Ultrasound-activated UiO-66 enables ROS-mediated antibacterial therapy and accelerates diabetic wound healing
Chuanwei Sun, Gaoquan Zheng, Yingying Zhao, Zuan Liu, Zhihui Jiang, Jiongliang Li, Chongquan Huang, Yuan Yan, Wen Lai, Zhifeng Huang, Lidan Liu, Feng Peng, Yu Zhang
TL;DR
This study shows that unmodified UiO-66, a metal-organic framework, can be used with ultrasound to kill bacteria and speed up wound healing in diabetic mice.
Contribution
Pristine UiO-66 is introduced as a first-of-its-kind sonodynamic therapy platform for dual antibacterial and pro-regenerative wound healing.
Findings
UiO-66 generates reactive oxygen species under ultrasound, effectively killing bacteria in vitro and in vivo.
UiO-66 promotes cell proliferation and migration of HUVECs and L929 fibroblasts, aiding tissue repair.
In diabetic mice, UiO-66 reduces bacterial load and accelerates wound closure in infected wounds.
Abstract
The development of noninvasive and dual-functional therapeutic strategies is of critical importance for the treatment of infected diabetic wounds, which require both effective bacterial eradication and enhanced tissue regeneration. Conventional approaches are frequently constrained by single-function modalities or adverse side effects, emphasizing the demand for safer and more versatile alternatives. In this study, we demonstrate for the first time that pristine UiO-66, a metal–organic framework without surface modification, can serve as a novel nanoplatform for sonodynamic therapy (SDT), simultaneously achieving antibacterial and pro-regenerative effects. Comprehensive structural and photochemical analyses reveal that UiO-66 efficiently generates reactive oxygen species (ROS) under ultrasound irradiation, which endow it with broad-spectrum antibacterial capacity. These effects are…
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Figure 10- —Guangdong Provincial Key Area R&D Program
- —National Natural Science Foundation of China10.13039/501100001809
- —Natural Science Foundation of Guangdong Province10.13039/501100003453
- —General guide project of Health Science and technology in Guangzhou
- —Key Project of Guangdong Basic and Applied Basic Reserch foundation
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Taxonomy
TopicsNanoplatforms for cancer theranostics · Wound Healing and Treatments · Ultrasound and Hyperthermia Applications
Introduction
The global surge in multidrug-resistant (MDR) bacterial infections—largely fueled by the overuse and misuse of antibiotics—has underscored the urgent need for novel antimicrobial strategies that bypass conventional antibiotic mechanisms [1–3]. In recent years, a variety of non-antibiotic approaches have been explored, including phage therapy [4, 5], antimicrobial peptides [6, 7], metal-ion-based treatments [8, 9] and immunomodulatory therapies [10]. While each of these strategies offers distinct advantages, they are often constrained by limitations such as narrow antibacterial spectra, potential cytotoxicity or complex fabrication procedures [11, 12].
Among the emerging alternatives, reactive oxygen species (ROS)-based antimicrobial therapies have garnered increasing attention due to their unique bactericidal mechanism and low potential for inducing bacterial resistance [13]. ROS, including singlet oxygen (^1^O_2_), hydroxyl radicals (•OH), superoxide anions ( ) and hydrogen peroxide (H_2_O_2_) [14], can irreversibly disrupt bacterial membranes, proteins and nucleic acids, leading to broad-spectrum antibacterial activity. Several strategies have been developed to generate ROS in situ, such as photodynamic therapy (PDT) [15], Fenton reactions [16], Fenton-like reactions [17] and sonodynamic therapy (SDT) [18]. Among these, SDT stands out for its ability to activate sonosensitizers using noninvasive ultrasound, enabling deep-tissue ROS generation without the drawbacks of phototoxicity or light penetration limitations [19]. The focused energy delivery and resistance to environmental interference make SDT particularly promising for precision antimicrobial treatment, including scenarios that are inaccessible to light-based or topical approaches [20].
The therapeutic efficacy of SDT, however, is intrinsically dependent on the properties of the sonosensitizer. To date, a broad range of nanomaterials—including noble metal nanoparticles [21], organic nanostructures [22], heterojunction-based composites [23] and two-dimensional materials [24]—have been investigated for this purpose [14]. Despite promising results, many of these systems suffer from poor structural stability, limited biocompatibility and complex synthesis, which collectively hinder their clinical translation. This has driven growing interest in developing robust, biocompatible and scalable sonosensitizers for next-generation SDT applications.
