Precision PEGylation of ZnO quantum dots enables selective intracellular killing of Uropathogenic E. coli via multimodal antibacterial mechanisms without inducing resistance

Abstract

The intracellular persistence and biofilm-forming capacity of uropathogenic Escherichia coli (UPEC) are major contributors to urinary tract infections (UTIs) recurrence and antibiotic failure. Here, we report a precisely engineered nanotherapeutic based on ZnO quantum dots (QDs) surface-functionalized with low-molecular-weight polyethylene glycol (PEG200), designed to enhance biocompatibility while preserving potent antibacterial activity. The optimized ZnO@PEG200 QDs exhibited excellent aqueous dispersibility, minimal cytotoxicity, and broad-spectrum efficacy against both drug-sensitive and multidrug-resistant Escherichia coli strains. Mechanistic studies revealed that the QDs exerted multimodal bactericidal effects, including Zn²⁺ ion release, membrane destabilization, intracellular reactive oxygen species (ROS) generation, genomic DNA fragmentation, and transcriptional repression of key virulence genes such as papG, FimH, and FliC. Notably, ZnO@PEG200 QDs disrupted bacterial motility and eradicated established biofilms even at sub-inhibitory concentrations. Long-term passaging assays demonstrated that sub-MIC exposure to ZnO@PEG200 QDs did not induce resistance development. In vivo, the QDs preferentially accumulated in the bladder and kidneys, significantly reduced intracellular bacterial burden, suppressed inflammatory cytokine expression, and promoted tissue repair in a murine UTIs model. Collectively, this work establishes ZnO@PEG200 QDs as a safe and effective nanoplatform for precision antimicrobial therapy, offering a resistance-free strategy for the treatment of intracellular and biofilm-associated bacterial infections.

Graphical Abstract

1. Introduction

Intracellular bacterial pathogens have evolved sophisticated mechanisms to evade host immune responses and resist conventional antimicrobial therapies, resulting in chronic and recurrent infections that pose significant clinical and public health challenges (Pizarro-Cerdá and Cossart, 2006; Bhavsar et al., 2007; Diacovich and Gorvel, 2010). Among them, Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of urinary tract infections (UTIs), accounting for nearly 80 % of community-acquired cases worldwide (Dielubanza and Schaeffer, 2011; Zyczynski et al., 2014; Liu et al., 2017). A hallmark of UPEC pathogenesis is its ability to invade bladder epithelial cells and establish intracellular bacterial communities (IBCs), which are protected from immune clearance and largely inaccessible to standard antibiotics (Abed and Couvreur, 2014; Cao et al., 2024). This intracellular reservoir not only contributes to treatment failure and frequent relapse, but also facilitates the emergence and dissemination of multidrug-resistant (MDR) strains (Mendez-Pfeiffer et al., 2024; Kamaruzzaman et al., 2017).

Effective management of intracellular UPEC infections necessitates the development of antimicrobial agents capable of penetrating host cells, selectively targeting internalized pathogens, and minimizing off-target cytotoxic effects. However, most conventional antibiotics exhibit limited intracellular bioavailability and rapidly drive resistance selection (Middendorf et al., 2001; Foxman, 2010; Aslam et al., 2020; Sarshar et al., 2022). In this context, nanoscale antimicrobial platforms have emerged as promising alternatives due to their tunable physicochemical properties, capacity for cellular entry, and potential for multimodal bactericidal activity (Rabiee et al., 2022; Akhavan, 2009; Gao et al., 2018; Rodríguez-González et al., 2020; Ebrahimi et al., 2022).

Zinc oxide quantum dots (ZnO QDs) have attracted particular attention owing to their broad-spectrum antimicrobial efficacy, high structural stability, and ability to generate reactive oxygen species (ROS), which facilitate bacterial membrane disruption and nucleic acid damage (Rosi and Mirkin, 2005; Akhavan et al., 2009; Lee et al., 2014; Wang et al., 2021; Sá et al., 2022; Amidani et al., 2025; Du et al., 2025). Their ultrasmall size and positively charged surfaces further enhance interactions with negatively charged bacterial membranes and promote cellular uptake. Despite these advantages, the clinical translation of ZnO QDs remains limited by their inherent cytotoxicity, primarily arising from uncontrolled ROS generation and colloidal instability under physiological conditions (Singh and Nalwa, 2007; Condello et al., 2016; Lu et al., 2019).

Surface functionalization strategies have been explored to mitigate the cytotoxic effects of ZnO QDs and improve their biocompatibility. Among them, polyethylene glycol (PEG) is a widely employed hydrophilic polymer that improves colloidal stability, enhances water dispersibility, and reduces nonspecific cellular interactions (Li et al., 2023; Li et al., 2024). While PEGylation has been shown to attenuate the toxicity of various nanomaterials, its impact on the delicate balance between cytocompatibility and antimicrobial efficacy—particularly in the context of intracellular bacterial eradication—remains insufficiently understood (Mishra et al., 2016; Oggero et al., 2023). Moreover, the influence of PEG molecular weight and surface density on therapeutic performance has not been systematically investigated.

In this study, we report the precision surface modification of ZnO QDs with low-molecular-weight PEG (PEG200) to engineer a nanoplatform with optimized intracellular antibacterial performance and minimal cytotoxicity. By fine-tuning the surface interface through PEGylation, we retained the intrinsic antimicrobial properties of ZnO QDs while significantly reducing their adverse effects on host cells. This rational design strategy enabled the selective and effective eradication of intracellular UPEC in both in vitro and in vivo models, without inducing bacterial resistance. Our findings underscore the potential of precision PEGylation as a versatile approach to enhance the biosafety–bioactivity profile of inorganic nanomaterials for intracellular infection therapy.

2. Experimental methods

2.1. Materials

Zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O, 99.9 %), potassium hydroxide (KOH, 95 %), polyethylene glycol (PEG200, 99 %), 8-anilino-1-naphthalenesulfonate (ANS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and glutaraldehyde (25 % in H₂O) were purchased from Macklin (China). Anhydrous ethanol (99.7 %) and sodium chloride (99.5 %) were obtained from Xilong Scientific (China). Yeast extract and tryptone were purchased from Oxoid (UK), and phosphate-buffered saline (PBS, pH 7.4) from Coolaber (China). Dihydroethidium was supplied by Beyotime (China), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from MedChemExpress (China). The Bacterial DNA Genome Kit was obtained from Sangon Biotech (China). All chemicals were of analytical grade and used without further purification. The human bladder epithelial cell line (5637) was provided by Pricella (China).

