Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2026 Mar 17;46(10):3437–3445. doi: 10.1002/jat.70160

Effect of Zinc Oxide Nanoparticles From Infant Formula Milk Powder on Colon Cells In Vitro

Gloria Salinas‐Lucero 1,2, Karla Juarez‐Moreno 3, Rafael Vazquez‐Duhalt 1,✉
PMCID: PMC13532849  PMID: 41840974

ABSTRACT

Zinc is an essential trace element that participates in numerous metabolic processes; however, excessive exposure may interfere with the absorption of other metals, such as copper. In recent years, nanotechnology has experienced rapid growth, and zinc oxide nanoparticles (ZnO NPs) have garnered significant attention due to their versatile physicochemical properties and increasing applications in industrial and consumer products. These nanoparticles are widely incorporated into plastics, ceramics, cosmetics, and even infant formula milk powder for their antimicrobial and nutritional properties. Given concerns about the presence of ZnO NPs in commercial infant formula milk powder, this work examines cytotoxicity, reactive oxygen species (ROS) generation, and effects on the cell cycle. The results show that ZnO NPs isolated from commercial infant formula milk powder induced concentration‐dependent cytotoxicity, triggered intracellular ROS overproduction, and cell‐cycle arrest (primarily in S and G2/M phases), suggesting that oxidative stress could be a key mechanism involved in ZnO NPs–induced toxicity. Although bulk ZnO is generally recognized as safe and despite approval by the U.S. FDA for certain applications, several studies have shown that ZnO NPs can induce cytotoxic effects depending on their size, shape, and concentration. The mechanisms underlying the toxicity of ZnO NPs remain unclear. Therefore, in this work, their biological effects on intestinal epithelial cell models, such as Caco‐2 and HT29 cells, were studied to assess the potential toxicity of ZnO nanoparticles present in infant milk formula. This is important for a better understanding of the potential health implications of ingesting zinc oxide nanoparticles through fortified food products.

Keywords: colon cells, infant milk formula, toxicity, ZnO nanoparticles

Short abstract

Zinc oxide nanoparticles (ZnO NPs) found in the infant formula milk powder were assayed for cytotoxicity, reactive oxygen species (ROS), and alteration of the cell‐cycle on colon cell lines. The results show that ZnO NPs induced concentration‐dependent cytotoxicity, triggered intracellular ROS overproduction, and cell‐cycle arrest, suggesting that oxidative stress is the mechanism underlying toxicity. Thus, it is imperative to better understand the potential health implications of ingesting zinc oxide nanoparticles through fortified food products, especially infant formula milk powder.

1. Introduction

Zinc (Zn) is a crucial trace element for adults (10 mg Zn2+ per day is recommended), and it is involved in numerous metabolic processes. Zinc may be stored in metallothionein reserves and also transported by protein‐based metal carriers (Valko et al. 2005; Rana et al. 2008; Osredkar and Sustar 2011). Zinc is commonly bonded and transported by albumin, and some remains freely available in the blood plasma. In addition, metallothioneins can regulate and take up zinc by 15%–40% in intestinal cells. Metallothioneins bind to both zinc and copper; thus, zinc concentration primarily impairs copper absorption (Aydemir et al. 2006). On the other hand, iron is transported by transferrin, and the excess iron can reduce zinc absorption, and vice versa. In normal zinc intake, its concentration in blood plasma remains relatively constant.

Over the last few decades, nanotechnology has advanced rapidly as one of the fastest‐growing domains of science and technology. Metal oxide nanoparticles (NPs) are increasingly being used in various industrial applications. Among the various metals, zinc attracts significant attention due to its strong reducing potential, moderate reactivity, and five stable isotopes. Among the different zinc‐based nanostructures, such as sulfide, ferrite, phosphide, selenide, and telluride, zinc oxide (ZnO) is the most attractive due to its extensive applicability, eco‐friendliness, and diverse physicochemical properties. Zinc oxide NPs are used as an additive in various industrial products and materials, including glass, ceramics, cement, rubbers, ointments, lubricants, paints, foods, fire retardants, plastics, pigments, adhesives, ferrites, and sealants, among others (Saraswathi et al. 2012). Bulk ZnO, compared to ZnO nanoparticles (ZnO NPs), is a bio‐safe material (Kołodziejczak‐Radzimska and Jesionowski 2014). Indeed, various studies have already shown the potential toxicity of ZnO NPs depending on their size, shape, and concentration (Liu et al. 2016; Cao et al. 2018). Although ZnO has been approved for cosmetic use by the U.S. FDA (Food and Drug Administration), the detailed toxicological profile and the mechanisms of cytotoxicity for ZnO nanomaterials remain incompletely understood (Wingett et al. 2016). Recently, in a previous work, we detected significant amounts of ZnO NPs in commercial infant formula milk powders (Salinas‐Lucero et al. 2024). Besides, ZnO NPs are also reported to have a good biocompatibility with human cells, and the ZnO has been documented as a safe material by the FDA (Khan et al. 2015; Jiang et al. 2018), although the intrinsic cytotoxicity of ZnO NPs against certain human cell lines has raised some concern as a potential health risk (Yang et al. 2013). On the other hand, their potential cytotoxicity toward pathogenic cells could be beneficial if their properties are precisely tailored (Akhtar et al. 2012; Wingett et al. 2016). The ZnO NPs have been proposed for therapeutic purposes in nanomedicine as a drug delivery system and an antibacterial agent (Cheng et al. 2024, 2025). In addition, the ZnO surface is chemically rich in ‐OH groups that can be readily modified by several surface‐adsorbing molecules (Ali et al. 2017).

In this work, the effect of ZnO NPs contained in infant formula milk powder on colon cells was determined in vitro cell cultures.

