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. 2026 Apr 20;98(17):12775–12789. doi: 10.1021/acs.analchem.6c00176

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

Tanja Dučić †,*, Manuel Algarra ‡, David Pérez Guaita §, Guillermo Quintas ∥, María I Castillo ⊥, Elena González-Muñoz ⊥,#,*
PMCID: PMC13150809  PMID: 42009312

Abstract

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.

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Introduction

Glioblastoma multiforme (GBM, WHO grade IV) is an aggressive brain cancer with rapid progression, therapeutic resistance, and poor prognosis, with a median survival of under 15 months. Its pronounced molecular and cellular heterogeneity challenges effective treatment and early diagnosis, highlighting the need for analytical methods capable of high-resolution biochemical profiling.

In our previous work, we demonstrated the application of synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy for label-free, live-cell biochemical profiling of GBM cells in comparison to noncancerous human astrocytes. We also employed complementary imaging techniques, including soft X-ray tomography and hard X-ray fluorescence, to characterize the ultrastructure and elemental composition of shock-frozen GBM cells.

Building on this foundation, the present study shifts focus toward three-dimensional (3D) multicellular models, specifically patient-derived GBM and astrocyte spheroids. Spheroids or 3D cell cultures, where cells cluster into spherical shapes, closely mimicking the structure of solid tumors. This makes them especially valuable in cancer research, providing more realistic models for studying tumor behavior and drug response. These higher-order cell culture systems offer a more physiologically relevant platform that closely mimics the in vivo tumor microenvironment. By integrating SR-FTIR microspectroscopy with advanced 3D models, we aim to investigate the molecular effects of riluzole-loaded AMPS-derived carbon dots (CDs) nanoparticles (AMPS-CDs@RZ), offering novel insights into their potential as selective therapeutic agents for GBM.

Traditional two-dimensional (2D) in vitro cell cultures, while widely used in cancer research, present critical limitations due to their low throughput and inability to replicate the structural and biochemical complexity of living tissues. These models often fail to capture essential physiological features, including intratumoral heterogeneity, cell–cell and cell–matrix interactions, and the influence of the tumor microenvironment. Although animal models serve as an important intermediate step, they are expensive, ethically constrained, and often exhibit poor translational correlation with results from 2D cultures. To address these limitations, there has been a growing shift toward three-dimensional (3D) culture systems and organ-on-a-chip technologies. These advanced models offer improved physiological relevance, enabling more accurate and reproducible simulation of disease progression and therapeutic responses. In particular, 3D spheroids and organoids derived from patient cells have emerged as valuable platforms that bridge the gap between basic in vitro assays and clinical applications. Engineered nanoparticles, with tunable properties, enhance drug solubility, enable targeted delivery, and controlled release, making nanochemistry transformative in cancer therapeutics. Among emerging medical candidates, riluzole, a small molecule originally approved by the FDA for the treatment of amyotrophic lateral sclerosis (ALS), has shown promising anticancer activity. Recent studies have demonstrated its ability to inhibit the proliferation of glioma and other cancer cell types through suppression of glutamate signaling, a pathway increasingly recognized as a driver of glioblastoma progression. Moreover, riluzole has been reported to potentiate the effects of chemotherapy and radiotherapy, highlighting its potential for combination treatment strategies. − These findings underscore the urgent need for innovative delivery systems, such as carbon-based nanocarriers, that can selectively enhance riluzole’s bioavailability and therapeutic index, especially in the context of complex 3D tumor models.

In this study, we employed CDs synthesized from 2-acrylamido-2-methylpropanesulfonic acid (AMPS-CDs), previously characterized and evaluated as nanocarriers for riluzole, to investigate their effect on 3D GBM and astrocyte cell cultures. AMPS-CDs belong to a class of carbon-based nanomaterials that have garnered significant attention for their potential in both therapeutic and diagnostic (theragnostic) applications. These nanoparticles were characterized by high biocompatibility, low cytotoxicity, and versatile surface chemistry, which allows for functionalization and interaction with a range of biological targets. Their ultrasmall size facilitates cellular uptake and intracellular transport, making them ideal candidates for drug delivery. In the context of cancer research, carbon dots not only act as efficient drug carriers but also possess intrinsic properties that may enhance the therapeutic effect of the loaded agents, a concept referred to as carrier drug therapy.

Therefore, here, riluzole-loaded AMPS-CDs (AMPS-CDs@RZ) were applied to 3D spheroid models of GBM and noncancerous astrocytes to evaluate their biochemical effects using SR-FTIR spectroscopy and microscopy. This approach enabled a label-free, high-resolution assessment of cellular macromolecular changes induced by the nanocarrier system. In this context, SR-FTIR microspectroscopy provides a powerful platform for probing molecular responses within complex three-dimensional cellular systems. Here, we employed SR-FTIR to investigate patient-derived 3D glioblastoma and astrocyte spheroids treated with AMPS-CDs@RZ, enabling spatially resolved profiling of nucleic acids, proteins, and lipids while preserving native cell–cell interactions and microenvironmental context. Combined with multivariate statistical analyses, this approach captures subtle, treatment-induced biochemical shifts, providing mechanistic insight into drug–nanocarrier interactions. By bridging physiologically relevant 3D models, including organoids and organ-on-chip systems, with high-content molecular analysis, SR-FTIR offers a powerful platform for guiding more selective and effective glioblastoma therapies. This approach extends previous applications of SR-FTIR for GBM single-cell analysis, where metabolic alterations in glioblastoma cell monolayers were induced. These compelling findings pave the way for personalized therapeutic strategies in glioblastoma, providing a solid foundation for developing more selective and effective treatments. By connecting the molecular specificity and high spatial resolution of SR-FTIR microspectroscopy, critical insights into the biochemical mechanisms underlying drug response in GBM. This work underscores the importance of studying cellular behavior within physiologically relevant 3D systems, such as organoid models, which more faithfully recapitulate native tissue architecture and microenvironmental factors. As analytical chemistry advances toward more biologically integrated approaches, SR-FTIR emerges as a powerful tool for probing complex disease models and guiding the development of next-generation therapeutics.