Metal–organic frameworks (MOFs)—a class of crystalline porous materials formed by metal clusters and organic linkers [25]—have recently emerged as highly tunable platforms owing to their ultrahigh surface areas, structural versatility and multifunctionality [26, 27]. Among them, UiO-66, a zirconium-based MOF constructed from Zr_6_O_4_(OH)4 clusters and terephthalic acid (BDC) linkers, has attracted considerable attention due to its exceptional thermal, chemical and aqueous stability. UiO-66 has been widely applied in gas storage and separation [28], photocatalysis and water source purification [29]. However, its potential as a sonosensitizer in SDT has not yet been systematically explored.
In parallel, increasing evidence suggests that effective infection control is closely linked to wound healing [30], as persistent bacterial colonization can severely impair the healing process [31]—particularly in immunocompromised individuals such as diabetic patients [32]. Therefore, an ideal therapeutic strategy should not only provide potent antibacterial effects but also facilitate tissue regeneration by restoring a favorable wound microenvironment. This highlights the need for dual-functional therapeutic materials that simultaneously exert antimicrobial and pro-regenerative effects, especially for treating infected chronic wounds. Moreover, although UiO-66 is primarily recognized for its structural robustness and tunable porosity, emerging studies suggest that certain MOF-based nanomaterials may also influence cellular behaviors relevant to tissue repair. These characteristics imply that pristine UiO-66 may not only produce ROS under ultrasound to exert antibacterial effects but may also support endothelial and fibroblast activities that are essential for wound regeneration. Guided by this hypothesis, we designed our study to comprehensively assess whether ultrasound-activated UiO-66 can simultaneously exhibit potent antibacterial activity and intrinsic pro-regenerative potential, integrating ROS analysis, antibacterial assays, cytocompatibility evaluation, cell migration and angiogenesis tests, as well as in vivo wound-healing assessments. This framework provides a coherent basis for exploring whether a single, unmodified MOF can function as a truly dual-functional sonosensitizer.
While several MOF-based platforms have been explored for sonodynamic therapy, most reported systems rely on structural modification—such as metal doping [27], heterojunction construction [33] or hybridization with conductive substrates [34]—to enhance ROS generation or therapeutic efficacy. These engineered designs significantly improve performance but also introduce additional components that obscure the intrinsic properties of the parent material. Notably, whether pristine, unmodified UiO-66 itself possesses inherent sonodynamic activity or regenerative potential has not been systematically examined. Clarifying the baseline bioactivity of UiO-66 is, therefore, essential, both for understanding its fundamental interactions with biological systems and for determining whether complex modifications are truly necessary.
In this study, we present the first systematic investigation of pristine UiO-66 nanoparticles as a dual-functional sonosensitizer for antibacterial and wound-healing sonodynamic therapy (Figure 1). We demonstrate that UiO-66, upon ultrasound activation, generates abundant ROS and effectively inhibits the growth of S. aureus. Genome sequencing further revealed that ROS-induced damage to bacterial membranes, proteins and nucleic acids underlies its bactericidal mechanism. To assess the therapeutic potential beyond antibacterial efficacy, we investigated the role of UiO-66-mediated SDT in tissue repair. In vitro studies showed that SDT promoted the proliferation of human umbilical vein endothelial cells (HUVECs) and murine fibroblasts (L929), while also enhancing angiogenesis in tube formation assays. Furthermore, in a diabetic mouse model of S. aureus-infected skin wounds, UiO-66-mediated SDT significantly accelerated wound closure and facilitated tissue regeneration. Collectively, our findings not only broaden the biomedical applicability of UiO-66 but also establish a new design paradigm for MOF-based sonosensitizers that integrate antibacterial and pro-healing functionalities for next-generation, antibiotic-free infection therapy.
Schematic diagram of the antibacterial and regenerative mechanisms of UiO-66.
Experimental section
Synthesis of UiO-66
A total of 0.212 g of ZrCl_4_ was dissolved in 40 mL of N, N-dimethylformamide (DMF). Subsequently, 0.136 g of 1,4-benzenedicarboxylic acid (H_2_BDC) and an additional 40 mL of DMF were added, and the mixture was stirred magnetically until fully dissolved. The resulting solution was heated at 120°C for 48 h. After naturally cooling to room temperature, the product was washed four times with absolute ethanol and centrifuged at 10 000 rpm for 5 min to obtain highly dispersible UiO-66 nanoparticles.
