3D Glioblastoma Molecular Responses to Carbon Dot-Delivered Riluzole Probed by Synchrotron FTIR

Engineered carbon-based nanocarriers provide a versatile platform for probing drug delivery-induced molecular responses in complex cellular models. Here, we report 2-acrylamido-2-methylpropanesulfonic acid (AMPS)-derived carbon dots (CDs) as vehicles for riluzole delivery in three-dimensional (3D) glioblastoma (GBM) spheroids, with 3D astrocyte spheroids employed as nontumoral control samples. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectro-microscopy was used to map subcellular biochemical responses, enabling label-free, high-resolution chemical imaging within intact 3D architectures. Multivariate analysis of SR-FTIR data revealed GBM-specific biomolecular alterations following treatment, including DNA conformational changes, lipid peroxidation, and protein secondary structure remodeling, whereas astrocyte spheroids exhibited minimal spectral perturbations, indicating a selective molecular response. Supervised classification using orthogonal partial least-squares-discriminant analysis (OPLS-DA) with constrained repeated random sampling cross-validation reliably differentiated treated and control spheroids, achieving high pixel-level classification accuracy and spatial consistency. These results demonstrate the capability of SR-FTIR microspectroscopy and imaging combined with multivariate analysis to resolve treatment-induced molecular responses in 3D tumor models while discriminating nontumoral controls. This integrated nanomaterial-spectroscopy approach provides a robust, data-driven framework for analytically evaluating nanocarrier-drug-cell interactions in complex biological systems.