2.2. Synthesis and characterization of ZnO@PEG200 QDs

ZnO@PEG200 QDs were synthesized via a modified sol-gel method (Huang et al., 2015). First, 10.0 mmol NaOH was dissolved in 100.0 mL of absolute ethanol at 50 °C under constant stirring for 20 min. Concurrently, 10.0 mmol of zinc acetate dihydrate was dissolved in 100.0 mL of ethanol at 65 °C and stirred for 20 min to form the zinc precursor solution. Upon cooling to 50 °C, PEG200 was added to the zinc precursor at final concentrations of 5, 10, 20, 40, and 80 μM, followed by stirring for an additional 10 min. The NaOH solution was then rapidly added to the PEG-containing zinc solution under vigorous stirring for 30 s. Different stirring durations were tested to facilitate nanoparticle formation.

Subsequently, 200 mL of distilled water was added to terminate the reaction, and the resulting mixture was centrifuged at 8000 rpm for 10 min. The supernatant was discarded, and the precipitate was washed twice with ethanol to remove residual reactants. The purified ZnO@PEG200 QDs were dried under vacuum at 60 °C for 48 h.

For aqueous dispersibility testing, ZnO@PEG200 QDs were dispersed in ultrapure water at a concentration of 1.25 mg/mL. The dispersions were examined under UV light and analyzed using a UV–Vis spectrophotometer (Thermo Fisher Scientific, USA). Unmodified ZnO QDs were prepared as control samples.

Characterization of QDs included transmission electron microscopy (TEM; JEOL JEM-2100F, Japan) for morphological observation, X-ray diffraction (XRD; Bruker D8 Advance, Germany) for phase structure, X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific, USA) for surface elemental composition and oxidation state, Fourier-transform infrared spectroscopy (FTIR; IRTracer-100, Shimadzu, Japan) for surface functional group analysis and thermogravimetric analysis (TGA; NETZSCH-STA 2500, Germany) for PEG200 surface coverage density.

2.3. Cytotoxicity evaluation by MTT assay

Cytotoxicity of ZnO@PEG200 QDs was assessed using the MTT assay. 5637 cells RAW264.7 and HK-2 were seeded in sterile 96-well plates at a density of 1 × 10^4 cells/well and allowed to adhere for 24 h at 37 °C in a humidified incubator with 5 % CO₂. Cells were treated with various concentrations of ZnO@PEG200 QDs or unmodified ZnO QDs and incubated for 24 h and 48 h. Subsequently, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well and incubated for an additional 4 h. The medium was then removed, and formazan crystals were solubilized in 150 μL of DMSO. Absorbance was measured at 490 nm using a microplate reader (BioTek, USA). Cell viability was calculated as a percentage of the control.

2.4. Antimicrobial activity evaluation

2.4.1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The MIC and MBC of ZnO@PEG200 QDs against E. coli CFT073 were determined using a standard broth microdilution method. A bacterial suspension of 2 × 10^5 CFU/mL in Mueller-Hinton broth was added to each well of a sterile 96-well plate (100 μL/well). ZnO@PEG200 QDs were added to achieve final concentrations ranging from 8 to 1024 μg/mL. After incubation at 37 °C for 16–18 h, MIC was defined as the lowest concentration that inhibited visible bacterial growth. For MBC, 100 μL from wells with no visible growth was plated onto MacConkey agar and incubated for 18–24 h at 37 °C. The MBC was defined as the lowest concentration yielding no bacterial colonies.

2.4.2. Time-kill kinetics

To assess time-dependent bactericidal activity, bacterial suspensions (1 × 10^5 CFU/mL) were treated with various concentrations of ZnO@PEG200 QDs and incubated at 37 °C with shaking (180 rpm). Samples were collected every 2 h over a 24 h period, and bacterial growth was quantified by measuring optical density at 600 nm (OD600). All experiments were performed in triplicate.

2.4.3. Biofilm eradication assay

Biofilms were formed in 24-well plates by inoculating 2 mL of tryptic soy broth (TSB) with 1 % (v/v) of an overnight culture of E. coli CFT073 (∼10^8 CFU/mL) and incubating at 37 °C for 24 h. After biofilm formation, wells were gently washed three times with sterile 0.9 % NaCl to remove planktonic cells. ZnO@PEG200 QDs were prepared in TSB at various concentrations (8–1024 μg/mL) and added to the wells. Plates were incubated for 12, 24, or 48 h. After treatment, wells were washed three times with saline, fixed with 95 % ethanol for 1 h, and air-dried. Biofilms were stained with 0.1 % (w/v) crystal violet for 10 min, washed, and solubilized with absolute ethanol. Absorbance was measured at 595 nm using a microplate reader (Thermo Fisher Scientific MK3).

2.4.4. Bacterial motility assay

Bacterial motility was evaluated using swimming and swarming assays. For swimming, semi-solid LB agar plates (0.3 % and 0.5 % agar) supplemented with ZnO@PEG200 QDs at ½ MIC, MIC, and 2× MIC were inoculated with 10 μL of mid-log phase E. coli CFT073 (OD600 = 0.8) at the center and incubated for 24 h at 37 °C. For swarming, LB agar containing 0.1 % agar and the same QD concentrations was used. Bacteria were inoculated at the interface using a sterile loop. Colony diameters were measured using digital calipers (Mitutoyo, Japan), and motility inhibition was calculated as: Inhibition (%) = [(Dcontrol − Dtreated)/Dcontrol] × 100 %.

2.4.5. Morphological characterization

To visualize the effect of QDs on bacterial flagella, E. coli CFT073 was cultured on LB agar plates containing 0, 64, or 128 μg/mL of ZnO@PEG200 QDs at 37 °C. After incubation, cells were subjected to negative staining and analyzed via E-test imaging system (manufacturer, China).

