Critical parameters for accurate monitoring of caffeine penetration in porcine skin using confocal Raman spectroscopy

Abstract

In this research, we addressed a challenge while measuring the penetration performance of caffeine (CAF) using confocal Raman spectroscopy (CRS). Normally in the process of CRS analysis, skin sample was moved from an incubation setup to a specified CRS-measuring sample holder. Accurate data collection may be questioned due to the variation of the environment the skin placed in. Therefore, two critical parameters including the CRS measuring temperature and proper skin hydration were focused; accordingly, four different conditions were designed. First, the skin was incubated in a real-time device with the skin placing onto PBS-filled chamber where the temperature was adjusted to 32℃. This device can be fixed under the CRS microscope, enabling simultaneous skin incubation and dynamic CRS measurements (condition i, reference). The other conditions referred to skins incubated in Franz diffusion cells for simulating the common experimental procedures. In order to control variables of CRS measuring condition, skins were transferred from cells to real-time device and open device. In real-time device, proper skin hydration was maintained and the skin temperature was adjusted to 32℃ (condition ii) and room temperature (condition iii). In open device, the skin was in a less hydrated state by moving onto a PBS-soaked filter paper and wrapped with aluminum foil at room temperature (condition iv). The skin penetration performances measured in these conditions were compared with reference. Caffeine solution and gel formulation were separately applied to the skin. The results showed in both cases that the decrease of skin temperature and hydration in condition iii and iv would apparently induce the decrease of detected caffeine signal, resulting in the inaccurate data collection. To this point, it indicates the reduction of solubilized caffeine in skin layer. We suggest the forming of caffeine crystallization at varied skin conditions to be the factor. Achieved video image, CRS spectrum collection and surface scan demonstrated the caffeine crystallization process on superficial skin layer. Polarized microscopic images exemplified the crystalline drug on tape stripped skin layers. It can be a potential support of caffeine crystallization inside skin. In short, we suggest the consideration of these parameters during CRS measurements for accurate monitoring of topical drug delivery. Meanwhile, the use of real-time device for dynamic skin incubation and data collection provides advantages in saving time and efforts in this study.

Keywords: Caffeine penetration; Confocal Raman spectroscopy; Crystallization; Porcine skin; Skin-placed environment.