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Povzetek: Graphene quantum dots (GQDs) are nanoscale carbon-based materials characterized by a tunable bandgap and unique physicochemical features. Typically composed of only a few atomic layers and measuring under 10 nm in lateral dimension, they show excellent biocompatibility and low toxicity, making them attractive for biomedical uses. Their strong photoluminescent properties support applications in optical and electrical sensing, bioimaging, cancer therapy, and the development of high‑performance nanocomposites. In this study, two types of GQDs—green‑emitting (G‑GQD) and blue‑emitting (B‑GQD)—were examined, differing primarily in their optical properties, particularly the colour of emitted light dictated by their bandgap. Potential genotoxicity was evaluated using a 3D human hepatocellular carcinoma (HepG2) spheroid model. DNA damage induction was assessed with the comet assay, after 24‑hour exposure to 12.5, 25, 50, and 100 µg/mL of B‑ and G‑GQDs. Single-cell suspensions from spheroids were obtained using a combination of mechanical disruption and enzymatic digestion (Štempar et al., 2019). Following 24-hour exposure to B- and G-GQD at concentrations of 12.5, 25, 50, and 100 µg/mL (corresponding to 5, 10, 20, and 40 µg/cm2) and 30 μg/mL BaP as the positive control, spheroids were collected and treated with a mixture of collagenase and TrypLE, diluted in serum-free medium (1:20:9), for 10 minutes. Spheroids were then mechanically dissociated into a single-cell suspension by pipetting. The subsequent steps followed the standard monolayer cell culture protocol. Briefly, 30 μL of the cell suspension was mixed with 70 μL of 1% low-melting-point (LMP) agarose and applied to fully frosted slides pre-coated with a layer of 1% normal-melting-point (NMP) agarose. The slides were lysed in a solution containing 0.1 M EDTA, 2.5 M NaOH (pH 10), 0.01 M Tris, and 1% Triton X-100 for 1 hour at 4°C. DNA was unwound and electrophoresed in an alkaline solution (300 mM NaOH, 1 mM EDTA, pH 13) for 20 minutes at 25 V and 300 mA (0.5–1 V/cm). The slides were then neutralised using 0.4 M Tris buffer (pH 7.5), and the gels were stained with GelRed. Images were captured and analysed using an Eclipse 800 fluorescence microscope (Nikon, Japan) equipped with a Basler camera and the Comet IV image analysis software (Perceptive Instruments, UK). Three independent experiments were conducted, with 50 randomly selected nuclei analysed per experimental condition. Results were expressed as the percentage of tail DNA.
Ključne besede: nanomaterials, graphene quantum dots, HepG2 spheroids, toxicity
Objavljeno v DiRROS: 31.07.2026; Ogledov: 278; Prenosov: 201
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Povzetek: Iron-oxide magnetic nanoparticles (MNPs) have been extensively investigated as magnetically actuated nanocatalysts for diagnostic and therapeutic applications. However, because wüstite/magnetite/maghemite phases can interconvert, coexisting Fe2+/Fe3+ species may redirect Fenton-like chemistry and generate reactive oxygen species (ROS) profiles that differ from the intended biocatalytic pathway. Here, we investigate monodisperse biphasic FeO@Fe3O4 core-shell MNPs with an average particle size ⟨d⟩ = 9.6(5) nm, and their glucose-coated analogue, combining EPR radical analysis with toxicity testing in a 3D HepG2 hepatic spheroid model. Naked particles exhibited conventional Fenton-like behavior dominated by hydroxyl radicals (⋅OH), whereas glucose coating markedly suppressed ⋅OH while increasing hydroperoxyl radicals (⋅OOH; ≈55 pM at 60 min), demonstrating ligand-controlled rerouting of the radical pathway. TEM mapping across spheroid cross-sections showed preferential MNP accumulation in the outer layer, with most observed events confined to the outer ≈10–15 μm, corresponding to an approximately one-cell-thick rim; sparse deeper events were observed up to ≈30–35 μm. MNPs produced dose- and time-dependent cytotoxicity in HepG2 spheroids, with IC50 values of 29.3 (24 h) and 10.8 (96 h) µg·cm− 2, without evidence of lipid peroxidation or genotoxicity. MDA levels remained unchanged, the comet assay showed no increase in DNA damage, and γH2AX and phospho-H3 (p-H3) positive events were not detected. Our results show that glucose functionalization provides a simple route to modulate radical pathways and define operational windows for redox-active FeO@Fe3O4 nano-reactors in oxidative nanomedicine.