2. Materials and Methods

2.1. Preparation and Characterization of ZnO NPs

Zinc oxide NPs (ZnO NPs) were obtained from commercial infant formula samples following the cloud point extraction (CPE) method previously described (Salinas‐Lucero et al. 2024). Briefly, the CPE technique employs the nonionic surfactant Triton X‐100 and humic acid as a complexing agent to selectively isolate ZnO NPs from the powdered formula. This procedure effectively separates NPs into a surfactant‐rich phase while minimizing interference from soluble zinc ions. Comprehensive details of the isolation, validation, and characterization steps, including Inductively Coupled Plasma Optical Emission Spectroscopy (ICP‐OES) quantification, transmission electron microscopy (TEM), and thermogravimetric analysis (TGA), are provided in the cited work.

2.2. Removal of Surfactant and NP Purification

Before their use in vitro biological assays, the surfactant layer remaining on the NP surface was carefully removed to avoid interference with cytotoxicity and oxidative stress measurements. The Triton X‐100 coating was removed by washing the NP suspension with a 1:1 (v/v) acetone/isopropyl alcohol mixture (Rojas et al. 2019), followed by centrifugation and redispersion in deionized water. After this purification step, transmission electron microscopy (TEM) confirmed the successful removal of the organic corona, revealing well‐dispersed ZnO NPs with morphology and size consistent with those previously reported (average diameter ∼6–9 nm) (Salinas‐Lucero et al. 2024).

2.3. Cell Culture

To investigate the potential cytotoxic effects of ZnO NPs derived from infant formula, three representative mammalian cell lines were used: Caco‐2 (human intestinal epithelial), HT‐29 (human colorectal adenocarcinoma), and AML‐12 (murine hepatocytes). These cell models were selected to mimic two main biological barriers potentially exposed to ingested NPs, the intestinal epithelium and the liver. Caco‐2 and HT‐29 cells are well‐established in vitro models for intestinal absorption and barrier integrity (Collett et al. 1996; Pan et al. 2015), whereas AML‐12 cells are a mouse cell line that displays morphological and gene expression patterns characteristic of differentiated hepatocytes (Messner et al. 2013) and offer a hepatic model for assessing metabolic processing and oxidative stress response. Caco‐2 cells can acquire enterocyte‐like characteristics and form tight junctions upon differentiation, while HT‐29 cells can express mucins depending on culture conditions; together, these models provide complementary intestinal epithelial systems (Sambuy et al. 2005; Martínez‐Maqueda et al. 2015). Cells were cultured under standard conditions at 37°C in a humidified atmosphere containing 5% CO2. The following culture media were used: AML‐12 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1% insulin‐transferrin‐selenium (ITS), and 1% penicillin–streptomycin. Caco‐2 and HT‐29 were cultured in high‐glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin.

2.4. Cytotoxicity

Cell viability was evaluated using the MTT assay in the HT‐29 and Caco‐2 cancer cell lines, as well as the noncancerous AML‐12 cell line. Cells were seeded in 96‐well plates at densities of 8000 cells per well for the cancer cell lines and 10,000 cells per well for AML‐12, and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. These seeding densities were selected based on preliminary optimization experiments to ensure adequate cell attachment, reproducible metabolism, and physiologically stable cellular conditions during exposure. Before each experiment, NP suspensions were freshly prepared, ultrasonicated for 10 min, diluted to the desired concentrations, and immediately added to the cell cultures. Cells were then exposed, in triplicate, for 24 h to ZnO NPs isolated from infant formula milk (0–60 μg/mL). To compare the effects, commercial pristine ZnO NPs (0–200 μg/mL) were also tested. Untreated cells incubated with culture medium served as the negative control, while cells treated with 20% DMSO were used as the positive control. After exposure, MTT was added, and following solubilization of the formazan crystals, absorbance was measured at 570 nm with background correction at 690 nm, including a medium‐only blank. Results were expressed as a percentage of cell viability.

2.5. Reactive Oxygen Species Assay

Oxidative stress is one of the primary mechanisms by which metal oxide NPs exert toxicity. Intracellular reactive oxygen species (ROS) generation was quantified using the fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA). Upon oxidation by ROS, this nonfluorescent compound is transformed into highly fluorescent dichlorofluorescein (DCF), enabling quantitative estimation of oxidative stress levels. After exposure to ZnO NPs (10–90 μg/mL) for 24 h, AML‐12, Caco‐2, and HT‐29 cells were washed twice with phosphate‐buffered saline (PBS) and incubated with 10 μM DCFH‐DA in serum‐free medium for 30 min at 37 °C in the dark. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a microplate reader in a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies, Santa Clara, CA, USA). Data were normalized to untreated control cells and expressed as the relative percentage of ROS production.

2.6. Cell‐Cycle Analysis by Flow Cytometry

Cell proliferation dynamics and cell‐cycle distribution were analyzed in samples from all three cell lines using flow cytometry. This approach enables the detection of alterations in cell‐cycle dynamics by quantifying the proportion of cells in the G0/G1, S, and G2/M phases, thereby revealing cell cycle arrest or replication stress that may be associated with cytotoxic effects. Cells were seeded in 6‐well plates and exposed to increasing ZnO NP concentrations (0, 10, 30, and 60 μg/mL) for 24 h. Then, after treatment, the cells were harvested, washed twice with cold PBS, and fixed overnight in 70% ethanol at 4 °C. Fixed cells were incubated at 37 °C for 30 min with 100 μg/mL RNase A in PBS, then rinsed and stained with 50 μg/mL propidium iodide (PI). Samples were then incubated for 30 min at room temperature in the dark prior to analysis. Fluorescence data were acquired using an Attune Acoustic Focusing Cytometer (Applied Biosystems, Thermo Fisher Scientific, USA). Data acquisition and compensation were performed using the Attune Cytometric Software, and at least 10,000 events were collected per sample. Statistical significance among treatments was assessed using one‐way ANOVA followed by Tukey's HSD post hoc test, with p < 0.05 considered significant.