Experimental Section

Cell Culture and 3D Spheroid Generation

Glioblastoma (GBM) cell line #11 (provided by the University Medical Center Göttingen, Germany; Ethics Committee approval number 11/8/13) was cultured in complete medium consisting of high-glucose DMEM (500 mL, Gibco), 10% fetal bovine serum (Gibco), 2 mM l-glutamine (Gibco), and penicillin–streptomycin (Thermo Fisher).

Primary human astrocytes, isolated from healthy cerebral cortex tissue and purchased from Innoprot (Derio, Bilbao, Spain; #P10251), were maintained in complete astrocyte medium containing high-glucose DMEM (Gibco), 10% fetal bovine serum (Gibco), 100 U·mL–1 penicillin–streptomycin (Gibco), 2 mM l-glutamine (Gibco), 1× nonessential amino acids (Gibco), and 1× N2 supplement (Gibco).

For 3D spheroid generation, GBM and astrocyte cells were detached using trypsin/EDTA and seeded into ultralow attachment 6-well plates at a density of 140,000 cells/well in DMEM supplemented with N2 and B27 supplements, 20 ng·mL–1 bFGF, 10 ng·mL–1 EGF, and 10 μM iROCK. On day 2, floating spheroids were identified, and the medium was replaced with the same spheroid medium lacking iROCK. Medium changes were performed every other day.

On day 7, GBM and astrocyte spheroids were treated.

Spheroids Treatments

AMPS-based carbon nanoparticles (AMPS-CDs) with an average size of approximately 5 nm were synthesized following the method previously described. The cooled reaction mixture was filtered to remove carbonized residues, then lyophilized to obtain a bright white powder, designated as AMPS-CDs nanoparticles. Spheroids were treated for 24 h with 50 μM riluzole, 0.1 mg·mL–1 AMPS-CDs, or a combination of both (AMPS-CDs@RZ). Untreated spheroids served as controls. Treatment concentrations were previously optimized for single-cell SR-FTIR analysis. In our previous studies, we selected the lowest riluzole concentration (50 μM), which, in combination with 0.1 mg/mL AMPS-CDs, yields an effect comparable to that produced by 100 μM riluzole alone.

Spheroid Fixation, Embedding, and Sectioning

Three-dimensional GBM and astrocyte spheroids were fixed in 4% paraformaldehyde for 20 min at 25 °C, followed by incubation in 30% sucrose at 4 °C for 12 h for cryoprotection. Subsequently, spheroids were embedded in OCT compound (Tissue-Tek OCT, 4583, Sakura Finetek). Samples were frozen at −80 °C, and 8 μm-thick sections were prepared using a Leica cryostat. Sections were collected onto BaF2 slides (13 × 0.5 mm; transparent to infrared and visible light) for subsequent analysis at ALBA synchrotron using synchrotron radiation-based Fourier-transform infrared spectroscopy (SR-FTIR). Although sucrose stabilization was necessary for the small (∼200–400 μm) spheroids, prefixation with PFA and identical processing ensured any minimal sucrose-related signals in the 1200–900 cm–1 region are negligible and do not affect comparative spectral analysis.

SR-FTIR Measurements

SR-FTIR measurements were performed at the MIRAS BL01 beamline of the ALBA synchrotron in Spain, utilizing the synchrotron radiation source coupled to a Hyperion 3000 microscope and Vertex 70 spectrometer end-station equipped with a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector. Data acquisition was conducted using the OPUS 8.2 software package (Bruker, Germany).

Spectra were collected in transmission mode using a 36× Schwarzschild objective and a 10 μm × 10 μm aperture, with a spatial step size of 5 μm. For seven individual cells, spectral data were acquired over the 3700–1000 cm–1 mid-infrared range at a spectral resolution of 4 cm–1, with 256 coadded scans per spectrum.

Data Analysis

Spectral preprocessing involved calculation of the second derivative using a Savitzky–Golay filter with 17 smoothing points and a third-order polynomial fit, complemented by vector normalization. The vector normalization step was applied postdifferentiation to avoid dependence on specific reference peaks. Principal component analysis (PCA) was subsequently performed on the second-derivative and normalized spectra to elucidate patterns of variance within the data set. PCA computations were carried out using Orange software (Bioinformatics Laboratory, University of Ljubljana, Version 3.20.1) with the spectroscopy add-on ( Version 0.4.1), including focused analyses on specific spectral regions.

Infrared Imaging

Infrared images were collected using a microscope equipped with a Schwarzschild 15× objective and condenser over 180 μm × 180 μm regions, using a full-field 64 × 64-pixel FPA detector and 128 scans. The setup provides a pixel size of 2.8 μm and a spectral resolution of 4 cm–1. Each pixel spectrum was averaged from 128 accumulations. The absorbance cartogram was generated by integrating spectral regions specific to biomolecules: 1480–1700 cm–1 for proteins (Amide I and II), 2800–2990 cm–1 for lipids, 1215–1265 cm–1 for DNA, 1720–1760 cm–1 for carbonyl groups, and 1505–1525 cm–1 for Tyrosine (Tyr).