2.5. Mechanistic studies of antibacterial action

2.5.1. Zeta potential measurement

Zeta potentials of ZnO@PEG200 QDs and E. coli CFT073 were measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) to evaluate surface charge interactions.

2.5.2. Zn²⁺ Ion release assay

ZnO@PEG200 quantum dots (QDs) were dispersed in 10 mL of MHB medium (pH = 7.4) to achieve a final concentration of 64 μg/mL. The dispersion was protected from light and incubated on a shaker at 37 °C with a rotation speed of 200 rpm for 12 h. At 0, 2, 4, 6, 8, 10, and 12 h, the supernatant was collected separately, centrifuged at 12,000 rpm for 5 min, and then filtered through a 0.22 μm filter. The concentration of Zn²⁺ ions in the filtrate was determined using inductively coupled plasma mass spectrometry (ICP-MS).

2.5.3. Membrane integrity assessment

Membrane permeability alterations were assessed using ANS fluorescence. Bacterial suspensions (10^8 CFU/mL) were washed thrice with PBS and treated with ZnO@PEG200 QDs at ½ MIC, MIC, and 2× MIC. After 12 h incubation at 37 °C, ANS was added to a final concentration of 4 μM and incubated for 15 min. Fluorescence intensity was measured at excitation/emission wavelengths of 385/473 nm using a microplate reader (MK3, Thermo Fisher).

2.5.4. DNA damage detection

Log-phase bacterial cultures (10^8 CFU/mL) were treated with ZnO@PEG200 QDs at ½ MIC, MIC, and 2× MIC for 12 h at 37 °C. Genomic DNA was extracted using the Bacterial DNA Genome Kit according to the manufacturer's protocol.

2.5.5. Intracellular ROS generation

Bacterial cells (∼10^8 CFU/mL) were incubated with ZnO@PEG200 QDs at 37 °C for 12 h. After washing, cells were stained with 10 μM DCFH-DA for 1 h at 37 °C in the dark. Fluorescence was recorded at excitation/emission wavelengths of 488/525 nm.

2.5.6. Superoxide anion quantification

Intracellular O₂⁻ levels were quantified using hydroxylamine-O-methanol. After treatment with QDs, bacterial suspensions were centrifuged and incubated with the probe at 37 °C for 30 min. The supernatant was collected, and fluorescence intensity was measured at 610 nm.

2.5.7. Resistance induction assay

To evaluate resistance development, E. coli CFT073 was cultured under continuous sub-MIC exposure to ZnO@PEG200 QDs or levofloxacin (as control). After each 24 h passage, MIC was re-evaluated and plotted across generations to assess resistance trends.

2.5.8. Quantitative PCR of virulence genes

Bacteria were cultured with ZnO@PEG200 QDs at MIC concentrations. Total RNA was extracted, reverse-transcribed into cDNA, and real-time PCR was performed using SYBR Green reagents in 20 μL reaction volumes. Primer sequences are listed in Supplementary Table S1.

2.6. In vitro and in vivo therapeutic evaluation

2.6.1. Cellular infection model

5637 cells were infected with E. coli CFT073 for 2 h, followed by gentamicin treatment to eliminate extracellular bacteria. Cells were then treated with ZnO@PEG200 QDs or levofloxacin for 12 h. Intracellular bacteria were quantified by lysing the cells and plating on MacConkey agar.

2.6.2. Murine urinary tract infection model

Female C57BL/6 J mice (6–8 weeks old) were anesthetized and transurethrally inoculated with 50 μL of E. coli CFT073 suspension (1 × 10^8 CFU/mL). At 24 h post-infection, mice were treated via intraperitoneal injection of ZnO@PEG200 QDs or control agents daily for 3 days. Urine samples were collected to quantify bacterial loads. Kidneys and bladders were harvested for histological evaluation (H&E and Masson's trichrome staining). Inflammatory cytokine levels (IL-1β and IL-6) in tissue homogenates were measured using ELISA kits (Elabscience, China) according to the manufacturer's protocol.

2.7. Statistical analysis

Statistical analysis was performed using GraphPad Prism 10 software (USA). All experiments were replicated three times, and a one-way ANOVA or Mann–Whitney test was employed to identify significant differences between samples. The results were presented as mean ± standard deviation. *p < 0.05 was considered statistically significant.

3. Results

3.1. Synthesis, characterization, and PEGylation optimization of ZnO@PEG200 QDs

ZnO@PEG200 QDs were firstly synthesized via a modified sol–gel method in ethanol. The resulting QDs exhibited excellent dispersibility in ethanol and emitted bright yellow-green fluorescence under 365 nm ultraviolet illumination(Fig. S1 A, B), confirming their size-dependent quantum confinement behavior (Zrazhevskiy et al., 2010).

Morphological characterization via TEM revealed a uniform quasi-spherical nanostructure with an average particle diameter of 5 nm (Fig. 1A, B), which was consistent with dynamic light scattering (DLS) measurements showing a hydrodynamic diameter of approximately 5 nm (Fig. S1C). High-resolution TEM further confirmed the characteristic lattice spacing of 245.5 pm (Fig. S1 D), indicative of the wurtzite phase of ZnO QDs. Elemental composition analysis using energy-dispersive X-ray spectroscopy (EDS) validated the presence of Zn and O, consistent with the expected stoichiometry, along with surface carbon signals attributable to PEG200 coating and residual solvent, as well as the underlying carbon film from the TEM grid (Fig. S1E—I).

Fig. 1.

Characterization and Biosafety Assessment of ZnO@PEG200 QDs.

(A) Particle size analysis by transmission electron microscopy (TEM). (B) Average Particle Size Histogram. (C) Water dispersibility comparison between unmodified and PEGylated ZnO QDs, measured by UV–Vis transmittance. (D) X-ray diffraction (XRD). (E) Fourier-transform infrared spectroscopy (FTIR). (F) X-ray photoelectron spectroscopy (XPS). (G) thermogravimetric analysis (TGA). (H) Cell viability of 5637 human bladder epithelial cells after 24 h and 48 h exposure to ZnO@PEG200 QDs, assessed by MTT assay. (I) PEG200-dependent antimicrobial and safety profiles of ZnO QDs.