3. Results and Discussion

ZnO NPs were isolated from infant formula milk powder and purified as previously described (Salinas‐Lucero et al. 2024). Figure 1 compares the NPs before and after surfactant removal. In the left panel (Figure 1A), the TEM image shows the ZnO NPs isolated by the CPE method, in which a thin layer of Triton X‐100 can be clearly visible surrounding the particles. In contrast, the right panel (Figure 1B) shows the NP suspension after the purification procedure, where the surfactant layer has been removed, revealing clean, spherical ZnO NPs obtained from the infant formula sample. Before surfactant removal, TEM images revealed well‐defined primary ZnO NPs together with small clusters, suggesting a moderate degree of interparticle association. This observation is consistent with DLS measurements, which showed a hydrodynamic diameter of ~289 nm and a polydispersity index of 0.361, reflecting a moderately polydisperse size distribution in suspension. However, after surfactant removal, the ZnO NPs showed an average diameter of 6–9 nm in a well‐dispersed suspension, showing a zeta potential of −25.5 mV.

FIGURE 1.

FIGURE 1

Open in a new tab

Comparison of ZnO nanoparticles before and after surfactant removal. (A) TEM images of ZnO NPs before surfactant removal. (B) TEM images of nanoparticle suspension after the surfactant removal procedure.

To evaluate the effect of ZnO NPs on colon cells, Caco‐2 (human intestinal epithelial) and HT‐29 (human colorectal adenocarcinoma) cells were used. These cell models were selected to mimic the main intestinal barrier (intestinal epithelium) potentially exposed to ingested NPs. Caco‐2 and HT‐29 cells are well‐established in vitro models for intestinal absorption and barrier integrity (Collett et al. 1996; Pan et al. 2015). These cell lines, Caco‐2 and HT29‐H, represent the two most abundant cell populations in the intestinal epithelium: absorptive cells and goblet cells (Artursson et al. 2012). On the other hand, AML‐12 cells are a mouse cell line that display morphological and gene expression characteristics (albumin, transferrin, and connexin 32) of differentiated hepatocytes (Messner et al. 2013) and provide a hepatic model for assessing metabolic processing and the oxidative stress response.

To assess the effect of ZnO NPs on mammalian cells, we compared NPs from two different sources: ZnO NPs isolated from commercial infant formula milk powder using the CPE method, and a commercial pristine ZnO NPs purchased from Sigma‐Aldrich (< 100 nm, cat. 544906). Besides the size differences, both types were purified and characterized prior to use, showing similar morphology and physicochemical properties. The CPE‐isolated NPs had average diameters of 6–9 nm, whereas the commercial particles showed larger aggregates (∼100 nm).

Figure 2A,B show the viability of intestinal Caco‐2 and hepatic AML‐12 cells, respectively, after 24‐h exposure to increasing concentrations of ZnO NPs isolated from infant formula milk powder. ZnO NPs induced a pronounced decrease in cell viability. Exposure of Caco‐2 cells to commercial ZnO NPs for 24 h induced also a concentration‐dependent decrease in cell viability (Figure 2C). At concentrations of 0–10 μg/mL, metabolic activity remained indistinguishable from that of the untreated control. At 30 μg/mL, viability decreased to approximately 80%, after which it declined consistently with increasing NP concentration, reaching approximately 25% at 150 μg/mL. These results indicate a clear concentration‐dependent loss of viability across the tested range. In the HT‐29 cell line, ZnO NPs exhibited a threshold‐dependent toxicity profile rather than a progressive concentration‐response (Figure 2D). Cell viability remained near control levels (95–100%) at concentrations up to 10 μg/mL, indicating tolerance to low‐concentration exposure. Above this threshold, viability declined sharply, reaching 50%–60% at concentrations ranging from 20 to 150 μg/mL. This pattern indicates a nonlinear threshold‐driven cytotoxic mechanism that is triggered only after exceeding a critical exposure level. AML‐12 hepatocytes displayed a clear threshold‐type response to ZnO NPs (Figure 2E). Cell viability was preserved near control levels (90%) up to 20 μg/mL but dropped precipitously at higher concentrations, reaching 20% in the mid‐to‐high concentration range (30–150 μg/mL). This pattern indicates that AML‐12 cells tolerate subthreshold exposures and that acute cytotoxicity occurs only when the critical concentration threshold is exceeded.

FIGURE 2.

FIGURE 2

Open in a new tab

Effect of commercial pristine ZnO nanoparticles (A, B) and ZnO nanoparticles isolated from infant formula milk powder (C, D, E) on cell viability of Caco‐2, HT‐29, and AML‐12 cells after 24‐h exposure to different concentrations. One‐way ANOVA with Tukey's HSD post hoc test versus control: asterisks above the bars indicate significance levels: *p ≥ 0.05, **p ≥ 0.01, ***p ≥ 0.001.

Both cell lines exhibited clear concentration‐dependent cytotoxicity. Caco‐2 cells exhibited moderate sensitivity, with viability decreasing gradually and reaching a plateau at approximately 50% at 60 μg/mL. In contrast, AML‐12 cells were markedly more susceptible to ZnO NP exposure, showing a sharp decline at 10 μg/mL and viability below 20% at 40–60 μg/mL. These results indicate that hepatic cells are more susceptible to ZnO NPs–induced cytotoxicity than intestinal epithelial cells, possibly due to differences in antioxidant capacity and metabolic activity. Previous studies have consistently reported similar concentration‐dependent toxicity of ZnO NPs mediated by oxidative stress, membrane damage, and mitochondrial dysfunction, leading to decreased metabolic activity (De Berardis et al. 2010). The heightened sensitivity of AML‐12 cells may be related to their hepatocytic phenotype, which facilitates greater uptake of Zn2+ ions released from dissolving NPs, thereby amplifying intracellular ROS production and lipid peroxidation (Xia et al. 2008; Vandebriel and De Jong 2012). Overall, ZnO NPs isolated from infant formula milk powder exert concentration‐dependent cytotoxicity, with pronounced cell‐type‐specific differences. Intestinal Caco‐2 cells exhibit relative resistance, suggesting that the epithelial barrier may confer partial protection. Conversely, hepatic AML‐12 cells, which play a central role in detoxification, appear particularly susceptible to ZnO‐NP–induced oxidative stress and metabolic impairment.

