Raman Spectroscopic Studies On The Hepatocytes And The Interactions With The Drugs

Raman spectroscopy, based on vibrational spectroscopy, is a rapid and nondestructive technique for studying biological system. Raman spectroscopy has the advantages for studying the cells in vitro, namely that no labels and fixation are required. Furthermore, Raman measurements on cells can be performed in physiological- like conditions. In this thesis, we applied Raman spectroscopy to study the character of the hepatocytes and discriminate the normal (HL-7702) and malignant (BEL-7402) hepatocytes, investigate the interactions between the antitumor drugs and hepatocytes, and explore the malignant hepatocytes by surface enhanced Raman spectroscopy (SERS). The details are the followings:(1) Micro-Raman spectroscopy was used to study the characters of single normal and malignant hepatocyte. The study indicates the Raman spectra are different for the different point in the cells, which suggests that the chemical component and structure are different in the different positions of cells. In addition, statistical analysis, including of T-test, principal component analysis (PCA), and linear discriminant analysis (LDA), was performed on the Raman spectra of malignant and normal hepatocytes. The T-test-LDA results show the bands at 785 cm^-1 and 1004 cm^-1 are significantly different between the malignant and normal hepatocytes and are the most crucial in distinguishing between the malignant and normal hepatocytes. The best results with the differentiation accuracy of 100% can be obtained with PCA-LDA.(2) Raman spectroscopy was used to explore the interactions between doxorubicin (DOX) and hepatocytes in vitro. And the interaction mechanism of DOX with malignant hepatocytes and side-effect of DOX to the normal hepatocytes were studied. The changes in the Raman bands for guanine (1313 and 1585 cm^-1) of DNA suggest that DOX may interact with DNA by the hydrogen bond to the N7 position of guanine to kill the cells. (3) The peptide A54 (AGKGTPSLETTP), a kind of hepatocarcinoma-binding peptide, was coupled with chemical toxin doxorubicin. We investigate the killing effect of A54-DOX to malignant hepatocytes and side-effect to normal hepatocytes by Raman spectroscopy. The results indicate that A54 can improve the cytotoxicity of DOX to malignant hepatocytes and decrease the side-effect of DOX to normal hepatocytes.(4) Ultrasensitive Raman measurements in single living malignant hepatocyte are possible through exploiting the effect of surface-enhanced Raman scattering. Colloidal gold particles (35 nm in size) that are deposited inside cells as "SERS-active nanostructures" result in strongly enhanced Raman signals of the native chemical constituents of the malignant hepatocytes in relatively short collection times (1 second for one mapping point). SERS mapping over a cell shows different Raman spectra at different places, reflecting the very inhomogeneous chemical constitution of the cells.