Biochemical Characterization of Human Salivary Extracellular Vesicles as a Valuable Source of Biomarkers

SIMPLE SUMMARY: Extracellular vesicles (EVs) are an emerging source of biomarkers for a plethora of human disorders, ranging from neurodegenerative to cancer disorders. Most works focusing on biomarker discovery use blood as the EV source; however, although informative, EVs from blood are not easily separated from other particles and proteins circulating in blood vessels. Other authors have proposed the use of saliva as an EV source, while also finding limitations in the downstream analysis due to the limited amount of salivary EVs. Herein, we propose the isolation of salivary EVs and their subsequent characterization by Raman spectroscopy to obtain a comprehensive biochemical characterization that can be easily translated to diagnostics. The proposed method was successfully applied. The sensitivity of the Raman characterization of EVs was not limited by the low concentration of salivary EVs compared to serum EVs, and was able to provide a comprehensive characterization of EVs in a high throughput and repeatable manner. Consequently, our data suggest a new perspective on the use of salivary EVs when the interference of lipoproteins might hinder biomarker detection or when blood withdrawal is hampered. Raman spectroscopy can represent a turning point in the application of salivary EVs in clinics. ABSTRACT: Extracellular vesicles (EVs) are natural nanoparticles secreted under physiological and pathological conditions. Thanks to their diagnostic potential, EVs are increasingly being studied as biomarkers of a variety of diseases, including neurological disorders. To date, most studies on EV biomarkers use blood as the source, despite different disadvantages that may cause an impure isolation of the EVs. In the present article, we propose the use of saliva as a valuable source of EVs that could be studied as biomarkers in an easily accessible biofluid. Using a comparable protocol for the isolation of EVs from both liquid biopsies, salivary EVs showed greater purity in terms of co-isolates (evaluated by nanoparticle tracking analysis and Conan test). In addition, Raman spectroscopy was used for the identification of the overall biochemical composition of EVs coming from the two different biofluids. Even considering the limited amount of EVs that can be isolated from saliva, the use of Raman spectroscopy was not hampered, and it was able to provide a comprehensive characterization of EVs in a high throughput and repeatable manner. Raman spectroscopy can thus represent a turning point in the application of salivary EVs in clinics, taking advantage of the simple method of collection of the liquid biopsy and of the quick, sensitive and label-free biophotonics-based approach.