Supervised Hyperspectral Image Analysis

Supervised analysis was performed in Matlab from Mathworks (Natick USA), using in-house written functions based on the PLS_Toolbox. Orthogonalized partial least squares-discriminant analysis (PLS-DA) models were performed by comparing each treated image with their corresponding control. To avoid overestimated effects related to the correlation of the spectra from adjacent pixels, we used constrained repeated random sampling-Cross-validation (CORRS-CV) for validation of the models as described elsewhere. In brief, 1/3 of the pixels were randomly selected for the test set, and then a filter was applied to exclude the pixels within a cutoff ∂ value proximity (in this study ∂ = 5, which corresponds to 2.8 μm). The remaining pixels outside the ∂ range of the test sets were placed in the calibration set (a minimum value of 200 pixels was established), which was used to create a model. An internal 2-fold CV was used for selecting the optimal number of Latent Variables (LVs). Then, the test set selected early was used to evaluate the generalization performance of the optimal model. This process was repeated several times, until at least 99% and 95% of labeled pixels were included at least one time in a training set and once in a test, respectively. Analysis was performed over the first derivative of the spectra, and oPLS-DA loadings were integrated using the cumsum in Matlab to obtain bands connected to the underivative spectrum.

Results

Building on our previous characterization of glioblastoma and nontumorous astrocyte cells using synchrotron-based X-ray imaging and live-cell FTIR spectroscopy, here we analyzed these cells organized into three-dimensional (3D) spheroid structures to capture a more physiologically relevant in vivo tumor microenvironment. Using SR-FTIR spectro-microscopy, we investigated the molecular alterations induced by riluzole and AMPS-CDs within the 3D GBM and control astrocyte spheroids. This approach enabled us to detect dynamic changes in key biomacromolecules, including proteins, lipids, and nucleic acids, while preserving the native cellular environment. As shown in Figure , SR-FTIR spectroscopy successfully revealed complex biochemical responses to treatment, demonstrating its utility in assessing the efficacy and molecular impact of potential therapeutic agents in a more physiologically relevant model than classical 2D cell culture.

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Second derivative of the FTIR averaged spectra of cells of astrocyte 3D-spheroids (A) and 11ST primary glioblastoma spheroids (E) (blue), after treatment with riluzole (red), after treatment with AMPS-CDs NPs (green) and AMPS-CDs@riluzole (orange) in the spectral region of the lipids, 3500–950 cm–1. The inset shows the t-SNE analysis (B,F). PCAs (loading values) of the first three principal components, PC1 (blue), PC2 (red) and PC3 (green), showing the contribution of individual absorbance of corresponding areas in the graphs above, are shown in (C,G). PCA Scatter plot of PC1 and PC2 and (D,H). N = 25–238.

SR-FTIR Microspectroscopy

As shown in Figure , the FTIR spectra revealed the main biomolecular components of the 3D spheroids derived from healthy astrocytes and the 11ST GBM cell line. By comparing the spectral regions corresponding to lipids, proteins, esters, carbohydrates, and nucleic acids, the average spectra depicted in Figure A,E illustrate the molecular changes induced by the treatments.

To further dissect the molecular differences, we employed t-SNE (t-Distributed Stochastic Neighbor Embedding) analysis based on second derivative spectra, revealing striking separations among the treatment groups, as shown in the insets of Figure B,F. This clear clustering suggests that each treatment elicits distinct biochemical fingerprints in both astrocyte and GBM spheroids. This visual distinction motivated a deeper investigation using principal component analysis (PCA), which identified the most relevant molecular variations across conditions. The loading plots for the first three principal components (PCs), shown in Figure C,G, highlight the specific spectral regions contributing to treatment-induced changes, and point out how these treatments alter the cellular molecular landscapes in 3D. The most pronounced biomacromolecule changes observed along PC1 included DNA-associated regions, followed by proteins and lipids. The distinct segregation among PCs underscores the complexity of the cellular response under 3D culture conditions, offering valuable insights into the molecular reprogramming triggered in GBM spheroids upon treatment.

In GBM spheroids (Figure E), PC1 captured the most prominent alterations, particularly in the nucleic acid region (∼900–1250 cm–1), as indicated in the loading plot (Figure G), and highlighted by the blue arrow in Figure H. PC2 (red line in Figure G) reflected additional notable changes in protein secondary structure, while PC3 primarily emphasized differences in lipid-related bands. Specifically, PC1 (blue line) revealed significant differences around the 1105–1113 cm–1 region, assigned to phospho-sugar backbone vibration (PO–C–O), commonly attributed to RNA and DNA structural features. , Modifications of bases, sugar moieties, and hydrogen-bonding networks in nucleic acidsoften observed upon radiation damagemay similarly occur here due to riluzole-induced stress, or as part of the nanocarrier’s effect on chromatin structure. These shifts are consistent with reported epigenetic alterations, including changes in DNA backbone conformation and DNA methylation state, − as well as modifications in RNA structure and content. Additionally, in the lipid region, a notable increase in intensity at ∼2926 cm–1 was observed in AMPS and AMPS-CDS@RZ treated groups, suggesting enhanced lipid reorganization, potentially associated with membrane fluidity changes or peroxidation events.

Overall, PCA provides a powerful overview of macromolecular changes, revealing that AMPS-CDS@RZ treatment primarily alters nucleic acid architecture, followed by alterations in protein conformation and lipid dynamics, expected as part of a coordinated treatment response. This hierarchy of molecular impact is particularly evident in GBM spheroids, where nucleic acid changes dominate, consistent with earlier single-cell analyses of riluzole-loaded CDs in 2D cultures, , and highlights the added value of 3D culture for revealing more physiologically relevant responses. Subsequent effects on protein secondary structure and lipid rearrangement were also detected. All these macromolecular alterations in lipids, and the fingerprint part, including proteins and DNA, were further analyzed in detail.