Intracellular ROS generation was assessed in Caco‐2 and AML‐12 cells following exposure to ZnO NPs isolated from infant formula milk powder or commercial pristine ZnO NPs (Figure 3). The intracellular generation of ROS in intestinal Caco‐2 and hepatic AML‐12 cells exposed to increasing concentrations of ZnO NPs isolated from infant milk formula was evaluated (Figure 3A,B). Both cell lines exhibited significantly higher ROS levels relative to untreated controls. In Caco‐2 cells, ROS production approximately doubled at low concentrations (5–10 μg/mL) and remained elevated up to 60 μg/mL, reaching a maximum induction of ~320%, comparable to the positive control (H2O2) (Figure 3A). In contrast, AML‐12 cells showed a marked ROS increase at 5–20 μg/mL, followed by a progressive decline at 40–60 μg/mL, consistent with extensive cell death and loss of metabolic activity (Figure 3B). These results confirm that ZnO NPs extracted from infant formula milk powder induce oxidative stress in both intestinal and hepatic cell models. However, the concentration‐response kinetics differ markedly: Caco‐2 cells display prolonged ROS elevation, whereas AML‐12 cells exhibit an early peak followed by a sharp decline, reflecting differences in NP uptake, antioxidant capacity, and metabolic resilience between the two cell lines.

FIGURE 3.

FIGURE 3

Open in a new tab

Reactive oxygen species (ROS) generation induced by isolated from infant formula milk powder (A and B) and by commercial ZnO nanoparticles (C, D, and D) in Caco‐2, HT29, and AML‐12 cells after 24‐h exposure. One‐way ANOVA with Tukey's HSD post hoc test versus control: asterisks above the bars indicate significance levels: *p ≥ 0.05, **p ≥ 0.01, ***p ≥ 0.001.

In the case of commercial ZnO NPs in Caco‐2 cells, ROS levels peaked at 10–50 μg/mL (~160%–180% of control) and slightly declined at 70–90 μg/mL, suggesting reduced metabolic activity or the onset of cytotoxicity (Figure 3C). A similar biphasic pattern was observed in HT29 cells, with moderate ROS elevation at 10–30 μg/mL (~180%–200%) of control and return near baseline at higher concentrations (Figure 3D). In AML‐12 cells, ROS production was more pronounced, reaching ~180%–220% of control at 10–30 μg/mL before progressively decreasing at higher concentrations, consistent with substantial loss of cell viability (Figure 3E). In all cell lines, the positive control H2O2 induced the highest ROS levels (> 300% of control), confirming the sensitivity of the assay.

Overall, these results demonstrate that both types of ZnO NPs trigger oxidative stress across all cell models, albeit with cell‐type–specific magnitude and concentration‐response profiles. This early and marked ROS induction, followed by a decline at cytotoxic concentrations, supports oxidative stress as a primary mechanism of ZnO NPs toxicity, modulated by the inherent physiological and antioxidant capacities of each cell.

The results indicate that ZnO NPs induce intense oxidative stress in both intestinal (Caco‐2) and hepatic (AML‐12) cell lines in a concentration‐ and time‐dependent manner. This finding is consistent with previous reports, which describe ROS overproduction as a key mechanism underlying ZnO NPs cytotoxicity (Kang et al. 2013; Cao et al. 2015). Excessive ROS levels can lead to lipid peroxidation, mitochondrial dysfunction, and activation of apoptotic pathways (Wang et al. 2015). In Caco‐2 cells, the marked increase in ROS levels observed after 48 h suggests that prolonged exposure exacerbates oxidative damage, likely due to progressive NP accumulation or delayed antioxidant response. In AML‐12 hepatocytes, the pronounced ROS generation is consistent with earlier findings showing that ZnO NPs disrupt mitochondrial redox balance and induce oxidative DNA damage (Almansour et al. 2017; Guan et al. 2012; Mittag et al. 2022). Together, these findings confirm that oxidative stress plays a central role in linking ZnO NPs exposure to the cytotoxicity previously observed in cell viability assays (Figure 2). The resulting ROS overproduction directly contributes to loss of metabolic activity and cell death in both intestinal and hepatic models (Anjum et al. 2021; Fernández‐Cruz et al. 2013).

In addition, these results are consistent with previous reports demonstrating that ZnO NPs promote ROS generation through surface‐catalyzed redox reactions and the intracellular release of Zn2+ ions, both of which disrupt mitochondrial function and overwhelm antioxidant defenses (Applerot et al. 2012; De Berardis et al. 2010; Premanathan et al. 2011). The sustained higher ROS generation in Caco‐2 cells may reflect greater NP–membrane interaction and higher endocytic uptake. In contrast, the reduction in ROS levels observed at cytotoxic concentrations of ZnO NPs in AML‐12 cells might result from extensive oxidative damage and the consequent loss of metabolically active cells (Xia et al. 2008; Vandebriel and De Jong 2012; Mittag et al. 2022). Overall, the data confirm that ZnO NPs isolated from infant formula induce a strong oxidative response, supporting the hypothesis that ROS generation is a primary mechanism of ZnO‐NP–induced cytotoxicity.

The cell cycle distribution of Caco‐2 and AML‐12 cells exposed to ZnO NPs isolated from infant formula milk powder was investigated using flow cytometry (Figure 4A,B), which revealed induced cell‐cycle alterations. In Caco‐2 cells, exposure reduced the G0/G1 population and increased the proportions of cells in S and G2/M phases. In Caco‐2 cells (Figure 4A), untreated control cells (0 μg/mL) exhibited a typical distribution, with a predominant fraction in the G0/G1 phase (75.6% ± 1.7%), a small fraction in the G2/M phase (4.2% ± 0.3%), and a fraction in the S phase (13.2% ± 0.2%). Exposure to 10 μg/mL ZnO NPs significantly decreased the G0/G1 population (to 41.6% ± 0.3%), while markedly increasing the S‐phase (20.3% ± 0.3%) and, in particular, the G2/M‐phase (33.5% ± 0.3%) populations, suggesting an initial block at the G2/M checkpoint for DNA synthesis and mitotic entry. Similar distributions were observed at 30 and 60 μg/mL concentrations. Induced cell‐cycle alterations were also observed in AML‐12 hepatocytes, where G0/G1 phase was reduced at low ZnO NPs concentration and significantly increased at 30 and 60 μg/mL concentrations, while S and G2/M populations were reduced (Figure 4B).

