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Nanomaterials have been associated with adverse effects on human health due to structural alterations following cellular internalization. However, from a therapeutic perspective, they offer advantages for cancer treatment by inhibiting processes such as cell division, motility, and invasion, which are key functions regulated by the cytoskeleton. Based on that, we aimed to examine the potential impact of titanium dioxide nanofibers (TiO2-NF) on the cytoskeleton disruption and their effects on cell and nuclear morphology, motility, cell cycle, and mitotic index in lung adenocarcinoma cells. Results showed that TiO2-NF exposure (1, 10, or 50 μg/cm2 TiO2-NF for 24 h) increased cell granularity and reduced cell size, consistent with nanofiber uptake. The cytoskeletal architecture was markedly disrupted, as evidenced by alterations in both the actin and microtubule networks associated with impaired cell motility. TiO2-NF predominantly accumulated near the nuclei, leading to their deformation and a slight increase in the proportion of cells in the G2/M phase, which was accompanied by an increased mitotic index. These structural disruptions were also associated with impaired cell motility and cell cycle progression. The findings of this study highlight the potential usage of TiO2-NF as a candidate for targeted cytoskeleton-based cancer therapy in lung adenocarcinoma cells.
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Introduction
Louis S. Goodman and Alfred Gilman were pioneers in chemotherapy using nitrogen mustard against advanced non-Hodgkin lymphoma, almost at the end of the Second World War. Since then, chemotherapy has achieved long-term survival or cure rates of millions of patients worldwide each year, according to the International Agency for Research on Cancer (IARC). However, chemotherapy still has to face the toxicity of healthy tissues, low selectivity, tumor heterogeneity, resistance, poor quality of life, and high costs, among others.
The spectrum of action modes of drugs used in chemotherapy is broad, but as soon as the role of the cytoskeleton in cell division and migration was described, drugs for cytoskeleton disruption were used against cancer cells. Nowadays, some cytoskeletal disruptors have become part of chemotherapy agents, including paclitaxel, nocodazole, and colchicine [1, 2]. Nevertheless, resistance is one of the greatest complications in the therapy of several types of cancer. For this reason, some nanomaterials that target the cytoskeleton have been suggested as candidates for therapy against cell division in cancer cells [3].
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Through toxicological studies, the alterations induced by fibrous inorganic nanoparticles in the cytoskeleton became evident. Multi-walled carbon nanotubes (MWCNTs), single-walled carbon nanotubes (SWCNTs), graphite carbon nanofibers (GC-NFs), silicon (SiO2-NFs), and titanium dioxide nanofibers (TiO2-NF) are among the most studied. For instance, MWCNTs deregulate some miRNAs related to actin expression, which is one of the most important proteins of the cytoskeleton, in lung adenocarcinoma cells [4]; SWCNTs induced cytoskeleton rearrangement in macrophages [5]; GC-NFs caused cytoskeleton disruption in lung adenocarcinoma cells [6]. In this regard, we have the eye on titanium dioxide nanofibers (TiO2-NF), which is a type of nanoparticle used for the unique combination of photocatalytic activity, high surface area, and structural stability relevant for applications related to water purification systems by degrading pollutants and microorganisms through photocatalysis [7, 8], incorporation into solar cells and batteries where their high aspect ratio enhances electron transport and storage efficiency [9, 10], and also, in the biomedical field as tissue engineering scaffolds [11, 12], drug delivery systems [13], and antibacterial coatings [14]. Ilmenite is the mineral source of TiO2, which has been used for more than five decades in the paint industry. Additionally, a food-grade TiO2 is used in the preparation of sweets, confectionery, baked goods, and pharmaceuticals as an excipient. However, in recent decades, the nanoform has experienced a surge in innovative designs being into shaped nanofibers. This has led to novel applications, including photocatalytic degradation, solar cells, lithium-ion batteries, water splitting, and sensors, among others [15, 16].
However, TiO2 has been classified as a possible carcinogen to humans by inhalation [17], and the shape resembles TiO2-NF and asbestos, which is a well-known carcinogen to humans [18]. Then, some studies have demonstrated adverse effects including deregulation of over 400 proteins in the Sprague Dawley rats receiving TiO2-NF by oral gavage [19]. TiO2-NF, compared to some other nanoparticles such as silicon dioxide (SiO2) or indium tin oxide NPs, exhibited greater cellular alterations in lung adenocarcinoma cells, including cytoskeletal microfilament disruption [20]. Similarly, when compared with TiO2 nanospheres, TiO2-NF induced greater DNA damage, as evidenced by increased DNA double-strand break markers [21]. Based on the above information, we hypothesized that some effects of TiO2-NF are beneficial in lung adenocarcinoma cells by disrupting the cytoskeleton architecture, thereby impairing cell motility.