To enhance aqueous dispersibility and biocompatibility, the surface of hydrophobic ZnO QDs was modified using polyethylene glycol with a molecular weight of 200 Da (PEG200), a widely used amphiphilic surfactant (Oggero et al., 2023). Comparative dispersibility analysis demonstrated a marked increase in water dispersibility following PEGylation: PEG-modified ZnO QDs (1.25 mg/mL) displayed 86 % UV–visible transmittance, in contrast to only 6 % for unmodified QDs (Fig. 1C). This significant improvement indicates successful surface hydrophilization and colloidal stabilization through PEG200 grafting.

X-ray diffraction (XRD) analysis demonstrated that PEGylation did not alter the intrinsic crystallinity of ZnO QDs, as evidenced by diffraction peaks corresponding to the standard wurtzite ZnO structure (JCPDS #00–036-145) (Fig. 1D). Surface chemical functionalities were identified by Fourier-transform infrared spectroscopy (FTIR), which exhibited prominent peaks for PEG200, including O—H (3370.09 cm⁻¹), C—H (1586.89 cm⁻¹), and C–O–C stretching vibrations (1420.15 cm⁻¹) (Fig. 1E). Complementary X-ray photoelectron spectroscopy (XPS) confirmed successful PEG200 modified: Zn 2p peaks were located at 1020.3 eV and 1043.4 eV (Fig. 1F); C 1 s deconvolution revealed peaks at 284.8, 286.05, and 288.2 eV, corresponding to C—C, C—O, and C O bonds, respectively (Fig. S2A); and O 1 s spectra further supported the coexistence of Zn—O and C O species on the surface (Fig. S2B), aligning with previous reports (Mishra et al., 2016; Pranjali et al., 2019). However, the presence of C O may indicate the existence of acetate ions on the surface of ZnO@PEG200 QDs.

Thermogravimetric analysis (TGA) was used for the quantitative evaluation of the PEG200 grafting ratio on the surface of ZnO@PEG200 QDs, as shown in Fig. 1G. The mass loss percentages of ZnO QDs and ZnO@PEG200 QDs were 1.96 % and 32.95 %, respectively. ZnO QDs exhibited a mass loss of approximately 1.87 % between 200 and 400 °C, primarily due to the removal of physisorbed water and trace impurities from the synthesis process. Above 400 °C and up to 800 °C, no additional significant mass loss was observed, thereby confirming the excellent thermal stability and intact crystalline structure of the ZnO lattice. In contrast, the ZnO@PEG200 QDs demonstrated a total mass loss of 30.15 %, occurring in two distinct phases: an initial 3 % loss from 100 to 200 °C, which can be attributed to the evaporation of surface-adsorbed water and volatile components of PEG200, followed by a major loss of 25 % between 200 and 400 °C, resulting from the thermal degradation of the PEG200 polymer backbone. After reaching 600 °C, only the inorganic ZnO core remained, indicating the complete decomposition of the organic coating.

Cell viability assays (MTT) confirmed the low cytotoxic profile of ZnO@PEG200 QDs at this optimal PEGylation level. After treatment with 128 μg/mL QDs, cell viability of 5637 cell, RAW246.7 and HK-2 remained high above 90 % at 24 h and 48 h (Fig. S3,). In contrast, unmodified ZnO QDs significantly compromised viability even at a lower concentration (32 μg/mL), reducing cell survival to 50 % and 30 % at the same time points. These findings highlight the critical role of PEGylation in modulating QD biocompatibility and mitigating dose-dependent cytotoxicity (Fig. 1H).

To optimize biological performance, we systematically evaluated the influence of varying PEG200 concentrations (5–80 μM) on antibacterial activity and cytocompatibility. Notably, QDs modified with 20 μM PEG200 exhibited the highest antibacterial efficacy against E. coli and Staphylococcus aureus, while concurrently demonstrating minimal cytotoxicity toward 5637 cells (Fig. 1I). This concentration likely provided optimal surface coverage, promoting bacterial membrane interaction while minimizing nonspecific cellular uptake.

3.2. ZnO@PEG200 QDs exhibit Potent Antibacterial activity against Uropathogenic and Multidrug-Resistant E. coli

To systematically evaluate the antibacterial efficacy of ZnO@PEG200 QDs, we investigated their inhibitory effects against both the reference uropathogenic E coli strain CFT073 and a collection of clinically isolated multidrug-resistant (MDR) E. coli strains under dark conditions. Broth microdilution assays revealed that both PEGylated and unmodified ZnO QDs shared a MIC of 64 μg/mL against CFT073 (Fig. 2A), indicating that PEG200 surface functionalization did not impair the intrinsic antibacterial activity of the ZnO QDs nanomaterials. Complete eradication of bacterial populations was observed at the MBC of 256 μg/mL (Fig. 2B). Importantly, control treatments with PEG200 alone showed no detectable antibacterial activity at equivalent concentrations, thereby confirming that the observed antimicrobial effects were solely attributable to the ZnO QD component.

Fig. 2.

Antimicrobial Activity of ZnO@PEG200 QDs Against Uropathogenic E. coli CFT073.

(A) Minimum inhibitory concentration (MIC) determination. (B) Minimum bactericidal concentration (MBC) determination. (C) Concentration-dependent bacterial inhibition rate after 24 h treatment. (D) Time-kill kinetics showing growth suppression over 24 h, monitored by OD600 measurements.

Time-kill kinetic assays further confirmed a strong concentration-dependent bactericidal effect. ZnO@PEG200 QDs achieved >97 % reduction in viable bacterial counts within 24 h across a broad concentration range (64–1024 μg/mL) (Fig. 2C). Complementary optical density measurements at 600 nm (OD600), recorded every two hours, demonstrated a sustained and time-dependent inhibition of bacterial growth (Fig. 2D), however, the sudden drop in OD value at the 12-h time point is most likely attributed to operational issues during the shaking culture in 96-well plates, as evidenced by the recovery trend of OD value at the 14-h time point. In addition to the reference strain, ZnO@PEG200 QDs exhibited marked antibacterial activity against a panel of clinically isolated MDR E. coli strains (Table S1), highlighting their broad-spectrum efficacy and potential applicability against drug-resistant infections. These results are consistent with the well-documented antimicrobial mechanisms of ZnO nanomaterials. (Akhavan et al., 2009; Gudkov et al., 2021). Notably, PEGylation did not compromise these functional bactericidal properties and may further enhance the biocompatibility and colloidal stability of the nanomaterial system.