FIGURE 4.

FIGURE 4

Open in a new tab

Cell cycle distribution determined by flow cytometry of Caco‐2 and AML‐12 cells exposed to increasing concentrations (0, 10, 30, and 60 μg/mL) of ZnO nanoparticles isolated from infant formula milk powder (A and B), and commercial ZnO nanoparticles (C and D). One‐way ANOVA with Tukey's HSD post hoc test versus control: asterisks above the bars indicate significance levels: *p ≥ 0.05, **p ≥ 0.01, ***p ≥ 0.001.

Commercial pristine ZnO NPs showed qualitatively similar effects to those observed with infant formula milk powder, despite their markedly smaller size (~9 nm vs. ~100 nm). Caco‐2, AML‐12, and HT29 cells were also exposed to commercial ZnO NPs at increasing concentrations (0, 10, 30, and 60 μg/mL). In Caco‐2 cells, ZnO NPs induced a less pronounced but qualitatively similar cell‐cycle perturbation (Figure 4C). At 10 μg/mL, the G0/G1 population remained essentially unchanged (72.8% ± 0.62% vs. 77.6% ± 0.2% in controls). However, higher concentrations at 30 and 60 μg/mL triggered a sharp decline in G0/G1 cells, concomitant with a marked accumulation in the S‐phase, from 15.5% ± 0.4% (control) to 83.1 ± 1.4% at 30 μg/mL. In the case of AML‐12 hepatic cells, the effect was similar to that obtained with NPs from infant formula milk powder (Figure 4D).

In HT‐29 cells (Figure 4E), untreated control cells (0 μg/mL) exhibited a typical distribution, with a predominant fraction in the G0/G1 phase (60.2% ± 0.2%), a small fraction in the G2/M phase (15.1% ± 0.1%), and a small fraction in the S phase (25.1% ± 0.1%). Exposure to 10 μg/mL ZnO NPs significantly decreased the G0/G1 population (to 8.9% ± 1.0%) and S population (to 15.6% ± 0.2%), while markedly increasing the G2/M‐phase (to 66.1% ± 1.4%), suggesting an initial block at the G2/M checkpoint for DNA synthesis and mitotic entry. At 30 μg/mL, a significant accumulation of cells was observed in the S phase (82.9% ± 1.1%), accompanied by a marked reduction in G0/G1 and G2/M populations, suggesting that ZnO NPs induce S‐phase arrest. At 60 μg/mL of ZnO NPs, partial recovery of the G2/M population (62.5% ± 0.4%) was observed, although the G0/G1 population remained severely reduced (19.7% ± 2.0%), consistent with extensive DNA damage and irreversible impairment of cell‐cycle progression. These findings demonstrate that ZnO NPs disrupt cell‐cycle progression in HT‐29 cells in a concentration‐dependent manner, inducing sequential S‐phase and G2/M arrest. Similar cell‐cycle perturbations have been reported previously and attributed to ZnO NPs–induced oxidative stress, DNA strand breaks, and activation of checkpoint proteins such as p53 and p21, leading to inhibition of DNA replication and mitotic entry (Almansour et al. 2017; Sirelkhatim et al. 2016; Akhtar et al. 2012). Comparable effects in other epithelial cell lines further support the activation of DNA‐damage response pathways and replication stress following ZnO NPs exposure (Hanley et al. 2009). In addition, the observed arrests are therefore consistent with the pronounced ROS generation documented in Figure 3, reinforcing the mechanistic link between oxidative damage, genotoxic stress, and dysregulation of cell‐cycle control (Kumar et al. 2024; Malaviya et al. 2019).

The prominent S‐phase accumulation found in Caco‐2 cells at cytotoxic concentrations suggests interferences with DNA replication and repair processes, leading to replication stress (De Berardis et al. 2010; Vandebriel and De Jong 2012). This pattern aligns with previous reports demonstrating that ZnO NPs–derived ROS cause oxidative DNA damage, activation of the ATR–Chk1 signaling pathway, and subsequent stalling of the replication fork (SharmaV. 2012; Ng, 2017). Overall, these results suggest that commercial ZnO NPs impair the normal cell‐cycle progression in Caco‐2 cells through oxidative stress and genotoxic mechanisms, potentially leading to S‐phase arrest, impaired proliferation, and, at higher concentrations, potential commitment to apoptosis.

From a food‐safety perspective, our results show that both pristine ZnO NPs (~100 nm) and ZnO NPs extracted from infant formula milk powder (~9 nm) retain significant cytotoxic potential toward intestinal (Caco‐2 and HT29) and hepatic (AML‐12) cells, even after interaction with the complex food matrix. Both forms trigger oxidative stress, disrupt cell cycle progression, and reduce cell viability. Previous studies have established that ingested ZnO NPs can partially dissolve into Zn+2 ions during gastrointestinal digestion; however, a significant particulate fraction can translocate across the intestinal epithelium by transcellular and paracellular routes, reaching systemic circulation and distant organs, including the liver, through the portal system (Setyawati et al. 2015; Kermanizadeh et al. 2017).

Our findings gain additional dimension when contextualized with recent results reported by Zeng et al. (2025), who demonstrated that oral intake of soluble zinc can lead to intraluminal formation of ZnO nanoclusters in the intestine. In this study, Zn2+ ions released during digestion underwent spontaneous nucleation and growth, leading to the formation of ZnO nanoclusters stabilized by lumen proteins. These new nanoscale structures crossed the intestinal epithelium, distributed systemically, and accumulated in organs, including the kidney and renal tumors, where they triggered specific cellular responses. This mechanism is highly relevant for interpreting our results, as it suggests that, regardless of the initial size of the ingested particles, the ZnO‐NPs present in the infant formula (9 nm) can participate in processes of reorganization, aggregation, or reconstruction within the gastrointestinal tract, generating even more active secondary structures.