Ključne besede: iron-oxide nanoparticles, Fenton-like catalysis, cytotoxicity, genotoxicity, HepG2 spheroids
Objavljeno v DiRROS: 02.06.2026; Ogledov: 327; Prenosov: 278
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Povzetek: Nanosized spinel-type ferrites have gained recognition as a unique class of engineered nanomaterials with promising applications, but their safety profiles remain insufficiently explored. Although iron (Fe), zinc (Zn), and manganese (Mn) are biologically relevant elements, the use of Zn- and Mn-containing ferrite nanoparticles in biomedical contexts demands careful (geno)toxicity evaluation. In this study, three ferrite nanoparticles – γFe2O3 (FeNPs), Zn0.7Fe2.3O4 (ZnNPs), and Mn0.4Fe2.6O4 (MnNPs) – synthesised through a microwave-assisted polyol route, functionalized with citric acid to improve colloidal stability, were evaluated for their potential (geno)toxic effects in an advanced in vitro 3D cell model, HepG2 spheroids. Cellular stress responses upon exposure to the particle were assessed using toxicogenomic analysis.This approach allows the identification of early molecular events that may precede overt toxicity, supporting a mechanistic understanding of adverse outcomes and facilitating the development of predictive biomarkers for hazard assessment. In the present study, the expression of selected DNA damage-responsive genes (TP53, MDM2, GADD45a, CDKN1A, OGG1, and JUNB), apoptosis-related genes (BCL2 and BAX) and oxidative stress response genes (SOD1, CAT, GPX1, GCLC, and GSR) was evaluated. The expression of the selected genes after exposure to the tested nanoparticles was analysed by qPCR primer assays (Applied Biosystems, USA) and One 48.48 Dynamic Array IFC for Gene Expression (Fluidigm, USA). After 24 and 96 hours of exposure, the spheroids were collected, and total RNA was isolated using the RNeasy Mini Kit from Qiagen (Qiagen, Germany) according to the manufacturer's instructions. 10 µg/mL etoposide served as athe positive control for the toxicogenomic analysis. RNA concentration and purity were assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) by measuring absorbance at 260/280 nm and gele efectrophoresis (Figure 1). Reverse transcription of 1 µg total RNA per sample was performed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, MA, USA) on a BIO-RAD T100 thermal cycler under conditions listed in Table 3. For preamplification, 4 µL of each of the 24 selected TaqMan assays (SM2) were pooled into a primer mix. The reaction mixture was prepared using TATAA PreAmp GrandMasterMix (Tataa Biocenter, Sweden), the primer pool, and nuclease-free water, following manufacturer instructions. Negative controls (NTC for preamplification and NTCq for qPCR) were included. Each reaction contained 8 µL of mix and 2 µL of 5× diluted cDNA, processed in a 96-deep well plate, sealed, vortexed, and centrifuged (1000 g, 1 min). Preamplification was carried out on a BIO-RAD T100 thermal cycler under conditions in Table 4. Gene expression analysis used TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Table 6). Preamplified samples were diluted 10× with nuclease-free water. Assays were prepared by mixing equal volumes (6 µL) of each assay with Fluidigm Assay Loading Reagent Kit – 10IFCS. The reaction premix combined DNA Sample Loading Reagent and Fast Probe Master Mix (Biotium/Roche) and was added to each diluted cDNA sample. qPCR was performed on 48.48 Dynamic Array™ IFC chips using the Fluidigm BioMark™ HD System under conditions in Table 5. Data were analysed with Fluidigm Gene Expression Analysis Software and quantGenious. Fold changes >1.5 or <0.66 were considered biologically relevant. Statistical significance between NP-exposed cells and solvent controls was assessed using ANOVA and Dunnett’s test in GraphPad Prism v9 (GraphPad Software, CA, USA).
Ključne besede: ferrite-based nanoparticles, HepG2 spheroids, toxicogenomics, changes in gene expression
Objavljeno v DiRROS: 24.03.2026; Ogledov: 467; Prenosov: 487
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Povzetek: Given the growing interest in nanosized spinel-type ferrite nanoparticles for biomedical applications and the limited information on their safety, this study aimed to assess their cellular and genotoxic effects in an in vitro 3D human hepatic cell model (HepG2 spheroids). Ferrite nanoparticles – γFe2O3 (FeNPs; 14 ± 4 nm), Zn0.7Fe2.3O4 (ZnNPs; 14 ± 5 nm), and Mn0.4Fe2.6O4 (MnNPs; 7 ± 2 nm) – were synthesised through a microwave-assisted polyol route, functionalized with citric acid, and characterised using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). Nanoparticle uptake was analysed using TEM, cytotoxicity was measured with CellTiter-Glo®, and oxidative stress induction was assessed using the 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) and malondialdehyde (MDA) assay. Genotoxic effects were evaluated using the comet, γH2AX and p-H3 assays. Cellular stress responses were assessed using toxicogenomic analysis. Significant cytotoxicity of the tested nanoparticles (0.1–250 µg/mL) was observed; however, TEM analysis revealed limited penetration to the outermost cell layers of spheroids. Notably, only FeNPs induced ROS generation, while MDA levels remained unchanged in all tested samples. Low DNA damage was detected at 24 h, but a significant increase was observed at 96 h (5–50 µg/mL). No increase in γH2AX or p-H3 was found. No substantial alterations in DNA damage or oxidative stress-response gene expression were detected. Altogether, our findings suggest that the effects of ferrite nanoparticles are time- and composition-dependent, underlining the importance of further mechanistic and chronic exposure evaluations in 3D cell models.
Ključne besede: DNA damage, genotoxicity, HepG2 spheroids, magnetic ferrite-based nanoparticles, ROS induction, safety assessment, toxicogenomics
Objavljeno v DiRROS: 27.01.2026; Ogledov: 699; Prenosov: 902
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Povzetek: 3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 μM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 μM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool.
Ključne besede: in vitro 3D cell model, HepG2, flow cytometry, cell cycle, proliferation, DNA strand, breaks
Objavljeno v DiRROS: 16.07.2024; Ogledov: 1750; Prenosov: 919
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