We highlight that cytoskeletal disorganization induces profound effects on cellular function, including changes in nuclear morphology, cell motility, and progression through the cell cycle [22, 23]. Furthermore, since TiO2 nanoparticles predominantly localize to the perinuclear region [24], we aimed to analyze the TiO2-NF effects on nuclear integrity and cell cycle progression. Overall, we investigated the effects of TiO2-NF on lung cell morphology and motility with a particular focus on cytoskeletal organization in lung adenocarcinoma cells.
Materials and methods
TiO2-NF physicochemical characterization
Synthesis of titanium dioxide nanofibers (TiO2-NF) was previously reported [25]. TiO2 from Sigma-Aldrich (637,254) was dispersed in a 10 M NaOH solution (0.5 g TiO2/35 mL). The solution was ultrasonicated for 10 min, poured into a steel container, and sealed. The container was heated at 200 °C for 24 h. Then, the TiO2 obtained after the hydrothermal treatment was repeatedly washed with distilled water and 0.1 M HCl until pH 7 was reached, and centrifuged between each wash at 3.5 × g for 15 min. To obtain the fiber shape, the TiO2 pellet was heated at 700 °C for 30 min at a ramp rate of 1 °C/min. Then, TiO2-NF morphology was analyzed by scanning electron microscopy (SEM; JEOL 5800-LV, Japan) using 5000 × magnification, 15 kV of electron acceleration, and the size of the fields was 26 pixels/nm. For cell culture exposure, 1 mg of TiO2-NF was suspended in 1 mL of F-12 K (Invitro, ME-038) supplemented with 10% fetal bovine serum (FBS; Cytiva, SH30910.03). F12-K cell culture media contains 4.5 g/L of glucose, 2 mM glutamine, 1 mM pyruvate, 10 mM Hepes, and 1.5 g/L sodium bicarbonate. TiO2-NF suspensions were sonicated at 40 Hz for 30 min to obtain a correct dispersion. Afterward, the suspensions were measured using the Zetasizer Nano-ZS90 equipment (Malvern Instruments). A dynamic light scattering system was used to measure the hydrodynamic size, in which the intensity size distribution peaks were quantified. The zeta potential was measured by analyzing the degree of repulsion or attraction between particles. Hydrodynamic size and zeta potential measurements were carried out under conditions simulating the exposure of the cell culture at 1 µg, 10 µg, and 50 µg to TiO₂-NF in 12-well plates (1 mL as a final volume). Three independent experiments were performed, each with three biological replicates.
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Cell culture and TiO2-NF exposure
Lung adenocarcinoma cells (A549 cell line, CCL-185, ATCC) were cultured in cell culture medium F-12 K supplemented with 10% FBS and 1% antibiotic–antimycotic (A-07, InVitro). Cells were maintained at 37 °C, 5% of CO2, and 90% of relative humidity. For all the experiments, once reaching 80% confluence, the cells were detached with Trypsin–EDTA solution, centrifuged at 1500 rpm for 5 min. After that, 5 × 104 cells per cm2 were seeded for all experiments, and then the cells were exposed to 1, 10, and 50 μg/cm2 of TiO2-NF (previously sonicated) for 24 h.
Cell viability
The viability of cells exposed to TiO2-NF was performed on a 96-well plate by MTT (3-[4,5-dimethylthiazol-2-yl]−2,5 diphenyl tetrazolium bromide) reduction. Briefly, after exposure to TiO2-NF, the cell culture medium was removed and the cells were washed with PBS. Then, the cells were incubated with 0.5 mg/mL of MTT reagent (M2128, Sigma-Aldrich) suspended in FBS-free cell culture medium for 1 h. After that, the MTT was removed, and the formazan crystals formed were dissolved in 200 μL of isopropanol. Afterward, the isopropanol was recollected for each concentration of TiO2-NF and centrifuged at 1500 rpm for 5 min to remove remaining TiO2-NF. Finally, the samples were analyzed by the BioTek Epoch Multi-Volume System at 545 nm. Three independent experiments were performed, each with eight biological replicates.
