Application of single molecule fluorescence microscopy to characterize the penetration of a large amphiphilic molecule in the stratum corneum of human skin.

Pierre Volz,Janna Frombach,Alexander Boreham,Sabrina Hadam,Zahra Afraz,Fiorenza Rancan,Ulrike Blume-Peytavi,Alexander Wolf,Annika Vogt,Jens Balke,Ulrike Alexiev,Tai-Yang Kim

doi:10.3390/ijms16046960

Abstract

We report here on the application of laser-based single molecule total internal reflection fluorescence microscopy (TIRFM) to study the penetration of molecules through the skin. Penetration of topically applied drug molecules is often observed to be limited by the size of the respective drug. However, the molecular mechanisms which govern the penetration of molecules through the outermost layer of the skin are still largely unknown. As a model compound we have chosen a larger amphiphilic molecule (fluorescent dye ATTO-Oxa12) with a molecular weight >700 Da that was applied to excised human skin. ATTO-Oxa12 penetrated through the stratum corneum (SC) into the viable epidermis as revealed by TIRFM of cryosections. Single particle tracking of ATTO-Oxa12 within SC sheets obtained by tape stripping allowed us to gain information on the localization as well as the lateral diffusion dynamics of these molecules. ATTO-Oxa12 appeared to be highly confined in the SC lipid region between (intercellular space) or close to the envelope of the corneocytes. Three main distinct confinement sizes of 52 ± 6, 118 ± 4, and 205 ± 5 nm were determined. We conclude that for this amphiphilic model compound several pathways through the skin exist.

Highlights

Treatment of skin disorders by topical therapy, i.e., topical application of active molecules on the skin surface, is challenging because the stratum corneum (SC) represents an effective physical and biochemical barrier [1]
Conventional fluorescence microscopy of cryosections obtained from human skin samples treated with the positively charged, lipophilic fluorescent dye ATTO-Oxa12 suggested that a vast amount of substance remained on the skin surface with no or minimal penetration to deeper layers (Figure 2C,E)
TIRF microscopy of longitudinal sections of human skin confirmed the presence of ATTO-Oxa12 molecules throughout the SC, in the subcorneal compartments and viable epidermis

Summary

Introduction

Treatment of skin disorders by topical therapy, i.e., topical application of active molecules on the skin surface, is challenging because the stratum corneum (SC) represents an effective physical and biochemical barrier [1]. The observation that molecules >500 Da usually do not reach therapeutic concentrations is frequently referred to as the “500 Dalton rule” [2]. Some therapeutic effects can be observed even with larger molecules such as the macrolide lactone tacrolimus (822 Dalton), which is an immunosuppressive drug used, for instance, in the treatment of atopic dermatitis [3]. Increasing experimental evidence suggests that large molecules can be recognized by the skin immune system through scanning and uptake activities of dendrites, which occur right below the SC [4]. A better understanding of how larger compounds cross the SC could help to develop strategies to facilitate penetration

Methods

Results

Conclusion