Optical photothermal infrared spectroscopy can differentiate equine osteoarthritic plasma extracellular vesicles from healthy controls

Background Equine osteoarthritis is a chronic degenerative disease of the articular joint, characterised by cartilage degradation resulting in pain and reduced mobility and thus is a prominent equine welfare concern. Diagnosis is usually at a late stage through radiographic examination, whilst treatment is symptomatic not curative. Extracellular vesicles are small nanoparticles that are involved in intercellular communication. The objective of this study was to investigate the feasibility of Raman and optical photothermal infrared spectroscopy to detect osteoarthritis using plasma-derived extracellular vesicles. Methods Plasma samples were derived from thoroughbred racehorses. A total of 14 samples were selected (control; n= 6 and diseased; n=8). Extracellular vesicles were isolated using differential ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy, and human tetraspanin chips. Samples were then analysed using Raman and optical photothermal infrared spectroscopy. Results Infrared spectra were analysed between 950-1800 cm-1. Raman spectra had bands between the wavelengths of 900-1800 cm-1 analysed. Bands below 900 cm-1. Spectral data for both Raman and optical photothermal infrared spectroscopy was used to obtain a classification model and confusion matrices, characterising the techniques ability to distinguish diseased samples. Optical photothermal infrared spectroscopy could differentiate osteoarthritic extracellular vesicles from healthy with good classification (93.4%) whereas Raman displayed poor classification (64.3%). Plasma-derived extracellular vesicles from osteoarthritic horses contained increased signal for proteins, lipids and nucleic acids. Discussion/ conclusion For the first time we demonstrated the ability to use optical photothermal infrared spectroscopy to interrogate extracellular vesicles and osteoarthritis-related samples. Optical photothermal infrared spectroscopy was superior to Raman in this study, and could distinguish osteoarthritis samples, suggestive of its potential use diagnostically to identify osteoarthritis in equine patients. This study demonstrates the potential of Raman and optical photothermal infrared spectroscopy to be used as a diagnostic tool in clinical practice, with the capacity to detect changes in extracellular vesicles from clinically derived samples. ### Competing Interest Statement The authors have declared no competing interest.