Characterization and Specific Detection of Lactobacillus paracasei-Derived Extracellular Vesicles Using Anti-p40-Modified Au Thin Film

Background/Objectives: Extracellular vesicles (EVs) are nanoscale, membrane-enclosed structures that play key roles in intercellular communication and biological regulation. Among them, Lactobacillus paracasei-derived EVs (Lp-EVs) have attracted attention for their anti-inflammatory and anti-aging properties, making them promising candidates for therapeutic and cosmetic use. However, methods for specific detection and quantitative evaluation of Lp-EVs are still limited. This study aims to develop a surface plasmon resonance (SPR)-based sensor system for the precise and selective detection of Lp-EVs. Methods: Anti-p40 antibodies were immobilized on gold thin films to construct an SPR sensing platform. The overexpression of the p40 protein on Lp-EVs was confirmed using flow cytometry and Western blotting. For functional evaluation, Lp-EVs were applied to an artificial skin membrane mounted on a Franz diffusion cell, followed by SPR-based quantification and fluorescence imaging to assess their skin penetration behavior. Results: The developed SPR sensor demonstrated high specificity and a detection limit of 0.12 µg/mL, with a linear response range from 0.1 to 0.375 µg/mL. It successfully discriminated Lp-EVs from other bacterial EVs. In the skin diffusion assay, Lp-EVs accumulated predominantly in the epidermal layer without penetrating into the dermis, likely due to their negative surface charge and interaction with the hydrophobic epidermal lipid matrix. Fluorescence imaging confirmed this epidermal confinement, which increased over 24 h. Conclusions: This study presents a sensitive and selective SPR-based platform for detecting Lp-EVs and demonstrates their potential for targeted epidermal delivery. These findings support the use of Lp-EVs in skin-focused therapeutic and cosmetic applications. Future studies will explore strategies such as microneedle-assisted delivery to enhance transdermal penetration and efficacy.