Gold Nanoparticle Detection with Two-Photon Excitation\nFluorescence Lifetime Imaging of NAD(P)H in Cancer Cells: An Analytical\nApproach to Separate Nanoparticle and NAD(P)H Signals

Gold\nnanoparticles (AuNPs) have shown promise for applications\nin the diagnosis and treatment of different diseases, including cancer.\nUnderstanding the effect of AuNPs on metabolic reprogramming in cancer\ncells at the single cell level is of high importance for improving\nthe efficacy and safety. Fluorescence lifetime imaging microscopy\n(FLIM) of nicotinamide adenine dinucleotide (phosphate) hydrogen (NAD(P)H)\nas a main metabolic cofactor and an indicator of metabolic reprogramming\nin cancer cells enables real-time monitoring of cancer cell metabolism\nin response to different treatments, including AuNPs. However, NPs\nsuch as AuNPs can be a potential source of signals themselves, which\nprovides opportunities to measure the NP internalization, but it is\nalso important to minimize confounding effects on metabolic measurements.\nIn this study, we detected inherent photoluminescence (PL) from the\nAuNPs in treated prostate cancer cells (PC-3 cell line) as well as\nin solution at the NAD(P)H emission wavelength. We developed an analysis\napproach to minimize the confounding effect of the AuNPs’ PL\non metabolic measurements. On the other hand, we assessed the reliability\nof the intracellular AuNPs’ PL as an estimator of AuNP uptake.\nTo assess if intracellular AuNPs’ PL may be dependent on the\nexposed cell type, we performed NAD(P)H FLIM imaging of AuNP-exposed\nSKBR-3 breast cancer cells, where we observed a similar AuNP PL but\nat a much lower level compared to PC-3 cells. We proposed that this\ndifference can be attributed to the different levels of AuNP uptake\nor varying intracellular microenvironments.