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theses

Vibrational spectroscopy and imaging are advantageous for a number of dermatological applications, including characterization of endogenous components of skin or tracking penetration of pharmaceutical or cosmetic agents in skin tissue. Three applications illustrating the power of these techniques are described herein.

I. Effects of permeation enhancers on flufenamic acid delivery in ex vivo human skin by confocal raman microscopy
For topical delivery to be effective, a drug or active agent must cross the stratum corneum (SC) barrier into viable tissue. As such, barrier modification has been the target of many approaches for epidermal, dermal and transdermal delivery. The use of permeation enhancers in formulations is certainly one of the most widespread approaches. In the current study, flufenamic acid (FluA), a non-steroidal anti-inflammatory drug, is used as a model active agent to investigate the influence of hydrophobic versus hydrophilic permeation enhancers on its penetration and spatial distribution in ex vivo human skin using confocal Raman microscopy. In separate experiments, FluA in octanol (hydrophobic environment) or propylene glycol/ethanol (75/25) (hydrophilic environment) was applied to the SC surface for varying time periods followed by evaluation of penetration via confocal Raman mapping. Use of deuterated versions of the aforementioned enhancers permit us to spectroscopically distinguish the exogenous chemicals from the endogenous SC lipids without affecting penetration parameters. The FluA pathway is tracked by the C=C stretching mode at ~1618 cm-1. The spatial distribution and relative concentration of exogenous materials along with perturbation to the skin molecular and ultrastructure are imaged using both univariate and multivariate analysis. This, in turn, provides insight into mechanisms of delivery.

II. Infrared investigation of terbinafine interaction with stratum corneum constituents
Stratum corneum, the outermost layer of the epidermis, consists of keratin-filled corneocytes embedded in a highly ordered lipid matrix. This structure provides the main permeability barrier of the skin and also maintains water homeostasis. Efficient cutaneous delivery often introduces modifications to the SC. Terbinafine is a small, fungicidal, lipophilic drug commonly applied using topical formulations. Previous studies have utilized quantitative techniques to evaluate the amount of terbinafine in skin. In addition, others have used model systems such as defatted, powdered keratin to study binding parameters. Generally, there is a paucity of research evaluating the molecular nature of terbinafine interaction in skin environments. Current work evaluates molecular structure alterations to specific SC constituents upon interaction with terbinafine in a “physiological” environment, i.e. ex vivo intact SC. In this study, isolated human SC was incubated in an ethanol/buffer solution of terbinafine followed by the acquisition of temperature-dependent IR spectra. IR spectroscopy is a widely used, non-destructive technique for studies of lipid and keratin structure in intact human SC. IR bands are very sensitive to secondary structure alterations in keratin and to lipid acyl chain packing/conformational order. Terbinafine was found to perturb keratin secondary structure. Partial reversibility of the terbinafine-induced changes was observed upon soaking with ethanol/buffer while reversibility was more pronounced upon exposure to high relative humidity. Terbinafine-induced changes in endogenous lipid order, monitored by the symmetric CH2 stretching frequencies, were mostly masked by the effect of ethanol. Finally, we note that this method is not limited to studies of terbinafine interaction with skin, but can also be applied to monitor perturbations in intact SC when evaluating dosage levels and a variety of topical drugs or cosmetics. The ability to monitor the API (active pharmaceutical ingredient) allows formulations to be screened for delivery of APIs into the SC or across the SC depending on the desired location of action.

III. Comparison of hydration levels in human skin upon application of commercial moisturizers by confocal raman microscopy
The appearance of facial youthfulness and radiance results from hydrated, moisturized skin. As skin ages, it loses its natural ability to retain moisture and becomes rough, dry, while resulting in a clinically dull appearance with a loss of radiance, firmness, and suppleness. These symptoms can be delayed and improved with therapeutic use of a moisturizer that builds and maintains moisturization over time; however, most moisturizers that occlude the skin surface are perceived as heavy, greasy and are not consumer-preferred.
A unique, consumer-preferred natural moisturizing factor (NMF) formula was developed, which mimics SC naturally occurring components, with specific emulsifiers that deliver water deep into skin. This formulation (advanced NMF moisturizer) provides immediate hydration and protection (or enhancement) of the skin barrier over time to lock in moisture.
Confocal Raman spectroscopy studies assessed the distribution of water in ex vivo skin after topical application of the new formulation, advanced NMF moisturizer and a leading humectant moisturizer. In contrast to a leading humectant moisturizer that does not contain NMF components, the NMF-containing formulation significantly increased the relative water content and spatial distribution of water throughout epidermal regions of skin.
The results from these studies illustrate that increasing skin water content in both the stratum corneum and viable layers of the epidermis through therapeutic hydration with an NMF-containing formula can significantly improve skin hydration, barrier function, and the clinical appearance of skin radiance.

ETD_9926

Ph.D.

Includes bibliographical references

doi:10.7282/t3-54fa-h718

ETD doctoral

The author owns the copyright to this work.