obtained from an emulsification process, were preliminary formulated and suggested as model systems to mimic the stratum corneum. In this work, we define the key directions for the development of an innovative approach to study the penetration of formulations through the skin.

Phospholipids, which are amphiphilic biomolecules composed of a polar head group and two nonpolar fatty acid tails, play a central role in cellular and body functions. The most common phospholipid is phosphatidylcholine (PC), which is also widespread used as a surfactant in colloidal systems, such as oil-in-water (O/W) emulsions, for several applications ranging from food to cosmetics. In these systems, PC tends to arrange at the interface between polar and nonpolar phases. Specific identification of these molecules at the interface is still an ambitious task. In this work, we propose immunofluorescence and confocal microscopy as valuable tools for the localization of PC at the interface of O/W emulsions, where phospholipids are used as surfactants. The protocol required the use of a primary antibody (TEPC-15), specific for choline, and a secondary fluorescently-labelled antibody, which selectively binds to the primary antibody. By using this approach, we were able to visualize choline-based phospholipids in cell membranes, lipid-based emulsions and in PC films and to estimate molecule concentration by image analysis techniques. We also investigated the interference of proteins on the staining procedure. We attributed this interference to possible molecular interactions close to the interface, which were found to depend on protein concentration. This innovative approach can have a relevant impact in a wide range of interfacial engineering applications, such as tuning emulsion microstructure and stability.

Gelatin gels are increasingly involved in many industrial applications due to several advantages including cost efficiency and biocompatibility. Generally, their production requires the use of aqueous solvents, which cause significant swelling, due to the ability of solvent molecules to penetrate through the gel microstructure and increase its volume. Since swelling mechanisms and their effect on the gel structure are not fully understood, further investigations are required. In this work, we combine macroscopic measurements of the swelling ratio (SR) with Nuclear Magnetic Resonance (NMR) and Confocal Laser Scanning Microscopy (CLSM) to investigate changes in the gelatin structure as a function of both polymer concentration and swelling time. SR values increase as a function of time until a maximum is reached and then show a slight drop for all the gelatin concentrations after 24 h swelling time, probably due to a network relaxation process. NMR allows determination of mass transport and molecular dynamics of water inside the gelatin pores, while CLSM is used to visualize the penetration of tracers (polystyrene microbeads) with a diameter much larger than the gel pores. Structural parameters, such as average pore size and tortuosity, are estimated. In particular, the pore size decreases for higher polymer concentration and increases during swelling, until reaching a maximum, and then dropping at longer times. The penetration of tracers provides evidence of the heterogeneity of the gel structure and shows that single microcarriers can be loaded in gelatin gels upon swelling.