Improved experimental protocols for sample preparation and handling during confocal Raman microscopy for skin drug permeation analysis

Despite the skin's barrier function limiting substance permeation, transdermal drug delivery offers benefits such as reduced systemic side effects and first-pass metabolism. This study explores confocal Raman microscopy as a non-destructive, real-time, label-free method to analyze drug retention and distribution in skin samples. Previous research identified key challenges related to laser interactions with skin components, such as thermal damage, fluorescence, and sample shrinkage. To mitigate these issues, particularly pronounced under 532 nm excitation, we developed pre-measurement protocols involving three consecutive Raman measurements (XY Raman mapping) with gradually increasing laser exposure, improving spectral quality and spatial accuracy of subsequent Raman analysis. Skin samples were additionally analyzed after ex vivo Franz cell diffusion studies (hydrated state) and freeze-drying (dehydrated state). We found that elevated hydration levels of samples from diffusion studies were associated with increased shrinkage, while freeze-dried skin exhibited limitations due to unpredictable sample movements and considerably reduced spectral quality at greater depths. Using the developed protocols, we assessed the spatial distribution of 4-cyanophenol (4-CP) in skin samples using both XY imaging at different depths and skin cross-section imaging, revealing reduced 4-CP content with depth and increased concentration with longer exposure during diffusion studies. This study demonstrates the effectiveness of pre-measurement protocols in overcoming challenges with Raman microscopy using 532 nm excitation wavelength for porcine skin analysis. It provides a robust and broadly accessible strategy to minimize thermal damage, fluorescence, and shrinkage while optimizing Raman scattering intensity, improving the reliability of Raman-based analyses in skin research and transdermal drug delivery. • Confocal Raman microscopy allows determining the spatial drug distribution in skin. • Pre-measurements improve quality and spatial accuracy of subsequent Raman analysis. • Laser-induced photobleaching reduces fluorescence and thermal illumination damage. • Freeze-dried skin shows unpredictable movement, signal attenuation, thermal damage.