Cell size and granularity
The size and granularity of cells exposed to TiO2-NF were analyzed by flow cytometry. Briefly, cells were exposed to TiO2-NF as indicated previously, and then were detached and fixed with cold ethyl alcohol at 70% for 30 min. Then, ethyl alcohol was removed by centrifugation at 1500 rpm for 5 min, and the pellet was resuspended in BD FACSFlow Sheath Fluid. Finally, the cells were read on the flow cytometer (BD FACSCalibur), and forward scatter (FSC) and side scatter (SSC) were adjusted according to the control group. Ten thousand events were counted, and cell debris was excluded. Three independent experiments were performed, each with three biological replicates.
Cytoskeleton immunostaining
Cytoskeleton compounds, such as actin microfilaments and microtubules, were analyzed by confocal microscopy. The cells were seeded on a sterile coverslip and exposed to TiO2-NF for 24 h. After, the cells were washed exhaustively with PBS to remove the remaining TiO2-NF. Then the cells were fixed with paraformaldehyde, permeabilized with cold acetone, and the epitopes were blocked with serum albumin (1%) at 37 °C for 90 min. Actin microfilaments and microtubules were detected with rhodamine phalloidin (R415, ThermoFisher Scientific) and Alexa Fluor 488 anti-tubulin-α antibody (627,905, BioLengend), respectively. The nucleus was stained by Hoechst (62,249, ThermoFisher Scientific). The coverslips were mounted and sealed in a slide and samples were analyzed by confocal microscopy (Leica TCS SP8 microscope) at 63 ×. The data were normalized with mean fluorescence intensity of nuclei to correct for cell density differences or to correct signal variations [26, 27]. Three independent experiments were performed, each with eight biological replicates.
Cellular motility
Cell motility was assessed using both wound healing and transwell assays. For the wound healing assay, cells were cultured in a 12-well plate and after 24 h, a wound was created in the cell monolayer using a 20–200-µL sterile pipette tip. The culture was then washed with 1 × PBS to remove any detached cells after exposure to TiO2-NF for 24 h. The cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and then stained with Hoechst (1:1500 dilution) for nuclei detection. Cell motility was quantified by measuring the area covered by migrating cells using ImageJ software and the cell-covered area was expressed as a percentage of the initial wound area. The images were acquired by an AxioCam MCR/ZEISS Axio Vert A1 fluorescence microscope at 20 × magnification. For the transwell assay, cell motility was evaluated using a 24-transwell plate (3421, Corning). Briefly, cells were seeded on the apical side of the transwell and incubated for 24 h. Afterward, the cells were exposed to TiO2-NF for an additional 24 h in F-12 K cell culture medium supplemented with 1% FBS on the apical side, while the basolateral side contained F-12 K medium supplemented with 10% FBS to establish a chemoattractant gradient. The cells that migrated to the basolateral side were stained with crystal violet solution for 5 min, washed, and quantified with a counter under an optical microscope. The motility of cells in the control group was considered 100% to normalize the data. Three independent experiments were performed, with three biological replicates per experiment.
TiO2-NF localization and nuclear morphology analysis
To localize the TiO2-NF into the cells, the TiO2-NF were stained previously with Alizarin Red S (ARS) according to Deciga-Alcaraz et al. [20]. Briefly, 1 mg of TiO2-NF was suspended in 50 µM of ARS and incubated with shaking at room temperature overnight. Then, the suspension was centrifuged at 0.2 × g for 10 min. The supernatant was discarded, and the pellet was sterilized by autoclaving. Later, the pellet was suspended in 1 mL of F12-K + FBS and sonicated at 40 Hz for 30 min, and 50 μL of the solution was mounted on a coverslip to obtain Pearson’s Correlation between TiO2-NF-reflection and TiO2-NF incubated with ARS by confocal microscopy.
Subsequently, once the positive correlation was obtained, lung adenocarcinoma cells were exposed to ARS-labeled TiO2-NF at 1, 10, and 50 μg/cm2 for 24 h. The ARS-labeled TiO2-NF were suspended in F12-K + FBS and sonicated under the same conditions. For this experiment, non-stained TiO2-NF and 50 µM ARS were used as negative controls [20].
After exposure, the cells were washed with PBS, fixed, and incubated with Hoechst for nuclei visualization. To obtain the 3D images and observe the ARS-labeled TiO2-NF localization, 15 z-stacks from the basal to apical side of the cells were taken, and image reconstruction was obtained in the Leica TCS SP8 Software, where Pearson’s correlation between ARS-labeled TiO2-NF and nuclei was also obtained. For nuclear circularity analysis, 10 nuclei per image and 10 images per treatment were analyzed. This approach is based on previous reports that analyze 10 images per image to evaluate nuclear morphology [28, 29] as well as the fact that every image contains between 10 and 15 cells. The images were focused on the middle of the z-axis and were processed in ImageJ software into 8-bit photos, followed by automatic thresholding and outline filters. We performed three independent experiments, and a total of 300 nuclei per treatment were analyzed. The image processing was performed according to the ImageJ Software website (https://imagej.net/ij/docs/menus/analyze.html).
