Label-Free Tracking of Nanoprodrug Cellular Uptake and Metabolism Using Raman and Autofluorescence Imaging.

Nano-DDS, a drug delivery system using nanoparticles, is a promising tool to reduce adverse drug reactions and maximize drug efficiency. Understanding the intracellular dynamics following the accumulation of nanoparticles in tissues, such as cellular uptake, distribution, metabolism, and pharmacological effects, is essential to maximize drug efficiency; however, it remains elusive. In this study, we tracked the intracellular behavior of nanoparticles of a prodrug, cholesterol-linked SN-38 (CLS), in a label-free manner using Raman and autofluorescence imaging. Bright autofluorescent spots were observed in cells treated with CLS nanoparticles, and the color tone of the bright spots changed with incubation time. The Raman spectra of the bright spots showed that the autofluorescence came from the nanoparticles taken into cells, and the change in color of bright spots indicated that CLS turned into SN-38 via hydrolysis inside a cell. It was found that most of the SN-38 were localized in small regions in the cytoplasm even after the conversion from CLS, and only a small amount of SN-38 was dissolved and migrated into other cytoplasm regions and the nucleus. The massive size growth of cells was observed within several tens of hours after the treatment with CLS nanoparticles. Moreover, Raman images of cells using the cytochrome c band and the fluorescence images of cells stained with JC-1 showed that cellular uptake of CLS nanoparticles efficiently caused mitochondrial damage. These results show that the combination of Raman and autofluorescence imaging can provide insight into the intracellular behavior of prodrug nanoparticles and the cell response and facilitate the development of nano-DDSs.

Label-Free Imaging of Intracellular Temperature by Using the O-H Stretching Raman Band of Water.

We propose a label-free method for measuring intracellular temperature using a Raman image of a cell in the O-H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O-H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label-free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.