From this perspective, the similarity in toxic effects observed between ~100 nm pristine particles and ~9 nm NPs derived from infant formula milk powder can be explained not only by their larger surface area but also by the possible intraluminal biogenesis of nanoclusters during digestion. This phenomenon would increase the effective NP load to which the intestinal epithelium and liver are actually exposed, providing a coherent explanation for the marked cell‐cycle disruption, G0/G1‐phase accumulation, and reduced proliferative phases observed in AML‐12 cells. These findings underscore the need to consider not only the initial particle size in food but also its in vivo transformations, which can modify bioavailability, toxicity, and systemic distribution.

Overall, our results are consistent with the growing evidence that ZnO NPs can induce oxidative stress, disrupt the cell‐cycle progression, and exert cytotoxic effects in both gastrointestinal and hepatic cell models. Importantly, they also provide new evidence that these adverse effects persist even at lower concentrations and persist after the NPs have been extracted from a complex food matrix, such as infant formula, and that their effects are not limited to the gastrointestinal tract (Li et al. 2020). Moreover, the comparable increase in the toxicity of food‐derived ZnO NPs suggests that intraluminal transformation processes (e.g., partial dissolution, protein corona formation, and changes in aggregation state) during digestion do not mitigate their biological impact and may even exacerbate it. These findings highlight the importance of reassessing the safety of ZnO NPs in products intended for children. Regulatory evaluation should extend beyond total zinc content to explicitly consider the presence, size, and physicochemical transformations of nanostructured forms throughout the gastrointestinal tract, as well as their potential for systemic translocation and organ‐specific toxicity.

4. Conclusions

Zinc oxide NPs isolated from commercial infant formula milk powder induced concentration‐dependent cytotoxicity and oxidative stress in mammalian cells. After surfactant removal, the isolated NPs exhibited physicochemical properties that closely resembled those of pristine commercial ZnO NPs. They elicited measurable biological responses in intestinal (Caco‐2 and HT‐29) and hepatic (AML‐12) cell models. Both types of NPs reduced cell viability in a concentration‐dependent manner, with AML‐12 hepatocytes exhibiting greater sensitivity and Caco‐2 enterocyte cells showing greater resistance, likely due to their barrier‐like epithelial characteristics.

Cell exposure to ZnO NPs consistently triggered intracellular ROS overproduction, cell‐cycle arrest (primarily in S and G2/M phases), and reduced metabolic activity, indicating that oxidative stress is a key mechanism involved in ZnO NPs–induced toxicity. Emerging evidence suggests that ZnO NPs undergo substantial transformation during gastrointestinal digestion, including partial dissolution, protein corona formation, and aggregation into nanoclusters, which may increase the effective NP burden reaching the intestinal epithelium, hepatic cells, and systemic compartments. Such dynamic physicochemical changes likely contribute to the pronounced in vitro effects observed here.

Overall, these findings suggest that the biological impact of ZnO NPs is influenced not only by the total zinc content but also by their nanostructured form, potential transformations during digestion, and biological reactivity. The results underscore the urgent need for additional toxicological studies, particularly in vivo, to deepen understanding of NP absorption, biodistribution, metabolism, and potential long‐term health effects. They also underscore the importance of reassessing the safety of ZnO NPs in food products intended for vulnerable populations, such as infants.

Author Contributions

Gloria Salinas‐Lucero: investigation, formal analysis, data curation, and writing – original draft. Rafael Vazquez‐Duhalt: conceptualization, investigation, formal analysis, data curation, review and editing, visualization, project administration, and funding acquisition. Karla Juarez‐Moreno: conceptualization, review and editing, and visualization. All authors read and approved the final manuscript.

Funding

This work was supported by the Univerisdad Nacional Autónoma de México (PAPIIT‐ IN202225).