Cell cycle progression analysis and mitotic index
After cell exposure, the cells were harvested and fixed with cold ethyl alcohol. Then, the ethyl alcohol was removed, and the cells were stained with FxCycle PI/RNase Staining Solution (F10797, Invitrogen) according to the manufacturer’s instructions and the cell cycle was analyzed by a flow cytometer. The data were analyzed in FlowJo Software, and the three main cell cycle phases (G0-G1, S, and G2-M) were established with a control group. For each cell cycle phase, the percentage of the cell population was obtained. Additionally, we calculated the mitotic index by counting 3000 nuclei per independent experiment using a confocal microscope, and we applied the formula: mitotic index (%) = (number of dividing cells/total number of cells) × 100. Three independent experiments were performed, and the statistical analysis was performed vs the control group.
Statistical analysis
The data were presented as the mean ± standard deviation. Differences in cell viability, cell size and granularity, cytoskeleton immunostaining, cell motility, TiO2-NF colocalization, nuclear circularity, and mitotic index, among the groups, were compared by one-way analysis of variance ANOVA followed by Dunnett’s multiple comparisons test. For cell cycle analysis, the two-way ANOVA and Tukey’s multiple comparisons test were applied. A p-value ≤ 0.05 was considered statistically significant. All tests were analyzed and graphed in GraphPad Prism 9.2.0 (GraphPad Software, San Diego, CA).
Results
TiO2-NF physicochemical characterization
The physicochemical properties of nanoparticles are strongly related to the cell outcome, such as uptake, biomolecule and organelle interaction, and cell degradation. In human exposure and experimental models, the properties of nanoparticles influence their distribution, metabolism, and excretion. These properties include chemical composition, size, shape, charge, and catalytic features, among others. Additionally, in cell culture systems, dispersion media such as water, buffers, and culture media modify the hydrodynamic size and zeta potential, which are crucial since these features are linked to biological effects. Then the physicochemical properties of nanoparticles can be related to the specific biological effects.
In this study, the shape of TiO2-NF was analyzed by SEM microscopy, revealing nanoparticles in the form of fibers (Fig. 1A). These fibers were then dispersed in F-12 K cell culture media supplemented with 10% FBS, mimicking concentrations used in cell culture conditions. The lowest tested concentration of 1 µg/mL TiO2-NF showed a hydrodynamic size of 363.1 ± 18.9 nm; the intermediate concentration of 10 µg/mL TiO2-NF exhibited a hydrodynamic size of 909.9 ± 53.6 nm, and the highest concentration tested, 50 µg/mL TiO2-NF, presented a hydrodynamic size of 1064.1 ± 54 nm (Fig. 1B). The zeta potential values for the tested concentrations of 1 µg/mL, 10 µg/mL, and 50 µg/mL were − 5.7 ± 1.5, − 8.0 ± 0.9, and − 8.6 ± 1.8 mV, respectively (Table 1).
Table 1
Zeta potential of TiO2-NF suspended in F-12 K + FBS
Zeta potential (mV)
1 µg/mL TiO2-NF
− 5.7 ± 1.5
10 µg/mL TiO2-NF
− 8 ± 0.9
50 µg/mL TiO2-NF
− 8.6 ± 1.8
Data shown as mean ± standard deviation
Fig. 1
A Representative image of the primary size and shape of TiO2-NF bulk obtained by scanning electron microscopy. Scale bar = 1 µm. B Hydrodynamic size after sonication of 1 mg of TiO2-NF suspended in 1 mL of F-12 K + FBS and diluted according to the concentrations applied to the experiments. Three independent experiments were performed, and data are presented as mean ± standard deviation
Cell viability is the first critical parameter evaluated for nanomaterials. Unaltered cell viability indicates potential biocompatibility or even suggests proliferative effects, while reduced viability is a clear hallmark of cytotoxicity. Here, the cell viability was quantified by MTT reduction, and TiO2-NF did not affect the cell viability of exposed cells compared to the control group (Table 2). However, it cannot be dismissed that longer exposure has an impact on cell viability, and apoptosis or necrosis is activated. This is suggested, based on a previous report in which lung epithelial cells exposed to 10 μg/cm2 of TiO₂-NF had an increase of 5.3 ± 1.1% and 6.8 ± 1.0% after 48 h and 72 h [21].
