Visualization of mitochondrial molecular dynamics during mitophagy process by label-free surface-enhanced Raman scattering spectroscopy

Background: Mitophagy is a selective way to eliminate dysfunctional mitochondria and recycle their constituents, which plays an important role in regulating and maintaining intracellular homeostasis. Real-time monitoring mitophagy process is of great importance for cellular physiological and pathological processes related to mitochondria. Howbeit, most of the current methods only focus on single-parameter detection of mitochondrial microenvironmental changes such as pH, viscosity and polarity. The mitochondrial molecular responses under mitophagy are not clear. Therefore, developing a new and simple method for molecular profiling is of great importance for accurately and comprehensively visualizing mitophagy. Results: In this work, Au NPs-based mitochondria-targeting nanoprobe was developed and the nanoprobe-based label-free surface enhanced Raman spectroscopy (SERS) method was proposed to track starvation induced mitophagy process at molecular level. The nanoprobe displayed good SERS performance and low cytotoxicity. Based on the developed strategy, the molecular response within mitochondria under mitophagy was validated. Meanwhile, the protein denaturation, conformational change, lipid degradation and DNA fragmentation within mitochondria under mitophagy were revealed for the first time, which provides molecular evidence for mitophagy. The changes in reactive oxygen species level and mitochondrial membrane potential further confirmed the damage of mitochondria. Moreover, the developed label-free SERS strategy was used to detect mitophagy in drug (cisplatin)-induced liver injury (DILI) cell model, and obvious mitophagy in DILI cells was observed. Significance: The molecular biochemical signature dynamic changes within mitochondria during mitophagy process were revealed by SERS for the first time. Moreover, compared with the current research, our study can provide new insights into mitophagy and mitophagy-involved diseases at molecular level. This study will provide new insights into the molecular mechanism of mitophagy and offer a simple and effective method for mito- chondrial molecular event monitoring in mitophagy-involved cellular processes.