Lipid Analysis

Figure shows an in-depth analysis of the lipid region in astrocytes and GBM spheroids. In GBM spheroids, key differences were observed at ∼2926 cm–1 and ∼2853 cm–1, corresponding to the asymmetric and symmetric stretching vibrations of methylene (CH2) groups in acyl chains, respectively. These bands are known to shift due to lipid peroxidation and alterations in membrane structure under oxidative stress conditions. ,, These changes were more pronounced in the riluzole and AMPS-CDS@RZ treatment groups compared to the control and AMPS groups (PC2), as shown by the PCA separation along PC2 in Figure E. The finding suggests that riluzole and AMPS-CDS@RZ may trigger lipid peroxidation, a hallmark of oxidative stress, that could impact membrane integrity and overall cellular stress. This interpretation is supported by our previous observations in single GBM cells, where inner membrane alterations were identified via cryo-soft X-ray tomography.

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Second derivative FTIR averaged spectra of the lipid region (2800–3050 cm–1) of 3D astrocyte (A) and 11ST primary GBM spheroids (D), comparing untreated controls (blue), RZ-treated (red), AMPS-CDs treated (green) and AMPS-CDS@RZ-treated (orange) samples. PCA scatter plots of PC1 and PC2 for astrocytes (B) and GBM (E), and PCAs loading plots showing PC1 (blue) and PC2 (red) contributions for astrocytes (C) and GBM (F). Sample sizes: N = 25–238.

In astrocyte spheroids, lipid analysis revealed distinct biochemical responses to AMPS-CDS@RZ treatment compared to either RZ or AMPS treatments alone. PCA supported this observation, with separation along PC2 differentiating the AMPS-CDS@RZ and RZ groups, mainly driven by the AMPS-CDS@RZ treatment (Figure A–C). The AMPS-CDs@RZ-treated astrocyte spheroids showed prominent changes in the symmetric and asymmetric CH3 and CH2 stretching bands. PC1 captured these alterations, while PC2 specifically highlighted a reduction in the CH3 band (2954 cm–1) after AMPS-CDS@RZ treatment. This differential modulation between RZ alone and AMPS-CDS@RZ suggests that the nanocomposite contributes to membrane reorganization in astrocytes, although to a lesser extent than in GBM cells. In contrast, GBM spheroids showed a clearer separation between control and all treatment groups, with PC2 emphasizing shifts in CH3 and CH2 asymmetric bands in GBM in response to AMPS-CDS@RZ treatment (Figure D–F).

Further investigation focused on the carbonyl (CO) group stretching vibration (∼1740 cm–1), which reflects carbonylation in both lipid and protein fractions depending on the spectral context, as shown in Figure . To distinguish these biochemical contributions, the CO band area was integrated and normalized in two different ratios: relative to Amide I (∼1650 cm–1) to assess protein carbonylation, and relative to the combined area of asymmetric CH2 and CH3 stretching bands (with maxima at ∼2926 and ∼2954 cm–1) to evaluate lipid-associated CO signals. These ratios were calculated from baseline-corrected and normalized spectra (Figure ). Significant increases in CO values in both lipids and the amide regions were observed in GBM spheroids after AMPS-CDs@RZ-treatment (red asterisks, Figure B,D). Notably, no significant differences were observed between control and AMPS-CDs@RZ-treated astrocyte spheroids, highlighting the tumor-selective response. When comparing AMPS-CDs@RZ to riluzole-alone treatments, both spheroid types exhibited significant reductions in protein-normalized areas, suggesting protein backbone alterations. Moreover, the level of CO -to-lipid ratioused as a marker of lipid peroxidation − was significantly higher in GBM spheroids, but not in astrocytes (red asterisks, Figure C,D), indicating enhanced oxidative damage in the tumor model. This pattern aligns with previous results in riluzole-treated GBM single cells, and supports the interpretation that the AMPS-CDs@RZ combination exacerbates oxidative stress and membrane lipid damage in GBM. An increased abundance of carbonyl (νCO) vibrations relative to the CH2 and CH3 bands is a widely recognized marker of lipid peroxidation − and was detected in this system.

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Integrated ratios of carbonyl (CO) vs Amide I in astrocytes (A) and 11ST primary glioblastoma spheroids (B). CO vs asymmetric CH2 and CH3bands in astrocyte (C) and GBM (D) spheroids. *p < 0.05.

These findings suggest that AMPS-CDs@RZ treatment selectively amplifies lipid peroxidation specifically in glioblastoma (GBM) cells, in contrast to nontumor astrocytes (Figure ). This differential lipidomic modulation highlights the potential of AMPS-CDs@RZ, presenting a promising therapeutic strategy to target glioblastoma while preserving healthy astrocyte membrane function.

Fingerprint Region

The fingerprint region (1480–900 cm–1) of the SR-FTIR spectra provided critical insights into nucleic acid and carbohydrate structural changes induced by treatment. As shown in the second-derivative spectra (Figure A,D), the band near ∼1113 cm–1, related to symmetric P–O–C stretching in RNA backbones, exhibited notable changes following riluzole treatment in both astrocyte and GBM spheroids, whereas alterations were observed exclusively in GBM spheroids after AMPS-CDs@RZ treatment (Figure D). Principal component analysis (PCA) revealed clear segregation along PC1 and PC2 for astrocyte spheroids, with even more pronounced separation in GBM spheroids (Figure B,E), indicating distinct treatment-induced macromolecular changes in both models. Control cells of both types of spheroids showed similar absorbance in this region, consistent with stable nucleic acid conformations. In contrast, cells treated with Riluzole and AMPS-CDs@RZ showed altered phosphate-associated spectral profiles, indicative of conformational changes in RNA and DNA, and possibly in phosphorylated proteins, especially in the GBM spheroids (Figure D–F). In GBM spheroids, PC2 loadings revealed characteristic minima at ∼992 cm–1 (related to vibration of C–O and C–C in ribose), ∼1050 (related to carbohydrates), and ∼1138 cm–1 (associated with oligosaccharide C–OH stretching band) as shown in Figure E,F. These shifts suggest treatment-induced alterations in RNA structure and glycosylation states. In astrocytes, the PC2 loading plot showed minima at ∼967 cm–1 (C–C and C–O in deoxyribose of DNA) and at ∼1088 cm–1 (associated with symmetric PO2 stretching vibration in B-form DNA), , implying more subtle nucleic acid conformational changes (Figure C). These spectral variations reflect treatment-induced molecular alterations that may influence RNA folding, DNA packaging, or phosphate group availability. The magnitude and specificity of these changes were more pronounced in GBM spheroids, suggesting a higher sensitivity to RZ and, importantly, to AMPS-CDs@RZ exposure. The differential pattern of changes observed between astrocytes and GBM is consistent with the tumor-selective molecular response previously described for this nanocomposite in 2D systems. , These spectral shifts reflect treatment-induced molecular alterations at the RNA level, indicating changes in RNA structure and interactions that may propagate to downstream regulatory processes, ultimately influencing cellular behavior, stress responses, and the overall therapeutic outcome. Furthermore, since the observed bands overlap with regions assigned to phosphorylated amino acid residues (e.g., phosphotyrosine), phosphorylation-related modifications could also be involved in the observed spectral changes, especially in GBM spheroids.

