1. Genotoxic effects of graphene quantum dots in an advanced in vitro human hepatic 3D model : version v1Irma Durmišević, Anja Haverić, Sonja Žabkar, Alja Štern, Bojana Žegura, 2026, zaključena znanstvena zbirka raziskovalnih podatkov 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: 120; Prenosov: 81
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2. In vitro toxicity assessment of graphene quantum dots using a 3D HepG2 modelIrma Durmišević, Anja Haverić, Sonja Žabkar, Alja Štern, Katja Kološa, Petra Jenuš, Iza Rozman, Bojana Žegura, 2026, izvirni znanstveni članek Povzetek: In the present study, two types of graphene quantum dots (GQDs) were investigated: green-emitting (G-GQDs) and blue-emitting (B-GQDs). Physicochemical characterisation was performed using transmission electron microscopy (TEM), zeta potential, and hydrodynamic radius measurements to evaluate the morphology, particle size, aggregation behaviour, and colloidal stability of the GQDs in both water and cell culture medium. G-GQDs exhibited superior colloidal stability and more uniform dispersion than B-GQDs, whereas both types showed reduced aggregation and surface charge in cell culture medium due to protein corona formation. Toxicological characterisation was performed using an in vitro human hepatocellular carcinoma (HepG2) 3D spheroid model, with GQDs exposures up to 250 µg/mL (100 µg/cm2). Cytotoxicity was measured using the CellTiter-Glo luminometric assay, while genotoxicity was evaluated by the comet assay and flow cytometric analysis of γH2AX and phosphorylated histone H3 (p-H3) after 24 h of exposure. Both GQDs induced dose-dependent cytotoxic effects in HepG2 spheroids. At non-cytotoxic concentrations, a dose-dependent increase in DNA damage was observed, as determined by the comet assay. However, no evidence of DNA double-strand breaks (γH2AX) or elevated p-H3 levels was detected, suggesting the absence of clastogenic and aneugenic activity. The observed DNA single-strand breaks may be partly attributed to reactive oxygen species induction. These results indicate that, although GQDs induced cytotoxicity and single-strand DNA damage, no clear evidence of more severe genotoxic effects was observed under the tested conditions. Further studies are warranted to elucidate underlying mechanisms and comprehensively assess the safety profile of GQDs for biomedical applications.
Ključne besede: nanomaterials, graphene quantum dots, HepG2 spheroids, toxicity
Objavljeno v DiRROS: 19.03.2026; Ogledov: 518; Prenosov: 192
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3. Succinylation of polyallylamine: influence on biological efficacy and the formation of electrospun fibersLucija Jurko, Matej Bračič, Silvo Hribernik, Damjan Makuc, Janez Plavec, Filip Jerenec, Sonja Žabkar, Nenad Gubeljak, Alja Štern, Rupert Kargl, 2021, izvirni znanstveni članek Povzetek: Succinylation of proteins is a commonly encountered reaction in biology and introduces negatively charged carboxylates on previously basic primary amine groups of amino acid residues. In analogy, this work investigates the succinylation of primary amines of the synthetic polyelectrolyte polyallylamine (PAA). It investigates the influence of the degree of succinylation on the cytotoxicity and antibacterial activity of the resulting polymers. Succinylation was performed in water with varying amounts of succinic anhydride and at different pH values. The PAA derivatives were analyzed in detail with respect to molecular structure using nuclear magnetic resonance and infrared absorbance spectroscopy. Polyelectrolyte and potentiometric charge titrations were used to elucidate charge ratios between primary amines and carboxylates in the polymers. The obtained materials were then evaluated with respect to their minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa. The biocompatibility was assessed using mouse L929 fibroblasts. The degree of succinylation decreased cytotoxicity but more significantly reduced antibacterial efficacy, demonstrating the sensitivity of the fibroblast cells against this type of ampholytic polyelectrolytes. The obtained polymers were finally electrospun into microfiber webs in combination with neutral water-soluble polyvinyl alcohol. The resulting non-woven could have the potential to be used as wound dressing materials or coatings.
Ključne besede: polyallylamine hydrochloride, succinylation, aqueous chemistry, cytotoxicity, antimicrobial effect, electrospinning, nanofibers, mouse L929 fibroblasts, Staphylococcus aureus, Pseudomonas aeruginosa
Objavljeno v DiRROS: 19.07.2024; Ogledov: 1336; Prenosov: 1218
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4. HepG2 spheroids as a biosensor-like cell-based system for (geno)toxicity assessmentMartina Štampar, Sonja Žabkar, Metka Filipič, Bojana Žegura, 2022, izvirni znanstveni članek 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: 1641; Prenosov: 862
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5. Exploring the safety of cannabidiol (CBD) : a comprehensive in vitro evaluation of the genotoxic and mutagenic potential of a CBD isolate and extract from Cannabis sativa LAlja Štern, Matjaž Novak, Katja Kološa, Jurij Trontelj, Sonja Žabkar, Tjaša Šentjurc, Metka Filipič, Bojana Žegura, 2024, izvirni znanstveni članek Povzetek: Cannabidiol (CBD), a naturally occurring cyclic terpenoid found in Cannabis sativa L., is renowned for its diverse
pharmacological benefits. Marketed as a remedy for various health issues, CBD products are utilized by patients
as a supplementary therapy or post-treatment failure, as well as by healthy individuals seeking promised advantages. Despite its widespread use, information regarding potential adverse effects, especially genotoxic
properties, is limited. The present study is focused on the mutagenic and genotoxic activity of a CBD isolate
(99.4 % CBD content) and CBD-rich Cannabis sativa L extract (63.6 % CBD content) in vitro. Both CBD samples
were non-mutagenic, as determined by the AMES test (OECD 471) but exhibited cytotoxicity for HepG2 cells
(~IC50 (4 h) 26 µg/ml, ~IC50 (24 h) 6–8 µg/ml, MTT assay). Noncytotoxic concentrations induced upregulation of
genes encoding metabolic enzymes involved in CBD metabolism, and CBD oxidative as well as glucuronide
metabolites were found in cell culture media, demonstrating the ability of HepG2 cells to metabolize CBD. In this
study, the CBD samples were found non-genotoxic. No DNA damage was observed with the comet assay, and no
influence on genomic instability was observed with the cytokinesis block micronucleus and the γH2AX and p-H3
assays. Furthermore, no changes in the expression of genes involved in genotoxic stress response were detected in
the toxicogenomic analysis, after 4 and 24 h of exposure. Our comprehensive study contributes valuable insights
into CBD’s safety profile, paving the way for further exploration of CBD’s therapeutic applications and potential
adverse effects.
Ključne besede: cannabidiol, CBD, metabolism, cytotoxicity, genotoxicity, mutagenicity
Objavljeno v DiRROS: 09.07.2024; Ogledov: 1265; Prenosov: 920
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6. Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα : dynophore-derived discoveryBarbara Herlah, Matej Janežič, Iza Ogris, Simona Golič Grdadolnik, Katja Kološa, Sonja Žabkar, Bojana Žegura, Andrej Perdih, 2024, izvirni znanstveni članek Povzetek: The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.
Ključne besede: topoisomerase II, catalytic inhibitors, chemotherapy, DNA damage, cancer
Objavljeno v DiRROS: 03.06.2024; Ogledov: 1725; Prenosov: 1046
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