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors thank Dr. Lucia Perezgasga and Dr. Oscar Gonzalez‐Davis for their technical assistance. This work was funded by the National Autonomous University of Mexico (PAPIIT‐ IN202225).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Akhtar, M. J. , Ahamed M., Kumar S., Khan M. M., Ahmad J., and Alrokayan S. A.. 2012. “Zinc Oxide Nanoparticles Selectively Induce Apoptosis in Human Cancer Cells Through Reactive Oxygen Species.” International Journal of Nanomedicine 7: 845–857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ali, A. , Ambreen S., Javed R., Tabassum S., Ul Haq I., and Zia M.. 2017. “ZnO Nanostructure Fabrication in Different Solvents Transforms Physio‐ Chemical, Biological and Photodegradable Properties.” Materials Science and Engineering: C 74: 137–145. [DOI] [PubMed] [Google Scholar]
  3. Almansour, M. I. , Alferah M. A., Shraideh Z. A., and Jarrar B. M.. 2017. “Zinc Oxide Nanoparticles Hepatotoxicity: Histological and Histochemical Study.” Environmental Toxicology and Pharmacology 51: 124–130. [DOI] [PubMed] [Google Scholar]
  4. Anjum, S. , Hashim M., Malik S. A., et al. 2021. “Recent Advances in Zinc Oxide Nanoparticles (ZnO NPs) for Cancer Diagnosis, Target Drug Delivery, and Treatment.” Cancers 13, no. 18: 4570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Applerot, G. , Lellouche J., Perkas N., Nitzan Y., Gedanken A., and Banin E.. 2012. “ZnO Nanoparticle‐Coated Surface Inhibit Bacterial Biofilm Formation and Increase Antibiotic Susceptibility.” RCS Advances 2: 2314–2321. [Google Scholar]
  6. Artursson, P. , Palm K., and Luthman K.. 2012. “Caco‐2 Monolayers in Experimental and Theoretical Predictions of Drug Transport.” Advanced Drug Delivery Reviews 64, Supplement: 280–289. [DOI] [PubMed] [Google Scholar]
  7. Aydemir, T. B. , Blanchard R. K., and Cousins R. J.. 2006. “Zinc Supplementation of Young Men Alters Metallothionein, Zinc Transporter, and Cytokine Gene Expression in Leukocyte Populations.” Proceedings of the National Academy of Sciences 103, no. 6: 1699–1704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cao, Y. , Gong Y., Liao W., et al. 2018. “A Review of Cardiovascular Toxicity of TiO2, ZnO and Ag Nanoparticles (NPs).” Biometals 31: 457–476. [DOI] [PubMed] [Google Scholar]
  9. Cao, Y. , Roursgaard M., Kermanizadeh A., Loft S., and Møller P.. 2015. “Synergistic Effects of Zinc Oxide Nanoparticles and Fatty Acids on Toxicity to Caco‐2 Cells.” International Journal of Toxicology 34, no. 1: 67–76. [DOI] [PubMed] [Google Scholar]
  10. Cheng, Z. , Janakiraman A. K., Sundarapandian R., et al. 2025. “Precision Engineering of Vinblastine‐Loaded Zinc Oxide Nanoparticles Through Statistical Experimental Design.” Nano‐Structures & Nano‐Objects 42: 101472. [Google Scholar]
  11. Cheng, Z. , Wong Z. Y., Ramkanth S., et al. 2024. “Colchicine‐Capped ZnO Nanoparticles: Elucidation of Their Cytotoxicity Potential Against MDA‐MB 231 Cancer Cells.” Open Nano 18: 100210. [Google Scholar]
  12. Collett, A. , Sims E., Walker D., et al. 1996. “Comparison of HT29‐18‐C1 and Caco‐2 Cell Lines as Models for Studying Intestinal Paracellular Drug Absorption.” Pharmaceutical Research 13: 216–221. [DOI] [PubMed] [Google Scholar]
  13. De Berardis, B. , Civitelli G., Condello M., et al. 2010. “Exposure to ZnO Nanoparticles Induces Oxidative Stress and Cytotoxicity in Human Colon Carcinoma Cells.” Toxicology and Applied Pharmacology 246, no. 3: 116–127. [DOI] [PubMed] [Google Scholar]
  14. Fernández‐Cruz, M. L. , Lammel T., Connolly M., et al. 2013. “Comparative Cytotoxicity Induced by Bulk and Nanoparticulated ZnO in the Fish and Human Hepatoma Cell Lines PLHC‐1 and Hep G2.” Nanotoxicology 7, no. 5: 935–952. [DOI] [PubMed] [Google Scholar]
  15. Guan, R. , Kang T., Lu F., Zhang Z., Shen H., and Liu M.. 2012. “Cytotoxicity, Oxidative Stress, and Genotoxicity in Human Hepatocyte and Embryonic Kidney Cells Exposed to ZnO Nanoparticles.” Nanoscale Research Letters 7, no. 1: 602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hanley, C. , Thurber A., Hanna C., Punnoose A., Zhang J., and Wingett D. G.. 2009. “The Influences of Cell Type and ZnO Nanoparticle Size on Immune Cell Cytotoxicity and Cytokine Induction.” Nanoscale Research Letters 4, no. 12: 1409–1420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jiang, J. , Pi J., and Cai J.. 2018. “The Advancing of Zinc Oxide Nanoparticles for Biomedical Applications.” Bioinorganic Chemistry and Applications 2018, no. 1: 1062562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kang, T. , Guan R., Chen X., Song Y., Jiang H., and Zhao J.. 2013. “ In Vitro Toxicity of Different‐Sized ZnO Nanoparticles in Caco‐2 Cells.” Nanoscale Research Letters 8, no. 1: 496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kermanizadeh, A. , Jantzen K., Ward M. B., et al. 2017. “Nanomaterial‐Induced Cell Death in Pulmonary and Hepatic Cells Following Exposure to Three Different Metallic Materials: The Role of Autophagy and Apoptosis.” Nanotoxicology 11, no. 2: 184–200. [DOI] [PubMed] [Google Scholar]
  20. Khan, Y. A. , Singh B. R., Ullah R., Shoeb M., Naqvi A. H., and Abidi S. M.. 2015. “Anthelmintic Effect of Biocompatible Zinc Oxide Nanoparticles (ZnO NPs) on Gigantocotyle explanatum, a Neglected Parasite of Indian Water Buffalo.” PLoS ONE 10, no. 7: e0133086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kołodziejczak‐Radzimska, A. , and Jesionowski T.. 2014. “Zinc Oxide—From Synthesis to Application: A Review.” Materials 7, no. 4: 2833–2881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kumar, R. , Lakhani P., Kumar A. Y. N., and Ghosh M.. 2024. “Nanotoxicity and Mechanisms.” In Nanotechnology Theragnostic in Livestock Diseases and Management, 699–741. Springer. [Google Scholar]