Table 2
Cell viability after TiO2-NF exposure for 24 h
Cell viability (%)
Ct
100.26 ± 0.14
1 µg/cm2 TiO2-NF
100.21 ± 0.35
10 µg/cm2 TiO2-NF
100.02 ± 0.30
50 µg/cm2 TiO2-NF
100.00 ± 0.12
Data shown as mean ± standard deviation
Cell granularity and cell size analysis
Nanoparticle internalization depends on the cell type [30] and the uptake mechanisms, including clathrin-coated vesicles, vacuoles, micro and macropinocytosis, receptor-mediated uptake, and others. Through these mechanisms, nanoparticles are internalized and are tightly coupled to the cell size and granularity [31].
In cells treated with 1 µg/cm2, 10 µg/cm2, and 50 µg/cm2 of TiO2-NF for 24 h, the granularity increased 25%, 94.5%, and 96.5%, respectively (2A). The size of control cells had 7% of small cells, and this percentage was increased in cells exposed to 10 µg/cm2 and 50 µg/cm2 of TiO2-NF up to 30 and 85%, respectively (Fig. 2B). Granularity is characterized by the presence of granules that can be lysosomes or any molecule stored, such as enzymes, neurotransmitters, among others. Lysosomes decrease if their membranes are damaged or degraded by apoptosis activation and increase under autophagy activation. However, accumulation of some substances that cannot be degraded, such as certain nanomaterials, increases granularity, which was the measurement of the A549 cell line internal complexity and was detected by an increase in side-scatter (SSC) in flow cytometry [32]. This increase is often an indicator of nanoparticle internalization, and these changes can reveal information about the cell responses, such as uptake, intracellular trafficking, and potential cytotoxic effects [33].
Fig. 2
TiO2-NF increased cell granularity and decreased cell size after 24 h of exposure. According to the distribution of the cell population, the granularity was categorized as either normal or high granularity. For the cell size, the cells were classified into normal and smaller cells. Three independent experiments were performed, and data are presented as mean ± standard deviation (%). p**** < 0.0001 vs Ct group
The increased granularity in exposed cells to TiO2-NF provided evidence of cell uptake. Since TiO2 cannot be metabolized or degraded by enzymatic action, it accumulates leading to cytoskeleton disruption. F-actin and tubulin are the primary components of the cytoskeleton, and unexposed cells displayed an expected organization of cytoskeleton architecture. TiO2-NF was detected in cells exposed in the cytoplasm (white fibers), and exposed cells displayed some vesicle-like structures (red arrows), which were surrounded by tubulin foci (Fig. 3A). The fluorescence intensity quantification of microtubules in non-exposed cells showed 2.5 arbitrary units of fluorescence, while this value decreased to 0.72 arbitrary units for TiO2-NF treatments (Fig. 3B). On the other hand, control cells showed expected actin distribution in the cytoplasm but exposed cells to TiO2-NF showed F-stress actin fibers (Fig. 3C). The semi-quantification of fluorescence in control cells had a value of 2.7 arbitrary units but TiO2-NF treatment decreased the fluorescence to 0.89 arbitrary units. (Fig. 3D). Additional images of cytoskeleton arrangement are provided in SI 1 and S2 figures (supplementary material).
Fig. 3
Cytoskeleton disruption and alterations in mean fluorescence intensity (MFI) of A microtubules (green) and C actin filaments (red) in lung adenocarcinoma cells exposed to TiO2-NF. The red arrows indicate the vesicle-like structures surrounded by microtubules and located near the nuclei, while the white arrows show the actin stress fibers. The images were obtained at 63 × magnification and a digital zoom of 4 ×. The most representative images were presented of three independent experiments. Scale bar = 10 µm. Quantification of mean fluorescence intensity of B microtubules and D actin filaments, obtained from 9 images from three independent experiments, and presented in arbitrary units. p*** < 0.0004, p**** < 0.0001 vs Ct group
Cell motility is the result of protrusive and contractile forces through the regulation of cytoskeletal proteins. These two forces allow cell motility, which is required for morphogenesis to wound healing. However, during cancer development, aberrant cell motility triggers invasion and metastasis. As we found a cytoskeleton rearrangement after TiO₂-NF exposure, cell motility was analyzed. Cell motility of non-exposed cells was considered 100% of the motility measured by the wound healing assay. TiO2-NF exposure to 1 µg/cm2 did not influence cell motility; however, exposure to 10 µg/cm2 and 50 µg/cm2 decreased cell motility 45.39% and 14.15%, respectively (Fig. 4).
