Simultaneous Time-Dependent Surface-Enhanced Raman Spectroscopy, Metabolomics, and Proteomics Reveal Cancer Cell Death Mechanisms Associated with Gold Nanorod Photothermal Therapy

Abstract

In cancer plasmonic photothermal therapy (PPTT), plasmonic nanoparticles are used to convert light into localized heat, leading to cancer cell death. Among plasmonic nanoparticles, gold nanorods (AuNRs) with specific dimensions enabling them to absorb near-infrared laser light have been widely used. The detailed mechanism of PPTT therapy, however, still remains poorly understood. Typically, surface-enhanced Raman spectroscopy (SERS) has been used to detect time-dependent changes in the intensity of the vibration frequencies of molecules that appear or disappear during different cellular processes. A complete proven assignment of the molecular identity of these vibrations and their biological importance has not yet been accomplished. Mass spectrometry (MS) is a powerful technique that is able to accurately identify molecules in chemical mixtures by observing their m/z values and fragmentation patterns. Here, we complemented the study of changes in SERS spectra with MS-based metabolomics and proteomics to identify the chemical species responsible for the observed changes in SERS band intensities during PPTT. We observed an increase in intensity of the bands at around 1000, 1207, and 1580 cm, which were assigned in the literature to phenylalanine, albeit with dispute. Our metabolomics results showed increased levels of phenylalanine, its derivatives, and phenylalanine-containing peptides, providing evidence for more confidence in the SERS peak assignments. To better understand the mechanism of phenylalanine increase upon PPTT, we combined metabolomics and proteomics results through network analysis, which proved that phenylalanine metabolism was perturbed. Furthermore, several apoptosis pathways were activated via key proteins (e.g., HADHA and ACAT1), consistent with the proposed role of altered phenylalanine metabolism in inducing apoptosis. Our study shows that the integration of the SERS with MS-based metabolomics and proteomics can assist the assignment of signals in SERS spectra and further characterize the related molecular mechanisms of the cellular processes involved in PPTT.

Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins

“…After PEG modification, the AuNRs became negatively charged (-10.2 ± 6.73 mV) and then became positive again after further modification of the RGD peptides. The characterization results are consistent with previous reports (52,53), which indicates the successful conjugation of the RGD peptides to the surface of AuNRs.…”

“…The proteomics analysis was conducted using the previously reported method (53). Briefly, purified and dried peptide samples from the previous step were dissolved in 10 μL solvent with 5% acetonitrile and 4% FA, and 4 μL of the resulting solutions were loaded onto a microcapillary column packed with C18 beads (Magic C18AQ, 3 μm, 200 Å, 100 μm × 16 cm, Michrom Bioresources) by a Dionex WPS-3000TPLRS autosampler (UltiMate 3000 thermostated Rapid Separation Pulled Loop Well Plate Sampler).…”

Near Infrared Fluorescence Imaging in Nano-Therapeutics and Photo-Thermal Evaluation

“…Gold nanoparticles (AuNPs) can be formulated in different shapes such as nanospheres, nanorods, nanoshells, nanostars, and nanocages because they exhibit excellent size and shape tunability and physiochemical properties [42]. Although AuNPs illustrate different shapes, all possess certain unique properties such as biological inertness and biocompatibility, the ability to absorb high energy X-rays, and they can be fine-tuned to absorb light in the near infra-red region (700–1200 nm) due to inherent SPR [43]. Considerable progress has been made towards the development of NIRF sensitive nanoparticles for efficient cancer imaging, identification of residual cells during surgery, and for locating the surgical margins [44].…”

Intracellular and Cellular Detection by SERS‐Active Plasmonic Nanostructures

“…More accurate information of PTT was given by MS identification of chemical species responsible for specific SERS signals. It was discovered that phenylalanine metabolism was perturbed upon PTT, causing irregular levels of phenylalanine and its derivative molecules, which induced cellular apoptosis (Figure B) . Hyperoxia‐induced biomolecular modifications were studied by SERS in neonatal mouse lung fibroblasts (nMLF), and Raman vibrations corresponding to phenylalanine and type 1 collagen indicated that hyperoxia induced intracellular acidification in nMLF …”