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Second derivative FTIR averaged spectra of the fingerprint region (900–1480 cm–1) for 3D astrocyte (A) and 11ST GBM spheroids (D), comparing untreated controls (blue), RZ-treated (red), AMPS-CDs NPs-treated (green) and AMPS-CDs@RZ-treated (orange). PCA scatter plots of PC1 and PC2 for astrocytes (B) and GBM (E). PCA loading plots showing PC1 (blue) and PC2 (red) contributions for astrocytes (C) and GBM (F) spheroids. Sample sizes: N = 25–238.

Protein Analysis

Following the analysis of DNA and RNA changes, protein-level alterations were examined in detail. Figure A,B display the FTIR spectra of the Amide I and Amide II regions, primarily associated with protein secondary structures, in astrocyte and GBM spheroids, respectively. The insets display t-SNE clustering plots for both spheroid types, revealing clear segregation of treatment groups, particularly in GBM spheroids after AMPS-CDs@RZ and RZ treatments (Figure B), suggesting distinct proteomic responses. The second derivative spectra of the Amide I and II regions (Figure C,D) provide critical information about the conformational state of cellular proteins. To gain deeper insight into potential structural alterations induced by the different treatments, the spectra were deconvoluted to isolate the main subpeaks corresponding to specific secondary structures, such as α-helices, β-sheets, and amino acid side-chains such as glutamine and tyrosine (Figure E,F, for untreated astrocytes and GBM spheroids, respectively). The integrated areas under these subpeaks were then quantified for each treatment group. The resulting data were presented as violin plots to illustrate the distribution and variability of secondary structure components across samples.

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FTIR averaged second derivative spectra of the Amide I and II regions (1480–1700 cm–1) in astrocyte spheroids (A,C), respectively, and 11ST GBM spheroids (B,D) respectively. Deconvoluted spectra for astrocytes spheroids (E) and GBM non treated spheroids (F), show the integrated areas for α-helix (1655 cm–1, violet), β-sheet parallel conformation (1635 cm–1, green), amino acid side chains (1620 cm–1, pink), free amino acid side-chains such as glutamine (1572 cm–1, orange), tyrosine (1515 cm–1, blue), as well as α-helix in the Amide II region (1545 cm–1). Sample sizes: N = 25–238.

Specifically, the α-helix-associated band at ∼1655 cm–1 (violet in Figure E,F) was used to assess changes in helical content. Subsequently, bands associated with β-sheet parallel conformations (∼1635 cm–1, green), amino acid side chains (∼1620 cm–1, pink), free amino acid side-chains such as glutamine (∼1572 cm–1, orange), and tyrosine (∼1515 cm–1, blue) were integrated for comparative analysis. These components revealed distinct spectral signatures in GBM vs astrocyte spheroids. The arrows in Figure F highlight pronounced differences in the GBM group compared with astrocytes, particularly in the α-helix (violet line), amino acid side chains (pink), and aromatic residues such as glutamine (orange) and tyrosine (blue). The significant differences among treatment groups are summarized in Figure .

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Integrated area of β-sheet parallel conformation bands (maximum at 1630 cm–1), amino acid side chains (e.g., Tyr and Phe; maximum at 1620 cm–1), α-helix proteins (maximum at 1655 cm–1), free amino acid side-chains, such as tyrosine (1515 cm–1) and glutamine (1572 cm–1) in control 3D cells culture (blue), riluzole treated (red), AMPS CDs (green) and AMPS-CDs@RZ treated (orange). *p < 0.05 indicates significant differences between groups. Sample sizes: N = 25–238.

Importantly, the deconvolution strategy enables discrimination of subtle changes in secondary structure compositionparticularly relevant when evaluating the impact of riluzole and its nanocarrier formulation (AMPS-CDs@RZ) on protein folding. These results support previous findings from our 2D single-cell studies, where AMPS-CDs@RZ induced prominent modifications in α-helix protein structure and side-chain exposure in glioblastoma cells. ,

α-Helix and Tyrosine Elevation in GBM Cells after Treatment

Calculation of the integrated areas under the individual subpeaks revealed that the most prominent changes were observed in the spectral regions associated with α-helix structures and tyrosine residues within proteins. The integrated area of the α-helix band, with a maximum at ∼1655 cm–1, showed a significant decrease in GBM spheroids following treatment, in contrast to an increase observed in astrocyte spheroids, as shown in Figure . Additionally, tyrosine-associated bands indicated a significant rise in tyrosine content in GBM spheroids, while astrocytes exhibited a marked significant decrease.