Fig. 4
The motility of lung adenocarcinoma cells was decreased after TiO2-NF exposure by the wound healing and transwell assays. The data were normalized and the values of the control group were considered as 100% of cell motility. Three independent experiments were performed, and data are presented as mean ± standard deviation (%). p**** < 0.0001, p#### < 0.0001 vs Ct group
In addition, cell motility measured by the transwell assay showed that TiO2-NF exposure to 1 µg/cm2, 10 µg/cm2, and 50 µg/cm2 decreased the cell motility by 67.3%, 82%, and 88.9%, respectively (Fig. 4).
ARS-labeled TiO2-NF colocalization
In order to detect the subcellular location after cell uptake, coated TiO₂-NF with ARS was used, as this organic fluorescent molecule binds to TiO₂-NF [20]. ARS-labeled TiO2-NF were detected by reflection and with the laser 552 nm by confocal microscopy, while the visual colocalization was observed in the merge image. Pearson’s correlation was obtained in the Leica software, and the value obtained from ARS-labeled TiO2-NF reflection and the laser 552 nm was of r2 = 0.83 (SI 3). In addition, the ARS-labeled TiO2-NF are in red and located near the nuclei in both the 2D and 3D images (Fig. 5A). To know if the ARS-labeled TiO2-NF were located near the nuclei, the colocalization rate was obtained and significative differences were found to 10 and 50 µg/cm2 of TiO2-NF, where the colocalization rate between the TiO2-NF and nuclei was 3.5 and 4.8% (Fig. 5B). However, it cannot be excluded that TiO₂-NF adhere non-specifically to the bottom of the cell culture plate rather than remaining in the supernatant, and those NF are not internalized [34].
Fig. 5
ARS-labeled TiO2-NF (red) colocalized with nuclei (blue) of lung adenocarcinoma cells observed in A 2D and 3D images. B Colocalization rate between ARS-labeled TiO2-NF and nuclei. Three independent experiments were performed, in each experiment 10 images were analyzed and data are presented as mean ± standard deviation (%). p**** < 0.0001 vs Ct group. Scale bar = 10 µm
Nuclear deformation was induced by TiO2-NF exposure
The internalization analysis revealed a concentration-dependent perinuclear localization of TiO₂-NF, accompanied by irregular nuclear shapes. The nuclei of lung adenocarcinoma cells are typically spherical to oval under normal conditions. For this reason, the circularity parameter was used to analyze nuclear deformation. This parameter ranges from 0 to 1, and a value of 1 indicates a perfect circular shape, while values close to 0 describe irregular non-spherical objects. The images obtained from nuclei stained with Hoechst of lung adenocarcinoma cells were processed to 8-bit; threshold and outline images in the ImageJ Software and the circularity values were obtained. Unexposed cells displayed a circular to oval nuclear shape and a value of circularity less than 1 is expected. We observed nuclear deformation of lung adenocarcinoma cells after TiO2-NF (shown by the yellow ellipse; Fig. 6A). The control group showed a nuclear circularity of 0.79, and this value decreased to 0.69, 0.54, and 0.41 in cells exposed to 1 µg/cm2, 10 µg/cm2, and 50 µg/cm2 of TiO2-NF, respectively. These results showed nuclear shape after TiO2-NF treatments (Fig. 6B).
Fig. 6
Nuclear morphology alteration after exposure to TiO2-NF observed by A immunofluorescence images and marked in yellow ellipses the nuclei altered. B Nuclear circularity indices were obtained from 49 images per independent experiment and analyzed using ImageJ software. A value of 1 was considered as complete circularity. Three independent experiments were performed, and data are presented as mean ± standard deviation (arbitrary units). Scale bar = 50 µm. p**** < 0.0001 vs Ct
G2/M phase and mitotic index increase by TiO2-NF exposure
Nuclear deformities are caused by errors during the reassembly of the nuclear envelope after mitosis. However, mechanical forces impact nuclear shape, and perinuclear accumulation of TiO2-NF could disturb cell cycle progression. Based on this, the cell cycle was analyzed. The cell distribution of G0/G1, S, and G2/M in the control group was 54.7 ± 2%, 10.5 ± 1.4%, and 34.2 ± 3.9% respectively. Cells exposed to 1 µg/cm2 had no alterations in the cell cycle. In cells exposed to 10 µg/cm2 and 50 µg/cm2, the G0/G1 phase was reduced to 7.8% and 13.3%, respectively. Cells exposed to 1 µg/cm2 and 10 µg/cm2 of TiO2-NF had no alterations in G2/M cell distribution, but an increase of 8.3% in G2/M was detected in cells exposed to 50 µg/cm2 (Fig. 7A). Due to the alteration of the cell cycle in the G2/M phase, we analyzed the mitotic index after exposure to TiO2-NF. The mitotic index in the control group was 2.4 ± 0.4% and increased to 3.4 ± 0.6% in cells treated with 50 µg/cm2 of TiO2-NF. No significant changes were observed in cells exposed to 1 or 10 µg/cm2 of TiO2-NF (Fig. 7B).
