Evaluation of Skin Penetration of Fluorescent Dissolved Formulations Using Confocal Laser Scanning Microscopy

<b>Background/Objectives</b>: Transdermal formulations are widely utilized in the pharmaceutical and cosmetic fields because they enable non-invasive administration and sustained local drug delivery. Conventional ex vivo skin permeation experiments using Franz diffusion cells have limitations in capturing the spatial and temporal dynamics of skin penetration. This study aimed to develop a confocal laser scanning microscopy (CLSM)-based approach to visualize and semi-quantitatively assess the penetration behavior of fluorescent dyes with differing lipophilicities. <b>Methods</b>: Four fluorescent dyes with different Log P values-Rhodamine B (Rho-B), Rhodamine 123 (Rho-123), Fluorescein Sodium (Flu-Na), and Nile Red (NR)-were formulated into lotion-based vehicles and applied to excised human abdominal skin. CLSM imaging was performed from 10 min to 240 min post-application. Fluorescence intensities were extracted from depth-resolved regions (R1-R4, 30-μm intervals) to examine penetration kinetics and distribution. <b>Results</b>: CLSM imaging demonstrated that Rho-B penetrated through stratum corneum and entered deep into the skin via the hair follicles. Rho-123 and Flu-Na exhibited intercellular and follicular penetration; however, Flu-Na showed only a slight increase in intensity over time; NR showed negligible penetration into the deeper layers. The results of our analysis indicated that moderately lipophilic substances such as Rho-B and Rho-123 diffused deeply into the skin via both transdermal and follicular routes, whereas highly hydrophobic or lipophilic substances remained in the superficial layers. <b>Conclusions</b>: The CLSM-based approach enabled spatially and temporally resolved, semi-quantitative evaluation of transdermal penetration in a single, non-destructive experiment. Although restricted to fluorescent probes, this approach provides a practical early-stage screening tool for comparing route-specific and time-dependent penetration behaviors of compounds with different lipophilicities.