  23. Li, J. , Song Y., Vogt R. D., Liu Y., Luo J., and Li T.. 2020. “Bioavailability and Cytotoxicity Of Cerium‐(IV), Copper‐(II), and Zinc Oxide Nanoparticles to Human Intestinal and Liver Cells Through Food.” Science of the Total Environment 702: 134700. [DOI] [PubMed] [Google Scholar]
  24. Liu, J. , Feng X., Wei L., Chen L., Song B., and Shao L.. 2016. “The Toxicology of Ion‐Shedding Zinc Oxide Nanoparticles.” Critical Reviews in Toxicology 46, no. 4: 348–384. [DOI] [PubMed] [Google Scholar]
  25. Malaviya, P. , Shukal D., and Vasavada A. R.. 2019. “Nanotechnology‐Based Drug Delivery, Metabolism and Toxicity.” Current Drug Metabolism 20, no. 14: 1167–1190. [DOI] [PubMed] [Google Scholar]
  26. Martínez‐Maqueda, D. , Miralles B., and Recio I.. 2015. “HT29 Cell Line.” In The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models, edited by Verhoeckx K., 113–124. Springer International Publishing AG. [PubMed] [Google Scholar]
  27. Messner, D. J. , Rhieu B. H., and Kowdley K. V.. 2013. “Iron Overload Causes Oxidative Stress and Impaired Insulin Signaling in AML‐12 Hepatocytes.” Digestive Diseases and Sciences 58: 1899–1908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mittag, A. , Singer A., Hoera C., Westermann M., Kämpfe A., and Glei M.. 2022. “Impact of In Vitro Digested Zinc Oxide Nanoparticles on Intestinal Model Systems.” Particle and Fibre Toxicology 19, no. 1: 39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Osredkar, J. , and Sustar N.. 2011. “Copper and Zinc, Biological Role and Significance of Copper/Zinc Imbalance.” Journal of Clinical Toxicology S3: 1–18. [Google Scholar]
  30. Pan, F. , Han L., Zhang Y., Yu Y., and Liu J.. 2015. “Optimization of Caco‐2 and HT29 Co‐Culture In Vitro Cell Models for Permeability Studies.” International Journal of Food Sciences and Nutrition 66, no. 6: 680–685. [DOI] [PubMed] [Google Scholar]
  31. Premanathan, M. , Karthikeyan K., Jeyasubramanian K., and Manivannan G.. 2011. “Selective Toxicity of ZnO Nanoparticles Toward Gram‐Positive Bacteria and Cancer Cells by Apoptosis Through Lipid Peroxidation.” Nanomedicine: Nanotechnology, Biology and Medicine 7: 184–192. [DOI] [PubMed] [Google Scholar]
  32. Rana, U. , Kothinti R., Meeusen J., Tabatabai N. M., Krezoski S., and Petering D. H.. 2008. “Zinc Binding Ligands and Cellular Zinc Trafficking: Apo‐Metallothionein, Glutathione, TPEN, Proteomic Zinc, and Zn‐Sp1.” Journal of Inorganic Biochemistry 102, no. 3: 489–499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rojas, J. , Ardila‐Rodríguez L., Diniz M. F., Gonçalves M., Ribeiro B., and Rezende M. C.. 2019. “Optimization of Triton X‐100 Removal and Ultrasound Probe Parameters in the Preparation of Multiwalled Carbon Nanotube Buckypaper.” Materials & Design 166: 107612. [Google Scholar]
  34. Salinas‐Lucero, G. , Juarez‐Moreno K., and Vazquez‐Duhalt R.. 2024. “Cloud Point Extraction and Characterization of Zinc Oxide Nanoparticles Isolated From Infant Milk Formulas.” Journal of Food Measurement and Characterization 18, no. 11: 9330–9340. [Google Scholar]
  35. Sambuy, Y. , De Angelis I., Ranaldi G., Scarino M. L., Stammati A., and Zucco F.. 2005. “The Caco‐2 Cell Line as a Model of the Intestinal Barrier: Influence of Cell and Culture‐Related Factors on Caco‐2 Cell Functional Characteristics.” Cell Biology and Toxicology 21, no. 1: 1–26. [DOI] [PubMed] [Google Scholar]
  36. Saraswathi, M. , Claude A., Devi K. N., and Sevvanthi P.. 2012. “Growth of Zinc Oxide Crystals by Accelerated Evaporation Technique From Supersaturated Solutions.” Growth 4, no. 4: 1343–1349. [Google Scholar]
  37. Setyawati, M. I. , Tay C. Y., and Leong D. T.. 2015. “Mechanistic Investigation of the Biological Effects of SiO2, TiO2, and ZnO Nanoparticles on Intestinal Cells.” Small 11, no. 28: 3458–3468. [DOI] [PubMed] [Google Scholar]
  38. Sirelkhatim, A. , Mahmud S., Seeni A., and Kaus N. H. M.. 2016. “Preferential Cytotoxicity of ZnO Nanoparticle Towards Cervical Cancer Cells Induced by ROS‐Mediated Apoptosis and Cell Cycle Arrest for Cancer Therapy.” Journal of Nanoparticle Research 18, no. 8: 219. [Google Scholar]
  39. Valko, M. , Morris H., and Cronin M.. 2005. “Metals, Toxicity and Oxidative Stress.” Current Medicinal Chemistry 12, no. 10: 1161–1208. [DOI] [PubMed] [Google Scholar]
  40. Vandebriel, R. J. , and De Jong W. H.. 2012. “A Review of Mammalian Toxicity of ZnO Nanoparticles.” Nanotechnology, Science and Applications 5: 61–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang, C. , Hu X., Gao Y., and Ji Y.. 2015. “ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in Ltep‐a‐2 Cells.” BioMed Research International 2015, no. 1: 423287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wingett, D. , Louka P., Anders C. B., Zhang J., and Punnoose A.. 2016. “A Role of ZnO Nanoparticle Electrostatic Properties in Cancer Cell Cytotoxicity.” Nanotechnology, Science and Applications 9: 29–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Xia, T. , Kovochich M., Liong M., et al. 2008. “Comparison of the Mechanism of Toxicity of Zinc Oxide and Cerium Oxide Nanoparticles Based on Dissolution and Oxidative Stress Properties.” ACS Nano 2, no. 10: 2121–2134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yang, Y. , Zhang C., and Hu Z.. 2013. “Impact of Metallic and Metal Oxide Nanoparticles on Wastewater Treatment and Anaerobic Digestion.” Environmental Science: Processes & Impacts 15: 39–48. [DOI] [PubMed] [Google Scholar]
  45. Zeng, X. , Wang Z., Zhao A., et al. 2025. “Zinc Nanoparticles From Oral Supplements Accumulate in Renal Tumours and Stimulate Antitumour Immune Responses.” Nature Materials 24, no. 2: 287–296. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Journal of Applied Toxicology are provided here courtesy of Wiley

RESOURCES