Nanoparticle‑Induced Modulation of Cellular Mechanics: Comparative Analysis of SiO₂ and TiO₂ Effects on Caco‑2 and A549 Cells

Abstract

The increasing incorporation of nanoparticles (NPs) into consumer products has outpaced comprehensive safety evaluations. This study investigates how the physicochemical attributes of SiO₂ and TiO₂ NPs influence cellular uptake, intracellular trafficking, and mechanical perturbations in two clinically relevant in‑vitro models—Caco‑2 (intestinal epithelium) and A549 (alveolar epithelial). We quantified NP internalization via ICP‑AES, assessed cytotoxicity with WST‑8 and LDH assays, measured oxidative stress (ROS, SOD, MDA), and evaluated actin cytoskeleton remodeling through confocal microscopy. Atomic force microscopy (AFM) determined changes in Young’s modulus within nuclear and cytoplasmic compartments. TiO₂ NPs displayed higher cellular uptake, pronounced ROS generation, and significant actin reorganization, leading to increased stiffness in A549 cells and reduced stiffness in Caco‑2 cells. These biomechanical alterations correlate with NP‑induced cytotoxicity and highlight the importance of integrating mechanical endpoints in nanotoxicology protocols.

Background

Engineered nanoparticles (ENPs) are pervasive in pharmaceuticals, cosmetics, food additives, and industrial formulations, raising concerns about human and environmental exposure pathways (ingestion, inhalation, dermal). The toxicity of ENPs is strongly modulated by their size, shape, surface chemistry, and crystalline state, yet standardized assessment methods remain limited, contributing to conflicting literature [1–5]. Metal‑oxide NPs, particularly amorphous SiO₂ and crystalline TiO₂, are widely employed due to their optical and chemical stability, yet evidence suggests they can disrupt cellular functions, including cytokine release, intestinal microvilli integrity, ROS production, ATP synthesis inhibition, and genotoxicity [15–26]. However, their impact on cell mechanics—a key determinant of migration, differentiation, and tissue homeostasis—has received limited attention [27, 28]. This study bridges that gap by systematically evaluating the biomechanical effects of 20‑nm SiO₂ and TiO₂ NPs on Caco‑2 and A549 cells.

Methods

Synthesis of SiO₂ and TiO₂ Nanoparticles

SiO₂ NPs were prepared via a W/O microemulsion using TEOS, Triton X‑100, cyclohexane, and NH₄OH, yielding spherical particles of 20 ± 2 nm. TiO₂ NPs were synthesized by a modified sol‑gel route from TTIP under acidic conditions, calcined at 430 °C to produce a mixed anatase‑rutile phase with an average diameter of 25 ± 5 nm. Both NP types were characterized by TEM, DLS, ζ‑potential, and XRD to confirm morphology, hydrodynamic size, surface charge, and crystalline structure (Fig. 1).

Cell Culture and Exposure

Caco‑2 and A549 cells were maintained in DMEM supplemented with 10–20 % FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. For uptake studies, cells were exposed to 15 or 45 µg/mL of NPs for 48–96 h. Post‑exposure, cells were washed, trypsinized, counted, and lysed in HCl/HNO₃ for ICP‑AES analysis of Si and Ti content.

Cytotoxicity, Membrane Integrity, and Oxidative Stress

Viability was assessed with the WST‑8 assay; LDH release quantified membrane damage. ROS production was measured by DCF‑DA fluorescence, and antioxidant response was evaluated via SOD activity and MDA levels (lipid peroxidation). All assays were performed in triplicate with appropriate controls.

Actin Cytoskeleton and Morphometry

Cells were fixed, permeabilized, and stained with Phalloidin–ATTO 488 and DAPI. Confocal laser scanning microscopy (CLSM) captured cortical actin distribution. ImageJ quantified integrated density and coherency (OrientationJ plugin) to assess actin quantity and alignment. Nuclear‑to‑cytoplasmic (N/C) area ratios were calculated to detect morphological changes.

Atomic Force Microscopy

AFM in force‑volume mode measured Young’s modulus in nuclear and cytoplasmic regions of cells treated with 45 µg/mL NPs for 72 h. Force‑distance curves were fitted to a modified Sneddon model to extract elastic moduli (E). Data from 20 cells per condition were analyzed statistically.

Statistical Analysis

Results are expressed as mean ± SD. Student’s t‑test compared treated versus control groups, with significance thresholds at p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).

Results

Nanoparticle Characterization

TEM images confirm monodisperse, spherical SiO₂ NPs (~20 nm) and TiO₂ NPs (~25 nm) with mixed anatase‑rutile phases (Fig. 1). DLS shows hydrodynamic diameters of 21 ± 7 nm (SiO₂) and 27 ± 12 nm (TiO₂) in water; sizes increase to 29 ± 9 nm and 41 ± 14 nm, respectively, in DMEM with 20 % FBS after 96 h, indicating protein corona formation. ζ‑potentials are −45 ± 3 mV (SiO₂) and −50 ± 3 mV (TiO₂) in water, becoming more negative in culture media.

