Monitoring of betulin nanoemulsion treatment and molecular changes in mouse skin cancer using surface enhanced Raman spectroscopy

The aim of this study was to obtain in vivo and ex vivo reproducible surface enhanced Raman signal from mouse skin and to use it for the differentiation of skin pathologies. We induced skin carcinoma in mice models using a chemical treatment and tested the chemopreventive activity of a new formulation based on natural compound betulin extracted from the bark of birch trees. Using surface enhanced Raman spectroscopy (SERS) we identified in vivo and ex vivo the spectral signatures characteristic to the healthy skin, melanoma skin induced in mouse models, and to the pathology evolution when the betulin nanoemulsion formulation was topically applied on the cancerous skin of mice. SERS markers associated to each pathology were identified and the signal was distinguished from the classical Raman signal of skin based on several biomarkers, such as the disappearance of the amide I band of proteins, the amplification of the 1574 cm−1 band assigned to nucleic acid bases, and the appearance of the highly amplified band at 230 cm−1 characteristic to the metal-biomolecules complex. The various skin pathologies were differentiated using principal components analysis and K-means clustering. The effectiveness of the betulin nanoemulsion treatment was validated by the histological examination and the chemometrics methods, which successfully confirmed the direct SERS differentiation between the cancerous and the betulin nanoemulsion treated skin.