Material characterization
The morphology and elemental distribution of the samples were examined using transmission electron microscopy (TEM, JEM-1400, JEOL, Japan) operated at an accelerating voltage of 200 kV. The crystalline structure was characterized by X-ray diffraction (XRD; D2 Phaser, Bruker, Germany) employing Cu Kα radiation (λ = 0.154 nm) at 30 kV. The surface chemical composition and elemental oxidation states were analyzed by X-ray photoelectron spectroscopy (XPS; AXIS, Shimadzu, Japan) using Al Kα radiation with a pass energy of 160 eV. Fourier transform infrared spectroscopy (FTIR; Nicolet iN10, Thermo Scientific, USA) was performed to identify surface functional groups. Optical absorption spectra were recorded using a UV–Vis spectrophotometer (Lambda 750, PerkinElmer, USA). The particle size distribution was measured using dynamic light scattering (90Plus PALS, Brookhaven Instruments, USA). Zeta potential analysis was performed using dynamic light scattering (Bruker ZetaPALS, Germany).
In vitro ROS detection
Hydroxyl radical (•OH): UiO-66 was dispersed in PBS at a concentration of 100 μg/mL and mixed with methylene blue solution (MB, final concentration: 50 μM). The mixture was then subjected to ultrasound irradiation (1.5 W/cm^2^, 1 MHz) for 5 min. Four groups were established: PBS, US, UiO-66 and UiO-66 + US. After treatment, the absorbance of each sample was immediately measured at 665 nm using a UV–Vis spectrophotometer.
Superoxide anion ( ): UiO-66 was dispersed in PBS at a concentration of 100 μg/mL and incubated with WST-8 working solution. Four groups were established: PBS (control), US, UiO-66 and UiO-66 + US. Ultrasound irradiation was applied at a power density of 1.5 W/cm^2^ and a frequency of 1 MHz for 5 min. Immediately after treatment, the absorbance of each reaction system was measured at 450 nm using a microplate reader (BioTek, USA).
The total superoxide dismutase (SOD) assay was performed using the Total Superoxide Dismutase Assay Kit with WST-8 (S0101M, Beyotime, Shanghai, China). The working solution required for the assay is a freshly prepared mixture rather than a single fixed-concentration reagent. According to the manufacturer’s instructions, the working solution was prepared using three components supplied in the kit: Detection Buffer (providing the necessary pH environment and partial reaction substrates), WST-8 Solution (the chromogenic substrate reduced by superoxide anions to generate water-soluble formazan) and Enzyme Solution containing xanthine oxidase (which continuously produces superoxide anions during the reaction). These components were mixed at a ratio of 151 (Detection Buffer): 8 (WST-8 Solution): 1 (Enzyme Solution) immediately before use, and all samples were processed strictly following the manufacturer’s protocol. The exact concentrations of active ingredients such as WST-8 and xanthine oxidase are proprietary and not disclosed by the manufacturer; however, the preparation ratios provided are the validated conditions ensuring the accuracy and reproducibility of the assay.
Bacterial culture and cell culture
In this study, the Gram-positive bacterium S. aureus (ATCC 25293) was selected as the model strain. The bacteria were cultured in Luria–Bertani (LB) broth (Merck, USA) at 37°C under well-aerated conditions. Human Umbilical Vein Endothelial Cells (ATCC) and L929 cells (ATCC) were cultured in Dulbecco’s modified Eagle Medium (DMEM) containing 10% FBS and 1% penicillin/streptomycin (Gibco, USA) at 37°C in a humidified 5% CO_2_ atmosphere as described previously. Cells were selected during the mid-exponential growth phase for all experiments.
In vitro antibacterial assay
Spread Plate Method: Phosphate-buffered saline (PBS, 200 µl) and UiO-66 solution (200 µg/mL, 200 µl) were each added to the prepared bacterial suspension (1 × 10^6^ CFU/mL, 200 µl) in individual wells of a 24-well plate. The mixtures were then subjected to ultrasound (1.5 W/cm^2^, 1 MHz) for different durations (0 and 5 min). After treatment, the bacterial suspensions were collected, diluted 10-fold and plated on solid agar for colony counting. Following incubation at 37°C for 18 h, the number of colony-forming units (CFUs) was recorded to assess bacterial viability. The bacterial survival rate was calculated using the formula: Survival rate (%) = (C/C0) × 100, where C is the number of colonies in the treatment group and C0 is the number of colonies in the control group.