Fig. 7
TiO2-NF altered the cell cycle phases and increased the mitotic index in lung adenocarcinoma cells. A After TiO2-NF exposure, the cell cycle phases (G0/G1, S, and G2/M) were analyzed by propidium iodide staining. B Mitotic index was obtained based on the number of cells undergoing mitosis by analyzing 3000 cells per independent experiment. Three independent experiments were performed, and data are presented as mean ± standard deviation (%). p** < 0.0067, p**** < 0.0001 vs Ct
The physicochemical properties of nanomaterials, especially shape, are critical determinants of both their technological potential and toxicity, influencing absorption, distribution, metabolism, and excretion [35]. It has been shown that TiO2-NF can be internalized as early as 1.5 h after exposure in alveolar macrophages from C57BL/6 mice leading to the formation of unusually enlarged phagolysosomes. Interestingly, TiO2-NF were in direct contact with the phagolysosomal membrane rather than suspended in the lumen, suggesting membrane interaction and potential destabilization [36]. Additionally, amorphous-shaped TiO2 nanoparticles are rapidly internalized in the rat (C6) glial cells (within 30 min) and in the human (U373) glial cells (within 2 h), involving active processes such as macropinocytosis (particularly in U373 cells) and caveolae-mediated endocytosis [37]. However, internalization of nanoparticles is influenced by both agglomerate size and fiber aspect ratio. Small agglomerates and low-aspect-ratio fibers are more easily internalized due to their ability to interact with the cell membrane [25].
Thus, fibers or asbestos-like morphologies, due to their length and high surface area, interact more extensively with cells than spherical particles. This interaction raises toxicological concerns but also offers unique opportunities, such as in targeted cancer therapy. Additionally, TiO2-NF possesses intrinsic photocatalytic properties that are superior to those of their counterparts, TiO2 nanoparticles [38], but also compared to other nanomaterials. The high photocatalytic activity potentially can be harnessed for photodynamic-like therapy. Upon activation with UV or visible light (depending on surface modifications), they generate reactive oxygen species (ROS), triggering oxidative damage and cell death [39]. The broad spectrum of applications and the possibility of human exposure underscore the importance of understanding TiO2-NF interactions with biological systems. In this regard, our study revealed effects consistent with those that occur in occupational inhalation scenarios, yet some of these same properties are applied for promising biomedical uses.
Our findings show that TiO2-NF induced a distinguishing set of cellular alterations in lung adenocarcinoma cells, including disruption of actin and tubulin organization, decreased cell motility, a higher proportion of cells in the G2/M phase of the cell cycle that increased the mitotic index, reduced nuclear circularity likely due to perinuclear accumulation of the fibers, decreased cell size, and increased cellular granularity. From a toxicological perspective, these findings highlight potential adverse effects. However, when contextualized within the field of oncology and nanomedicine, they reveal a set of properties that are applied for therapeutic benefit if carefully controlled. The arrest of cells in G2/M is particularly relevant in cancer therapy because this phase is the most radiosensitive as observed in hepatic carcinoma [40], gastric cancer [41], esophageal cancer [42], and breast cancer cell [43]. Cells exposed to TiO2-NF are therefore more vulnerable to ionizing radiation, enhancing the efficacy of radiotherapy. However, the arrest is unlikely to result from interference of TiO₂-NF with mitosis or cytokinesis, as previous studies have shown that lung epithelial cells exposed to TiO₂-NF continued to undergo cell division 72 h post-exposure, exhibiting only mild cytotoxic effects [21]. Nevertheless, within 72 h post-exposure, an increase of pATM and γH2AX, both DNA damage markers, was observed, suggesting a potential compromise of DNA integrity. In this case, these cells become more susceptible to chemotherapy.