3.3. ZnO@PEG200 QDs Suppress Bacterial Motility and Disrupt established Biofilms

The antibiofilm capabilities of ZnO@PEG200 QDs were evaluated against mature E. coli CFT073 biofilms, which are notoriously resistant to both antibiotics and host immune defenses due to their protective extracellular polymeric substance (EPS) matrix (Jiang et al., 2024; Guo et al., 2023; Gebreyohannes et al., 2019). Under dark conditions, treatment with 128 μg/mL ZnO@PEG200 QDs for 12 h effectively disrupted preformed biofilms (Fig. 3A). Lower concentrations of 64 μg/mL and 32 μg/mL required 24 and 48 h, respectively, to achieve comparable levels of disruption, indicating a clear time- and dose-dependent antibiofilm effect. Quantitative biofilm assays corroborated these findings, showing progressive biofilm degradation with increased exposure duration.

Fig. 3.

Biofilm Disruption and Motility Inhibition by ZnO@PEG200 QDs.

(A) Time- and concentration-dependent elimination of mature CFT073 biofilms. (B) Overview of motility inhibition. (C-E) Quantitative assessment of: (C) swimming, (D) swarming, and (E) twitching motility. (f–h) Transmission electron microscopy (TEM) images of CFT073 after co-incubation: (F) untreated control (×8.0 k), (G) treated with 64 μg/mL ZnO@PEG200 QDs (×10.0 k), and (H) treated with 128 μg/mL ZnO@PEG200 QDs (×12.0 k). Red arrowheads indicate flagella. Scale bar = 1 μm.

Type 1 fimbriae mediated motility is a critical virulence trait of UPEC (Lane et al., 2007; Rodríguez-Serrano et al., 2020). We investigated the effects of ZnO@PEG200 on UPEC motility and found that ZnO@PEG200 significantly inhibited bacterial motility even at sub-inhibitory concentrations. Quantitative motility assays demonstrated a marked 92.79 % reduction in swimming motility, complete inhibition of swarming, and a modest 2.05 % reduction in twitching motility (Fig. 3B-E). At the MIC level of 64 μg/mL, swarming was inhibited by 41.93 %, underscoring a concentration-dependent suppression of collective locomotion.

Mechanistic insights into motility inhibition were obtained using TEM. Control E. coli CFT073 cells exhibited intact and well-structured flagella (Fig. 3F), while treatment with 64 μg/mL ZnO@PEG200 QDs led to visibly truncated flagellar filaments (Fig. 3G). At 128 μg/mL, flagella were almost entirely absent (Fig. 3H), suggesting that ZnO@PEG200 QDs disrupt the structural integrity of the flagellar assembly apparatus in a dose-dependent manner. These findings imply that ZnO@PEG200 QDs not only interfere with fimbrial-mediated adhesion but also directly compromise bacterial motility machinery, thereby targeting two pivotal virulence determinants of UPEC.

3.4. Antibacterial mechanism of ZnO@PEG200 QDs against E. coli CFT073

To investigate the antibacterial mechanism of ZnO@PEG200 QDs, a series of physicochemical and biological assays were performed to characterize their interactions with uropathogenic E coli CFT073.

Zeta potential measurements indicated a strong electrostatic interaction between the negatively charged bacterial cell surface (−27.57 mV) and the positively charged ZnO@PEG200 QDs (+30.47 mV, pH = 7) (Fig. 4A). This charge difference enabled close contact between nanoparticles and bacterial membranes.

Fig. 4.

Antibacterial mechanisms of ZnO@PEG200 QDs against Uropathogenic E. coli CFT073.

(A) Zeta potential analysis of electrostatic interaction between ZnO@PEG200 QDs and bacterial surfaces. (B) Zn²⁺ concentrations released by ZnO@PEG200 QDs at pH = 7.4 (C) Membrane fluidity disruption assessed by ANS fluorescence. (D) Agarose gel electrophoresis of CFT073 DNA after ZnO@PEG200 QD treatment. (E) DCFH-DA assay for intracellular ROS generation. (F) Dose-dependent O₂⁻ production measured by absorbance. (G) Relative mRNA expression of virulence genes (FimH, FliC, papG, gapA, FimA, motA) in CFT073, normalized to nusA, showing downregulation of adhesion/motility genes.

Under dark conditions, 64 μg/mL ZnO@PEG200 quantum dots (QDs) sustainably released Zn²⁺ ions in Mueller-Hinton Broth (MHB) medium (pH = 7.4), with the Zn²⁺ concentration in the solution reaching 7.5 μg/mL after 12 h (Fig. 4B). This demonstrates that ZnO@PEG200 QDs exert antibacterial effects through a synergistic action of the QDs themselves and the released Zn²⁺ ions, which is consistent with previous studies (Mendes et al., 2022).

Membrane integrity was assessed using 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectroscopy (Singh et al., 2019). After 12 h of treatment, ZnO@PEG200 QDs induced a concentration-dependent increase in ANS fluorescence, with a 1.17-fold elevation at 128 μg/mL compared to untreated controls (Fig. 4C). The increase in ANS binding indicates altered membrane fluidity and disrupted lipid packing.

DNA integrity was analyzed by agarose gel electrophoresis. In untreated controls, intact genomic DNA bands were observed, whereas ZnO@PEG200 QD-treated samples exhibited diffuse smear patterns, indicative of DNA fragmentation (Fig. 4D).

Oxidative stress responses were measured by quantifying both intracellular ROS levels and extracellular superoxide anion production. Under dark conditions, intracellular ROS levels increased by 1.1-fold at 128 μg/mL (Fig. 4E), while extracellular superoxide levels exhibited a concentration-dependent rise, with maximum absorbance at the highest tested concentration (Fig. 4F). These results indicate that ROS-mediated oxidative stress is a key contributor to the bactericidal activity of ZnO@PEG200 QDs, in addition to membrane disruption and DNA damage.

