Novel Approach for Assessment of Dermal Absorption of Extracellular Vesicle Products Using Immunoaffinity Fluorescent Nanodiamonds

INTRODUCTION: This study investigates the transdermal permeation efficiency and traceability of fluorescent nanodiamond-conjugated small extracellular vesicles (FND-sEVs) derived from umbilical cord mesenchymal stem cells. METHODS: First, the sEVs were isolated and characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis, confirming their typical cup-shaped morphology and size distribution (62.5-141.1 nm) consistent with canonical sEV properties. Subsequently, FNDs were conjugated to sEVs, forming FND-sEVs, whose composite structure - featuring a high-electron-density FND core surrounded by a vesicular sEV membrane - was verified by TEM. Conjugation efficiency was then validated via confocal microscopy, showing complete colocalization between FND fluorescence and Alexa Fluor 488-labeled wheat germ agglutinin-tagged sEVs. Transdermal assessment was conducted using a skin tissue model, with magnetically modulated fluorescence (MMF) spectroscopy applied for background-free quantification. RESULTS: Leveraging the unique nitrogen-vacancy centers in FNDs (fluorescence lifetime ∼20 ns), MMF eliminated interference from skin autofluorescence (∼3 ns) and enabled direct quantification in tissue digests without pre-separation. FND-sEVs were discovered to remain predominantly in the supernatant, with a measurable fraction permeating skin tissue layers, while their presence in the filtrate was negligible, thereby indicating limited transdermal passage. CONCLUSION: Collectively, these findings establish FND-sEVs as a traceable platform for transdermal studies and highlight MMF as a robust tool for quantifying nanoscale vesicle distributions in complex biological matrices.