On the other hand, the exposure to TiO2-NF caused pronounced actin stress fiber formation and microtubule depolymerization, indicating structural disorganization and loss of spatial arrangement. This is highly relevant since the cytoskeleton is known to consist of hundreds of different (associated) proteins cooperating in the organization of the complex machinery that is involved in essentially all structural and dynamic aspects of living cells, including maintenance of cell shape, cell movement, cell replication, apoptosis, cell differentiation, and cell signaling [22, 44]. Therefore, the loss of cytoskeletal integrity directly impairs the motility and invasive potential of tumor cells, processes essential for metastasis since the ability to extend lamellipodia and filopodia, adhere to the extracellular matrix, and invade surrounding tissue is diminished. This is confirmed in our results since the exposure to TiO2-NF decreased their capability to migrate, even under conditions where the cells had a gradient of chemotaxis. The mode of action of TiO₂-NF showed in the present study resembles chemotherapeutic agents like taxanes and vinca alkaloids, which are microtubule-targeting agents that disrupt cytoskeletal integrity leading to cell proliferation inhibition [45, 46]. The potential of TiO₂-NF as a nanomaterial-based therapeutic agent relies on the induction of F-actin fibers and microtubule depolymerization, because both are essential for mitosis. Explicitly, F-actin is disassembled for mitosis while microtubule dynamics during mitosis play a key role at metaphase when chromosomes become attached to the spindle fibers. The induction of actin stress fiber formation and tubulin depolymerization by TiO₂-NF exposure in tumor cells can disrupt cytokinesis, ultimately inhibiting successful cell division.
Additionally, the loss of nuclear circularity observed after exposure to TiO2-NF adds another layer of functional disruption. Although the precise mechanism remains unclear, nuclear shape is regulated by components such as lamin A and B, AKT2, the perinuclear cytoskeleton, and nuclear F-actin networks [47‐52]. This suggests that TiO2-NF-induced nuclear deformation is mediated by cytoskeletal disruption. However, it is not ruled out that nuclear deformation is an independent event in response to mechanical stress which influences the 3D organization of chromatin [53]. Changes in nuclear shape are not merely morphological artifacts; this reflects altered chromatin organization, mechanical stress on the nuclear envelope, and potential defects in nucleus-cytoskeletal coupling via the LINC complex [54]. Moreover, nuclear deformation interferes with transcriptional regulation and DNA repair, further contributing to cell cycle arrest or cell death [53, 55, 56]. The increased ROS generation, combined with the structural effects on the cytoskeleton, produces a synergistic anti-tumor outcome. This occurs where the mechanical destabilization of the cell architecture and the oxidative stress converge leading the cells to apoptosis.
For a potential therapy translation, toxicity reduction and tumor specificity are essential. Bare TiO2-NF exposure causes nonspecific interactions and triggers unintended damage; thus, functionalization is critical. Coating the TiO2-NF with biocompatible materials such as polyethylene glycol, phospholipids, or proteins reduces immune recognition, prolongs circulation time, and limits off-target interactions, as demonstrated for other types of nanoparticles [57]. Further, conjugating tumor-targeting ligands such as antibodies against overexpressed surface receptors or peptides that bind tumor-specific markers allows preferential accumulation within malignant cells [58], as activation should ideally be confined to the tumor site. Therefore, spatially confined activation through localized light exposure, ultrasound, or any microenvironment-responsive trigger, such as acidic pH or enzymatic activity, can enhance cytotoxic effects in the restricted tumor site [59, 60].
Despite the potential usage of TiO2-NF as a therapeutic agent, it has limitations. Administration of TiO2-NF causes inflammation [61] and long-term exposure causes interstitial fibrosis in the lung [62]. In addition, since these types of fibers are non-metabolized and do not degraded once they are internalized by cells, sustained inflammation and profibrotic activation are undesired effects during any medical treatment. By combining targeted delivery, localized activation, and dose control, TiO2-NF shifts from being purely a toxicological concern to becoming a multifunctional anticancer tool with applications in radiosensitization, photodynamic therapy, and anti-metastatic interventions. Indeed, oncology represents the wider field of nanoformulations approved or under clinical development by the FDA [63], possibly in combination with lower doses of conventional treatments in order to enhance treatment efficacy.
Conclusion
Our results provide evidence of alterations caused by TiO2-NF on the cytoskeleton network that impact cell motility, nuclear morphology, cell cycle, and mitotic index in lung adenocarcinoma cells. The impact of TiO2-NF in this cell type provides evidence of adverse effects in environmental settings during inhalation. However, TiO2-NF is also a potential therapeutic nanoparticle for cancer treatment, particularly when used in combination with targeted therapies or bioconjugated to biomolecules or antineoplastic agents.
Acknowledgements
We thank Sofía González Gallardo and Alejandra Sánchez Barrera from Facultad de Estudios Superiores Cuautitlán UNAM, for their support with TiO2-NF characterization.
Declarations
Ethics declarations
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Conflict of interest
The authors declare no competing interests.
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