To evaluate transcriptional changes in virulence-associated genes, qRT-PCR analysis was conducted after 24 h of exposure to ZnO@PEG200 QDs. Using nusA as a reference gene, significant downregulation was observed in FimH, FliC, papG, and gapA (Fig. 4G). Conversely, modest upregulation was observed for FimA and motA, likely reflecting a compensatory response to maintain basal levels of motility and adhesion under stress conditions.

These results indicate that ZnO@PEG200 QDs exert antibacterial effects through multiple mechanisms, including electrostatic interaction, zinc ions released, and membrane destabilization, induction of intracellular DNA fragmentation, generation of oxidative stress, and transcriptional suppression of several virulence-associated genes.

3.5. Long-term exposure to ZnO@PEG200 QDs does not induce antimicrobial resistance

To evaluate whether long-term exposure to ZnO@PEG200 QDs induces antimicrobial resistance, a 30-day serial passaging experiment was conducted using E. coli CFT073 under sub-inhibitory conditions (½ MIC), as Fig. 5A. For comparison, levofloxacin was included as a positive control. Bacterial cultures were passaged daily, and MICs were recorded at defined intervals (denoted as Gn for the nth generation).

Fig. 5.

Evaluation of antimicrobial resistance development under long-term exposure to ZnO@PEG200 QDs.

(A) Schematic diagram of the 30-day continuous sub-MIC exposure protocol. (B) MIC values of E. coli CFT073 across serial generations (G1–G30) following treatment with ZnO@PEG200 QDs or levofloxacin. (C) MIC fold-change rates showing resistance progression trends over time. (D) Comparison of cumulative MIC increases over 30 days, highlighting rapid resistance development in levofloxacin-treated strains and stability in QD-treated strains.

Throughout the experimental period, the MIC of ZnO@PEG200 QDs against E. coli CFT073 remained stable at 64 μg/mL from generation 5 through generation 30 (Fig. 5B). A transient increase to 128 μg/mL was observed at generation 20, but this elevation was reversed by generation 23, and the MIC returned to its original level. In contrast, exposure to levofloxacin resulted in a rapid increase in MIC, with a fivefold elevation observed by generation 5 and a continued upward trend in subsequent generations.

Quantitative analysis of MIC progression revealed that the resistance index for levofloxacin increased steadily over time, whereas ZnO@PEG200 QDs maintained a flat resistance profile across the 30-day period (Fig. 5C). A minor fluctuation in MIC was noted between generations 20–22 under ZnO@PEG200 QD exposure but resolved without intervention in later passages.

Phenotypic resistance assessments further corroborated the MIC data. Levofloxacin-treated strains exhibited a 30-fold increase in MIC by day 30, while ZnO@PEG200 QD-treated strains consistently maintained full sensitivity, with no shift from the baseline MIC of 64 μg/mL (Fig. 5D).

Taken together, these results demonstrate that prolonged, sub-inhibitory exposure to ZnO@PEG200 QDs does not induce antimicrobial resistance in E. coli CFT073 under the tested conditions. In contrast, exposure to a conventional antibiotic under similar conditions led to rapid and sustained resistance development. This distinction highlights the stability of bacterial susceptibility to ZnO@PEG200 QDs during long-term exposure.

3.6. Biosafety of ZnO@PEG200 QDs

In hemocompatibility evaluations, ZnO@PEG200 QDs induced less than 5 % hemolysis in murine erythrocytes at concentrations up to 1024 μg/mL, well within the acceptable threshold defined by ISO/TR 7405–2016 standards (Fig. 6A). Furthermore, in vivo toxicity assessments via intravenous injection in C57BL/6 J mice revealed a well-defined safety window. The no-observed-adverse-effect level (NOAEL) was established at 0.5 mg/mL, with higher doses (5 mg/mL and above) leading to dose-dependent renal pathology, and 50 mg/mL resulting in acute lethality (100 % mortality within 24 h) (Fig. 6B). Histopathological analysis of major organs at the NOAEL confirmed preserved tissue architecture (Fig. 6C), supporting the potential of ZnO@PEG200 QDs for safe systemic administration. Notably, renal-specific toxicity at higher doses aligns with known nanoparticle clearance routes (Cui et al., 2019; Li et al., 2020) while the absence of significant hepatic or splenic damage suggests a favorable biodistribution profile (Lu et al., 2019).

Fig. 6.

Biosafety assessment.

(A) Hemolysis of mouse red blood cells at different concentrations of ZnO@PEG200 QDs, (B) Survival rate of mice at different concentrations of ZnO@PEG200 QDs, (C) H&E-stained sections of various organs from mice treated with different concentrations of ZnO@PEG200 QDs, ×20.00. Red arrowheads indicate deep pigmented granular deposits. Bar = 100 μm.

3.7. ZnO@PEG200 QDs effectively treat Uropathogenic E. coli-induced urinary tract infections

To evaluate the therapeutic efficacy of ZnO@PEG200 QDs in treating intracellular infections caused by UPEC, a time-course infection assay was performed. The antibacterial effects of ZnO@PEG200 QDs at concentrations of 128 μg/mL and 64 μg/mL were compared with those of the clinical antibiotic levofloxacin at 50 μg/mL and 25 μg/mL, respectively (Fig. S4A, Fig. 7A). At 6 h post-treatment, all groups showed reductions in intracellular bacterial loads without significant differences among treatment conditions. By 12 h, treatment with 128 μg/mL ZnO@PEG200 QDs resulted in a statistically significant decrease in intracellular bacterial burden compared to the untreated control group, while the 64 μg/mL dose did not show a significant effect. Both levofloxacin treatment groups demonstrated marked reductions in bacterial counts over the same period. At 24 h, the antibacterial activity of ZnO@PEG200 QDs at 128 μg/mL was maintained, with continued suppression of intracellular bacterial viability

Fig. 7.

Therapeutic efficacy of ZnO@PEG200 QDs against UPEC-induced urinary tract infections.