Furthermore, spectral bands corresponding to protein side chains, particularly ∼1620 cm–1 and ∼1572 cm–1commonly associated with glutamine, phenylalanine, and tyrosine moietieswere significantly elevated only in GBM spheroids, suggesting enhanced exposure or structural alterations in aromatic residues. In contrast, astrocyte spheroids exhibited minor changes only in the β-parallel sheet region, indicating a more restricted or selective structural response to AMPS-CDs@RZ treatment and could reflect a preservation of protein folding integrity in nontumoral cells.

These findings highlight distinct, cell-type-specific alterations in protein secondary structures and side-chain environments following treatment, potentially reflecting differential stress responses, signaling regulation or adaptive mechanisms between tumorous and nontumorous cells.

FTIR Imaging

FTIR imaging revealed the spatial distribution of key biomacromolecules across treatment conditions. Figures and map proteins (Amide I and II: 1480–1700 cm–1), lipids (2800–3000 cm–1), DNA (1215–1264 cm–1), carbonyl (ester) groups (C=O:1720–1760 cm–1) and tyrosine (Tyr: 1505–1525 cm–1) in astrocyte and patient-derived GBM spheroids, respectively, highlighting striking, cell-type-specific molecular responses to the treatments. In astrocyte spheroids, total protein absorbance was highest in control samples and showed only minor changes across treatments (Figure ). Notably, both lipid and carbonyl group signals peaked under RZ treatment. Interestingly, while the CO (carbonyl) band increased with RZ alone, it was markedly reduced under AMPS-CDs@RZ treatment. This pattern suggests that AMPS-CDs@RZ mitigates RZ-induced carbonyl accumulation, potentially by modulating lipid oxidation or protein carbonylation levels. The DNA-associated phosphate band showed the highest intensity in AMPS-CDs@RZ–treated spheroids, indicating enhanced nucleic acid activity and suggesting treatment-induced modulation of DNA structure or function. Such spectral changes are consistent with previous observations of nucleic acid reorganization upon riluzole-nanocomposite treatment in 2D systems. , The distribution of tyrosine appeared largely uniform across all astrocyte-treated spheroids.

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Spatial distribution of Amide I and II-, lipids-, DNA- and carbonyl group (esters) and Tyrosine-related absorptions by FTIR imaging of astrocyte spheroids under control, AMPS, Riluzole and AMPS CD@Riluzole treatments. Integrated bands: Amide I and II (1700–1480 cm–1), lipids (3000– 2800 cm–1), DNA (1265–1215 cm–1), CO (1780–1700 cm–1), Tyr (1505–1525 cm–1). Scale bar 100 μm.

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Spatial distribution of Amide I and II-, lipids-, DNA- and carbonyl group (esters) and Tyrosine-related absorptions by FTIR imaging of GBM spheroids under control, AMPS, Riluzole and AMPS CD@Riluzole treatments. Integrated bands: Amide I and II (1700–1480 cm–1), lipids (3000–2800 cm–1), DNA (1265–1215 cm–1), CO (1780–1700 cm–1), Tyr (1505–1525 cm–1). Scale bar 100 μm.

In contrast, GBM spheroids exhibited the highest concentration of carbonyl groups following AMPS-CDs@RZ treatment (Figure ), indicating an opposite trend to that seen in astrocytes. The carbonyl signal was spatially widespread and showed higher intensity than in any other treatment group, aligning with previous results (Figure ) showing increased lipid peroxidation in GBM spheroids. The distribution of other biomacromolecules (proteins, lipids, and DNA) remained relatively stable across AMPS and control conditions, but the pronounced protein and DNA signal drop with RZ and the strong carbonyl signal with AMPS-CDs@RZ underscore distinct treatment-specific biochemical effects. Tyrosine levels in GBM cells were generally higher than in astrocyte-treated spheroids, suggesting that AMPS CDs may contribute to this alteration, whereas riluzole did not affect the distribution of this amino acid. Tyrosine distribution overlapped with amides I and II, lipids, and partially with DNA, suggesting a strong correlation between these molecules.

These observations reinforce the role of AMPS-CDs@RZ in amplifying oxidative damage selectively in tumor sheroides, while riluzole alone may trigger nucleic acid and protein reorganization without strong lipid oxidation.

Supervised Hyperspectral Analysis

Figure presents the results of the oPLS-DA analysis used to discriminate each treated spheroid’s image from the corresponding control image. In Figure , the average distribution of the predicted class values (Y = 1 for treated, Y = 0 for control) for GBM (Figure B) and astrocyte spheroids (Figure D), obtained from the CORRSCV analysis, is shown. The classification performance was high: the models correctly classified nearly 100% of the pixels according to the double cross-validation results from the CORRSCV process. This strong predictive accuracy demonstrates clear and robust dissimilarities between the control and treated images and the homogeneity of prepared spheroids. Moreover, the prediction maps reveal that pixel classifications are generally uniform across each image, without distinct regions or “hot spots”. This indicates that the spheroids do not display localized spatial features where treatment effects are either enhanced or diminished; instead, the treatment impact appears evenly distributed throughout the images. This uniformity supports the reproducibility of spheroid preparation and the consistency of treatment-induced biochemical changes, confirming that the observed spectral alterations are global rather than spatially restricted.

9.

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Three partial least squares discriminant analysis (PLS-DA) models classify each pixel of the hyperspectral spheroids of GBM cells (A,B) and astrocytes (C,D). Each model compares the control condition with A (AMPS-CDs), R (riluzole), or AR groups (AMPS-CDs@RZ), using CORRSCV for validation. Predicted Y-value images for each model showing a homogeneous classification across spheroids, without spatial clustering. (B: GBM and D: astrocytes) Average first loading of the oPLS-DA models, where negative bands correspond to controls and positive bands to treated cells, reflecting treatment-induced biochemical changes.

