Immunoaffinity-enriched salivary small extracellular vesicles in periodontitis

Abstract

Aim: Saliva extracellular vesicles (EVs) serve as a significant reservoir of biomarkers that may be of clinical use in disease diagnosis. Saliva, however, contains EVs of both host- and bacterial- origin. Identifying suitable EVs for disease diagnosis involves enriching host EVs and limiting non-host contamination with effective isolation methods. The objectives of this research were: (1) to evaluate the salivary EVs enrichment in 12 periodontally healthy patients by two different methods: size exclusion chromatography (SEC) and bead-based immunoaffinity capture (EXO-NET®); (2) to analyze the variance expression of inflammatory cytokines in EXO-NET-enriched EVs, comparing individuals with periodontitis (n = 20) to non-periodontitis (n = 12). Methods: Whole unstimulated saliva samples were collected from 12 periodontally healthy and 20 periodontitis patients. EVs were isolated from the 12 non-periodontitis patients using SEC (referred to as SEC-EVs) and EXO-NET (referred to as EXO-NET EVs), after which their total protein content, 37 EV surface markers, and bacterial pathogens expression were compared. Subsequently, the inflammatory cytokines expression levels (interleukin-IL-6, IL-1β, IL-8, and IL-10) in EXO-NET EVs were measured for non-periodontitis and periodontitis. Results: EXO-NET EVs contained more EV-specific protein and substantially higher expression of EV surface markers (CD9, CD81, CD63), but less pathogenic DNA was detected compared to that in SEC-EVs. Additionally, EXO-NET EVs from periodontitis patients contained higher amounts of IL-6 and IL-8, and decreased IL-10, compared to those from non-periodontitis patients. Conclusion: The findings suggest that immunoaffinity capture (EXO-NET) is a dependable method for salivary EVs enrichment, resulting in a higher yield of host EVs with reduced bacterial DNA detection compared to SEC. Furthermore, the research proposes that immunoaffinity capture enriched EVs can function as biomarkers for periodontitis, demonstrated by an increased expression of proinflammatory cytokines from periodontitis patients.