NP Uptake

ICP‑AES revealed dose‑ and time‑dependent internalization. TiO₂ NPs accumulated more readily, reaching 8.2 ± 0.4 µg per 3.6 × 10⁵ cells in Caco‑2 after 72 h and 9.7 ± 0.03 µg after 96 h. In A549, uptake was 5 ± 0.6 µg (72 h) and 7.1 ± 0.1 µg (96 h). SiO₂ NPs displayed lower accumulation, particularly in A549 (2.6 ± 0.05 µg at 72 h). Overall, TiO₂ NPs exhibit higher cellular uptake across both cell lines.

Cell Viability, Membrane Damage, and ROS

WST‑8 assays showed a modest viability reduction in both cell lines at 45 µg/mL. Caco‑2 viability dropped by ~40 % after 72 h and ~50 % after 96 h of TiO₂ exposure; A549 exhibited a ~30 % reduction only after 96 h. LDH release mirrored these trends, peaking at ~160 % of control after 96 h in Caco‑2. ROS generation increased dose‑dependently, with TiO₂ NPs inducing up to 165 % DCF‑DA fluorescence in Caco‑2 (Fig. 4).

Antioxidant Response and Lipid Peroxidation

SOD activity declined in a dose‑ and time‑dependent manner. In Caco‑2, SOD decreased from 4.1 ± 0.2 U/mL (control) to 1.0 ± 0.3 U/mL after 96 h of 45 µg/mL TiO₂. SiO₂ exposure reduced SOD to 1.4 ± 0.1 U/mL. MDA levels rose correspondingly in both cell lines, indicating enhanced lipid peroxidation.

Actin Cytoskeleton Remodeling

Confocal imaging revealed disrupted tight junctions and elongated morphology in Caco‑2, and diminished cell‑cell contacts in A549 upon NP treatment. Quantitative analysis showed no significant change in integrated density (actin amount) but a pronounced decrease in coherency: Caco‑2 coherency dropped from 0.26 ± 0.03 (control) to 0.09 ± 0.02 (TiO₂). A549 coherency decreased from 0.40 ± 0.03 to 0.16 ± 0.04. N/C ratios increased in Caco‑2 (0.40 → 0.62) and decreased in A549 (0.55 → 0.23), reflecting divergent morphological responses.

Mechanical Properties (Young’s Modulus)

AFM measurements indicated contrasting stiffness changes. In Caco‑2, Young’s modulus decreased from 105 ± 25 kPa (nucleus) to 27 ± 4 kPa after TiO₂ exposure; cytoplasmic modulus fell from 47 ± 21 kPa to 18 ± 4 kPa. Conversely, A549 stiffness increased dramatically: nuclear modulus rose from 129 ± 24 kPa to 372 ± 60 kPa, and cytoplasmic modulus from 147 ± 26 kPa to 549 ± 40 kPa under TiO₂ treatment.

Discussion

Our findings demonstrate that TiO₂ NPs, owing to their crystalline structure and surface reactivity, provoke stronger cellular responses than amorphous SiO₂ NPs. The enhanced uptake, ROS generation, and actin reorganization correlate with significant alterations in cell elasticity—softening of Caco‑2 cells and stiffening of A549 cells—suggesting distinct mechanistic pathways in epithelial versus alveolar contexts. Such biomechanical changes may influence cell migration, barrier integrity, and potentially tumor progression. Integrating mechanical endpoints with conventional cytotoxicity assays offers a more comprehensive nanotoxicological assessment.

Conclusion

SiO₂ NPs exhibit lower cytotoxicity compared to TiO₂ NPs. TiO₂ exposure induces actin remodeling, altered N/C ratios, and divergent mechanical responses: increased stiffness in A549 and reduced stiffness in Caco‑2. These biomechanical perturbations underscore the importance of evaluating cytoskeletal dynamics and cell mechanics in nanotoxicology studies to predict potential pathological outcomes.

Abbreviations

A549

Human adenocarcinoma alveolar basal epithelial cells

AFM

Atomic force microscopy

ATP

Adenosine triphosphate

Caco‑2

Human epithelial colorectal adenocarcinoma

CLSM

Confocal laser scanning microscopy

DAPI

4′,6′‑Diamidino‑2‑phenylindole

DCF‑DA

2′,7′‑Dichlorofluorescein diacetate

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified Eagle’s medium

ENPs

Engineered nanoparticles

FBS

Fetal bovine serum

HCl/HNO₃

Hydrochloric/nitric acid

ICP‑AES

Inductively coupled plasma atomic emission spectroscopy

LDH

Lactate dehydrogenase

MDA

Malondialdehyde

NH₄OH

Ammonium hydroxide

NPs

Nanoparticles

PBS

Phosphate buffered saline

ROIs

Regions of interest

ROS

Reactive oxygen species

SiO₂ NPs

Silicon dioxide nanoparticles

SOD

Superoxide dismutase

TBA

Thiobarbituric acid

TEM

Transmission electron microscopy

TEOS

Tetraethyl orthosilicate

TiO₂ NPs

Titanium dioxide nanoparticles

TTIP

Titanium (IV) isopropoxide

XRD

X‑ray diffraction