(A) Quantification of intracellular E. coli in infected epithelial cells following treatment with ZnO@PEG200 QDs or levofloxacin at different time points. (B—C) Bacterial counts in urine samples collected at designated time points. (D) Schematic diagram of treatment for mouse urinary tract infection model. (E-F) Bacterial burdens in bladder and kidney tissues, respectively. (G-H) Expression levels of inflammatory cytokines IL-1β and IL-6 in bladder tissue, assessed by ELISA. (I) Representative histological images of bladder and kidney tissues stained with H&E and Masson's trichrome.

To treat a mouse model of urinary tract infection (UTI), ZnO@PEG200 QDs and levofloxacin antibiotic were administered via tail vein injection for 7 consecutive days. Compared with the untreated group, both ZnO@PEG200 QDs and levofloxacin treatment groups showed significantly reduced bacterial counts in urine. By day 11, the levofloxacin group had almost no bacterial colonies in urine, while the ZnO@PEG200 QDs group still showed a significant reduction in bacterial load compared to the untreated group (Fig. 7B, C). As shown in Fig. 7B and C, the bacterial burden in the bladder and kidneys of mice was also significantly lower than that in the untreated group after treatment. Additionally, no significant differences were observed in the organ indices of the bladder and kidneys between the treatment groups and the healthy control group (Fig. S5A, B, and C). These results demonstrate that ZnO@PEG200 QDs have excellent therapeutic effects on UTI in mice.

To assess the inflammatory response associated with UTIs, levels of pro-inflammatory cytokines IL-1β and IL-6 were measured in bladder tissue samples. In the infected, untreated group, IL-1β expression was significantly elevated compared to healthy controls. In contrast, IL-1β levels in both the ZnO@PEG200 QD- and levofloxacin-treated groups were comparable to those in uninfected mice (Fig. 7G). IL-6 expression in the ZnO@PEG200 QDs group was moderately elevated compared to the healthy control but significantly lower than in the infected, untreated group. Levofloxacin treatment fully normalized IL-6 expression to baseline levels (Fig. 7H).

Histopathological evaluation of bladder and kidney tissues was conducted using hematoxylin and eosin (H&E) and Masson's trichrome staining (Fig. 7I). In the untreated infected group, bladder tissue exhibited smooth muscle hypertrophy, epithelial disorganization, and submucosal edema, while kidney tissue showed tubular dilation, inflammatory cell infiltration, and interstitial fibrosis. These pathological alterations were substantially reduced in both the ZnO@PEG200 QD- and levofloxacin-treated groups. Quantitative analysis of collagen deposition from Masson-stained sections confirmed lower levels of fibrosis in treated animals compared to the untreated control (Fig. S6).

These results demonstrate that ZnO@PEG200 QDs at therapeutic doses effectively reduce intracellular bacterial burden, suppress infection-induced pro-inflammatory cytokine expression, and attenuate histopathological damage in both bladder and kidney tissues. Treatment outcomes were comparable to those observed with levofloxacin under the tested conditions.

3.8. Antimicrobial activity and mechanism of ZnO@PEG200 QDs against UPEC

UPEC, the predominant causative agent of UTIs, is characterized by its capacity to invade uroepithelial cells and form IBCs. To assess the therapeutic potential of ZnO@PEG200 QDs against both planktonic and intracellular forms of UPEC, a series of in vivo and mechanistic evaluations were conducted.

ZnO@PEG200 QDs entered the bladder and kidney via the systemic blood circulation following systemic intravenous administration, which are precisely the primary colonization sites of UPEC.This organ-specific deposition enabled direct action of the QDs at the site of infection.

Mechanistic studies demonstrated that ZnO@PEG200 QDs exert antibacterial effects through multiple distinct modes of action. Against planktonic UPEC, ZnO QDs release zinc ions, which play a partial antibacterial role. Additionally, ZnO QDs can disrupt bacterial membrane fluidity, generated intracellular ROS, and induced genomic DNA fragmentation, resulting in cell lysis (Hassan et al., 2023; Rezaei et al., 2024; Shankar and Rhim, 2019). Within host cells, the QDs were able to penetrate infected epithelial cells and interact with IBCs. The nanoparticles traversed the biofilm-like protective matrix of IBCs and exerted bactericidal activity through quantum-scale tunneling, membrane destabilization, and interference with bacterial motility machinery.

This multimodal mechanism allowed simultaneous targeting of extracellular, surface-adhered, and intracellular bacterial populations. Quantitative assessment of host cytokine responses indicated effective infection control: animals treated with ZnO@PEG200 QDs exhibited IL-1β expression levels similar to those of healthy controls. IL-6 expression in the treatment group was moderately elevated compared to uninfected controls but significantly lower than in untreated infected animals.

Histopathological analysis of bladder and kidney tissues showed notable restoration of tissue structure in the ZnO@PEG200 QD-treated group. H&E staining indicated reduced inflammatory infiltration and tissue disorganization, while Masson's trichrome staining demonstrated reduced collagen deposition, suggesting effective mitigation of UTI-associated fibrosis. These observations were consistent with tissue recovery and decreased chronic inflammatory damage.

Together, the results indicate that ZnO@PEG200 QDs possess dual-targeted antibacterial activity against both extracellular and intracellular UPEC. The combined evidence from biodistribution, molecular mechanism studies, and histological evaluation confirms the capability of ZnO@PEG200 QDs to effectively localize at infection sites, eliminate pathogenic bacteria in multiple niches, and reduce infection-induced tissue pathology (Fig. 8, Scheme. 1).

Fig. 8.

Schematic representation of the in vivo antibacterial activity and mechanism of ZnO@PEG200 quantum dots against Uropathogenic E. coli (UPEC).

Scheme. 1.

Schematic mechanistic illustration of ZnO@PEG200 QDs in the treatment of UITs.

The schematic demonstrates the synthesis of ZnO@PEG200 QDs and their therapeutic mechanisms in vivo, including ROS generation, cell membrane disruption, and DNA damage, thereby effectively inhibiting the persistent pathogenic effects of UPEC in vivo.