Furthermore, the discriminating variables can be examined in the first oPLS-DA loading, where positive values indicate correlation with the treatment and negative values indicate correlation with the control. These loadings, shown in Figure A,C, reveal the biochemical changes induced by each treatment, reflected as differences in the corresponding spectral profiles. In GBM spheroids (Figure A), the regression vectors corresponding to AMPS-CDs@RZ and RZ treatments display a similar profile, with negative peaks around 1540 and 1650 cm–1 (Amide bands) and a positive peak near 1750 cm–1 (carbonyl region), indicating protein loss and increased lipid peroxidation. In contrast, the AMPS-only treatment displays predominantly positive loadings for protein bands, suggesting a different underlying response mechanism.

An equivalent supervised CORRSCV analysis was performed for the astrocyte spheroids (Figure C,D). A similar trend was observed, with high classification performance and homogeneous predicted Y- values across the maps, without any specific spatial features. This again indicates that the astrocyte spheroids did not display localized regions where treatment effects were enhanced or diminished, suggesting that the biochemical responses were uniformly distributed throughout the samples. Notably, in astrocytes, the spectral loadings were less pronounced than in GBM, reflecting the more modest molecular impact of the treatments in nontumoral cellsconsistent with the findings from the FTIR spectral and imaging analyses. In this case, the three treatments showed overall similar spectral behavior in astrocytes, reinforcing the cell-type specificity of the biochemical alterations induced by AMPS-CDs@RZ in GBM spheroids.

Discussion

Patient-derived glioblastoma (GBM) spheroids represent a robust and physiologically relevant 3D model for evaluating nanotherapeutics, combining the cellular heterogeneity, invasive potential, and therapy resistance of native tumors with experimental accessibility and compatibility with high-throughput assays. While more complex organoid models capture long-term tumor architecture, spheroids remain a widely adopted platform for early-phase drug screening and mechanistic studies, particularly when exploring material cell interactions in vitro.

In this study, AMPS-CDs were employed as nanocarriers for riluzole, enabling enhanced delivery and cellular uptake. In comparison to 2D classical cell culture, which was our first step in the CDs nanocarrier treatment of GBM cells, , we found similar, but more profound molecular changes in the response to the treatment. Unlike 2D cultures, 3D spheroids replicate tissue-like spatial organization, extracellular matrix interactions, and diffusion gradients that govern nanocarrier penetration, drug metabolism, and biomolecular responses. Comparative multivariate FTIR analyses and hyperspectral image classification revealed that AMPS-CDs@RZ induced more pronounced macromolecular rearrangements in GBM spheroids than riluzole treatment alone. These findings highlight the synergistic effect of combining AMPS-CDs with riluzole and establish SR-FTIR as a versatile tool for probing nanomaterial–cell interfaces in complex 3D systems.

Recently, live near-infrared microscopy of multicellular spheroids using nanoparticles to investigate oxygenation gradients was reported. In our study, all measurements were performed under near-physiological conditions, without staining or chemical modifications that could perturb the 3D spheroids, and together with our previous single-cell live FTIR measurements, this provides a comprehensive multilevel overview from individual cells to multicellular spheroids. By extending SR-FTIR analyses from 2D monolayers to 3D spheroids, we captured the influence of cellular architecture and intercellular organization on drug response. Although AMPS-CD nanoparticles alone were nontoxic for single cells, their combination with riluzole significantly enhanced the effect of GBM spheroids (i.e., 3D cell culture), suggesting further that AMPS-CD nanoparticles may serve as an effective carrier for riluzole in GBM therapeutic models. The combined treatment induced marked alterations in nucleic acid organization, protein secondary structure and lipid composition reflecting a coordinated molecular response. In particular, changes in the fingerprint region (e.g., ∼1113 cm–1 and ∼1050 cm–1), together with increased ratio of CO/CH2 + CH3 bands, supported the presence of treatment-induced nucleic acid reorganization and oxidative stress in GBM cells.

Alterations in the spatial distribution and intensity of carbonyl groups in AMPS-CDs@RZ–treated GBM spheroids point to enhanced lipid oxidation, indicative of oxidative stress responses. These data highlight that the carbon dot nanocomposite not only facilitates efficient riluzole delivery but also triggers distinct redox-sensitive molecular pathways, supporting its potential as a multifunctional platform for glioblastoma therapy.

Though CDs exposure is expected to trigger complex, multicomponent cellular responses involving metabolites, proteins emerge as one of the most sensitive molecular targets of AMPS-CDs@RZ in GBM. SR-FTIR revealed treatment-induced changes in protein secondary structure (α-helices), tyrosine and glutamine content, reflecting proteins’ molecular-level effects of AMPS-CDs@RZ. Notably, GBM spheroids exhibited increased tyrosine (Tyr) and glutamate (Gln) amino acids content and altered protein conformation, whereas astrocytes maintained more stable α-helical structures with reduced Tyr and Gln, indicating cell-type-specific pathways triggered by the nanocarrier–drug combination. Spectral changes associated with tyrosine and glutamate, as well as protein conformation, suggest that AMPS-CDs@RZ may selectively modulate stress edition and signaling in tumor spehroids, while sparing nontumor astrocytes, which is a feature highly desirable for precision nanomedicine. These alterations, particularly the drop in α-helix and rise in carbonyl and Tyr bands, indicate that the mechanism of cytotoxicity may involve protein misfolding, oxidative stress, and disrupted amino acid homeostasis in GBM spheroids. These pronounced alterations induced by AMPS-CDs@RZ treatment were observed at the level of protein secondary structure, indicating that the primary effect of the treatment is on protein folding and molecular packing rather than overall protein abundance, consistent with our previous single-cell data.