4. Discussion

UTIs caused by UPEC present a persistent clinical challenge, particularly due to the emergence of multidrug-resistant strains and the bacteria's ability to evade treatment through intracellular colonization and biofilm formation. Conventional antibiotics are often limited by poor cellular penetration, short half-lives, and the growing threat of resistance (Miao et al., 2024; Pang et al., 2022; Sharma et al., 2021; González et al., 2020). In this context, we developed and evaluated a nanotherapeutic platform based on ZnO QDs surface-modified with low-molecular-weight polyethylene glycol (PEG200), designed to enhance biocompatibility while preserving the intrinsic antimicrobial properties of ZnO.

PEGylation is a well-established strategy to improve nanomaterial stability and reduce host cytotoxicity, yet excessive PEG shielding can diminish antimicrobial activity by hindering bacterial contact (Xu et al., 2023; Runliang et al., 2015; Kobylinska et al., 2018). In this study, we systematically optimized PEG200 surface coverage and identified 20 μM as the optimal modification condition, yielding ZnO@PEG200 QDs with high aqueous dispersibility, minimal cytotoxicity, and preserved antibacterial activity. Compared with unmodified QDs, PEGylated particles showed significantly reduced cytotoxic effects in vitro and in vivo, including <5 % hemolysis and no histopathological abnormalities at the established NOAEL. These results demonstrate that precise PEG200 functionalization effectively improves safety profiles without compromising antibacterial efficacy.

Mechanistic investigations revealed that ZnO@PEG200 QDs exert antibacterial effects through a combination of physical and biochemical pathways. Against planktonic bacteria, QDs adhered to negatively charged bacterial surfaces via electrostatic interactions, disrupting membrane fluidity and integrity, inducing oxidative stress, and promoting DNA fragmentation. This multifaceted mode of action bypasses single-target resistance mechanisms typically exploited by bacteria against conventional antibiotics (Akhavan et al., 2009; Gudkov et al., 2021). Notably, long-term serial passaging under sub-MIC levels of ZnO@PEG200 QDs did not induce any stable resistance phenotype in E. coli CFT073, in contrast to levofloxacin which rapidly selected for resistant strains. C. R. Mendes et al. (Mendes et al., 2022) demonstrated that ZnO exerts synergistic antibacterial effects through multiple mechanisms, including electrostatic adsorption, Zn²⁺ release, reactive oxygen species (ROS) generation, and direct physical damage. It is characterized by rapid action, broad targets, and the predominance of nonspecific membrane disruption—these characteristics make it difficult for bacteria to develop effective drug resistance via single or a few gene mutations. Their findings are consistent with the results of the present study, which also explains why long-term induction with ZnO@PEG200 does not induce drug resistance in UPEC. This finding supports the platform's potential for long-term antimicrobial use without exacerbating resistance pressure (Zhu et al., 2025; Prabhu et al., 2025; Niño-Martínez et al., 2019).

Beyond planktonic forms, ZnO@PEG200 QDs were also shown to penetrate host epithelial cells and disrupt IBCs, a critical niche contributing to UTIs persistence and recurrence. The particles accumulated preferentially in bladder and kidney tissues following systemic administration, enabling targeted delivery to infection sites. In infected mice, ZnO@PEG200 QD treatment significantly reduced intracellular UPEC loads, suppressed IL-1β and IL-6-mediated inflammatory responses, and restored bladder and renal tissue architecture (Cao et al., 2024). These effects were comparable to those of levofloxacin, highlighting the QDs' therapeutic potential in treating intracellular bacterial infections.

Moreover, ZnO@PEG200 QDs significantly impaired UPEC motility and biofilm formation—two additional virulence strategies that facilitate tissue colonization and antibiotic evasion. At sub-inhibitory concentrations, QDs disrupted swimming, swarming, and twitching motility in a dose-dependent manner, accompanied by ultrastructural damage to flagellar filaments. Simultaneously, preformed biofilms were degraded within 12–48 h depending on dose, demonstrating potent antibiofilm activity. Downregulation of key virulence genes (Paniagua-Contreras et al., 2017; Kurabayashi et al., 2016) (e.g., papG, FimH, FliC) further supports the QDs' capacity to interfere with UPEC pathogenic mechanisms.

Taken together, ZnO@PEG200 QDs represent a multifunctional nanoplatform that combines high biosafety, broad-spectrum antimicrobial activity, intracellular accessibility, and virulence suppression. Their non-specific, multimodal antibacterial mechanism reduces the risk of resistance development while preserving therapeutic efficacy against drug-resistant and intracellular UPEC.

5. Conclusion

In summary, we have developed a precisely PEGylated ZnO QDs nanoplatform that combines enhanced biocompatibility with potent, multimodal antibacterial activity. Through systematic surface engineering with PEG200, the ZnO@PEG200 QDs exhibit selective and effective eradication of both planktonic and intracellular E. coli, including drug-resistant strains, while demonstrating minimal cytotoxicity and hemolytic potential. Mechanistic studies revealed that the QDs exert their antibacterial effects via membrane disruption, ROS generation, DNA damage, and downregulation of key virulence genes. Notably, ZnO@PEG200 QDs also inhibit bacterial motility, disrupt mature biofilms, and do not induce antimicrobial resistance under prolonged exposure. In vivo experiments confirmed their targeted accumulation in infected urinary tissues, efficient clearance of intracellular bacteria, attenuation of inflammatory responses, and promotion of tissue recovery in a murine UTIs model. These findings position ZnO@PEG200 QDs as a safe and effective nanotherapeutic platform with significant translational potential for the treatment of intracellular and biofilm-associated bacterial infections.

CRediT authorship contribution statement

Jingqi Niu: Writing – review & editing, Writing – original draft, Investigation, Data curation. Fangyuan Du: Writing – review & editing, Methodology, Data curation. Mingxuan Zhang: Methodology, Writing – review & editing. Beiliang Miao: Methodology. Yu Hong: Methodology. Heyujia Yu: Methodology. Xiaohua Liang: Methodology, Writing – review & editing. Mengqi Gao: Investigation. Qifan Chen: Methodology. Shiwei Liu: Funding acquisition, Conceptualization. Baoshan Liu: Supervision, Funding acquisition. Hongwei Xin: Funding acquisition, Resources. Zeliang Chen: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.