Additionally, changes in the fingerprint and Amide II regions support a potential impact on protein phosphorylation status, particularly involving tyrosine residues, which could be further validated using complementary phosphoproteomic approaches. Because tyrosine phosphorylation drives critical conformational transitions, , these structural alterations suggest that CDs directly interfere with tyrosine-dependent signaling and protein function. AMPS-CDs bearing SO3 –, COOH, and NH2 groups, principally keeping SO3 –-riluzole interactions, could actively compete with Gln amino acid on protein surfaces, destabilizing their local environment and compromising GBM protein functionality. Besides protein conformation changes, we observed significantly higher Tyr and Gln amino acid levels in GBM spheroids versus healthy astrocytes, reflecting tumor dependence on amino acid-driven signaling, bioenergetics, and drug interaction. Perturbation of Tyr and Gln could further trigger mitochondrial stress, ROS accumulation, and energy depletion, amplifying GBM vulnerability, leading to cell death. Given the chemical diversity of binding sites in phosphoproteins, even subtle changes in amino acid availability or functional group presentation can disproportionately impact phosphorylation-dependent interactions and downstream signaling cascades, mostly by Tyr. − Besides, significantly increased Gln reflects the tumor’s dependence on glutamine to support bioenergetics, redox balance, and biosynthesis. , Both Tyr and Gln are critical for cancer cell survival, supporting signaling, metabolism, and redox balance, and AMPS-CDs@RZ treatment simultaneously disrupts Tyr- and Gln-dependent structural and metabolic pathways, amplifying stress and pushing GBM cells toward nonviability. These biochemical alterations may involve redox-sensitive proteins such as protein tyrosine phosphatase sigma (PTPσ), which is modulated through cysteine oxidation and interacts with sulfated glycosaminoglycans to regulate signaling, invasiveness, , and therapy response in GBM.

Conformational changes in α-helix disruption could impair syntaxin-1 (Stx1)–mediated vesicular glutamate release , while modulating SNAT3 −mediated glutamine transport. Elevated Gln fuels excessive glutamate production, causing excitotoxic neuronal loss and promoting tumor expansion via the system Xc– antiporter and glutamine–glutamate cycle, as reviewed in, with upregulated SNAT3 sustaining imbalance. On the other hand, GBM cells are also connected with elevated intracellular iron, − that we observed in the same cell type in a previous study, which could drives ROS production and sensitizes tumors to ferroptosis and radiation. CD-mediated RZ-induced perturbation of protein structure likely amplifies its oxidative stress, revealing a tumor-specific weakness. Together, these results support a materials-driven, multipronged mechanism: AMPS-CDs@RZ destabilize protein structure, disrupt amino acid–dependent signaling, impair glutamine–glutamate metabolism, likely hinder vesicle-mediated glutamate release, and amplify iron-driven oxidative stress. This coordinated attack selectively weakens GBM cells while sparing healthy astrocytes, highlighting the potential of CDs as nanoscale modulators of tumor-specific metabolic and signaling vulnerabilities.

The observed macromolecular changes further underscore the value of label-free SR-FTIR imaging for probing nanocarrier–cell interactions at the molecular level. From an analytical standpoint, SR-FTIR combined with chemometric approaches offers a high-resolution, nondestructive method for characterizing nanomaterial-mediated drug effects at the molecular level. The oPLS-DA combined with CORRSCV analysis reliably discriminated treated spheroids from controls, achieving near-perfect pixel-level classification and demonstrating the homogeneity of both GBM and astrocyte spheroids. The analyses revealed that treatment effects are uniformly distributed across the spheroids, without localized “hot spots,” indicating consistent biochemical responses throughout each sample. Spectral loadings highlighted distinct biochemical signatures for different treatments: AMPS-CDs@RZ and RZ induced similar effects with decreased Amide bands and increased lipid signals, whereas AMPS primarily affected protein bands, reflecting different underlying mechanisms. Overall, these results demonstrate that the combined imaging and multivariate analysis approach provides a robust and sensitive method to evaluate spheroid responses to treatment, capturing both global and molecular-level changes.

Conclusion

In summary, AMPS-derived carbon dots enabled the analytical interrogation of riluzole-induced molecular responses in three-dimensional glioblastoma spheroids, while SR-FTIR spectroscopy and microscopy provided a robust, label-free framework for characterizing material–cell interactions in physiologically relevant 3D models. The multiscale application of SR-FTIR spectro-microscopy afforded sensitive, spatially resolved mapping of macromolecular alterations induced by CDs–mediated drug delivery in glioblastoma spheroids, in comparison to astrocyte spheroids, serving as nontumoral controls.

By generating nondestructive, subcellular chemical fingerprints, SR-FTIR enabled direct correlation between nanocarrier formulation, cellular uptake, and biomolecular responses without the need for exogenous labels. Advanced spectral processing, including multivariate classification and image-based analysis, facilitated discrimination of subtle macromolecular rearrangements across both 2D and 3D cellular models. In particular, OPLS-DA with constrained repeated random sampling cross-validation reliably differentiated treated and control spheroids and captured treatment-specific biochemical variations with high spatial uniformity.

Together, this work establishes SR-FTIR microspectroscopy as a powerful analytical platform for the chemically precise, spatially resolved investigation of nanomaterial–cell interactions. The approach provides a generalizable, data-driven strategy for high-content analysis of nanocarrier systems in complex biological architectures, supporting the development and validation of advanced analytical methodologies for nanoscale biointeractions.

Acknowledgments

This work is supported by the Spanish Ministry of Science and Innovation (MCIN/AEI/10. 13039/501100011033) through projects PID2021-122613OB-I00 and PID2024-155480OB-100. The authors gratefully acknowledge Prof. Dr. Veit Rohde, Dr. Milena Ninković, and Swetlana Sperling, University Medical Center Göttingen, Germany, for providing the cells.

Conceptualization: TD; Investigation: TD, MA, MIC, EGM; Formal analysis: TD, DPG, GQ; Visualization: TD; writing original draft: TD; writing review and editing: all authors. All authors read and approved the final manuscript.

The authors declare no competing financial interest.

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