ROS detection: S. aureus were cultured in four experimental groups as mentioned above, followed by staining with a bacterial reactive oxygen species (ROS) detection kit (BestBio, China) according to the manufacturer’s instructions. The stained samples were then observed under a Nikon 80i fluorescence microscope (Nikon, Japan).
Live & Dead staining: S. aureus were cultured in four experimental groups as mentioned above. After sonication, the bacterial suspensions were dropped onto titanium discs and co-cultured for 4 h. The samples were then rinsed three times with PBS to remove nonadherent bacteria. The adherent bacteria on the surface of the titanium discs were stained using BBcellProbe N01/PI dyes (Biosen, Shanghai). After staining, the samples were washed again with PBS and live (green fluorescence) and dead (red fluorescence) bacteria were visualized using a Nikon 80i microscope (Nikon, Japan).
SEM observation: The four groups (PBS, US, UiO-66 and UiO-66 + US) were treated as described above. Approximately 1 × 10^7^ CFU of bacteria were collected from each group, and after treatment with an electron microscope fixative, the morphology of the bacteria was observed using a SEM (Hitachi, Japan).
Protein Leakage Assay: The disruption of bacterial membrane integrity was evaluated by quantifying extracellular protein leakage using a BCA Protein Assay Kit (Beyotime Biotech, China). Specifically, S. aureus suspensions (1 × 10^6^ CFU) were treated with either PBS or UiO-66 (100 μg/mL) under ultrasound stimulation (1.5 W/cm^2^, 1 MHz) for 5 min. Following treatment, samples were centrifuged to remove bacterial cells, and the protein concentration in the resulting supernatants was determined in accordance with the manufacturer’s protocol. An increase in extracellular protein levels was considered indicative of compromised bacterial membrane integrity.
RNA-Seq analysis: Following treatment with UiO-66 combined with ultrasound, S. aureus cultures were incubated at 37°C for 2 h, after which approximately 1 × 10^8^ CFU were collected. Untreated S. aureus served as the control group. Total bacterial RNA was extracted using the HiPure Bacterial RNA Kit (Magen, China) according to the manufacturer’s instructions. Prokaryotic RNA sequencing was performed on the Illumina NovaSeq 6000 platform using paired-end 150 bp (PE150) sequencing mode (CHI BIOTECH Co., LTD, Guangzhou, China). Cutadapt (v4.3) was employed to remove adapter sequences and low-quality reads. The filtered reads were aligned to the reference genome using STAR (v2.7.10b), and gene quantification was performed using featureCounts (v2.0.4). Differential gene expression analysis was conducted using EdgeR (v3.40.2), with significance thresholds set at false discovery rate (FDR) < 0.01 and |log_2_ fold change| ≥ 2. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was carried out using TopGO (v2.50.0), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using ClusterProfiler (v1.2.14).
Cell viability
CCK-8: HUVECs were seeded into 96-well plates at a density of 3000 cells/well and co-cultured with UiO-66 nanoparticles for 1 and 3 days. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Abbkine, USA) following the manufacturer’s protocol. The absorbance at 450 nm was measured to determine metabolic activity, enabling quantitative analysis of cell proliferation in response to UiO-66 exposure.
Live/dead staining: HUVECs were also seeded into 6-well plates at a density of 1 × 10^6^ cells/well and treated with UiO-66 for 3 days. A live/dead viability/cytotoxicity assay was performed using the Calcein-AM/PI Cell Viability Kit (Biosharp, China). Viable cells emitted green fluorescence (Calcein-AM), while nonviable cells fluoresced red (PI). Images were captured using a Nikon TI-S fluorescence microscope (Nikon, Japan) to visually assess cell membrane integrity and cytotoxic effects.
L929 fibroblasts were subjected to the same procedures to validate results across cell types.
Cell migration assays
To investigate the effects of UiO-66 on cell migration, both Transwell and scratch assays were conducted. L929 fibroblasts and HUVECs were seeded into 6-well plates at a density of 1 × 10^6^ cells/well and cultured for 24 h. Transwell migration assays were performed exclusively with L929 cells, while scratch assays were applied to both L929 cells and HUVECs.
Transwell Assay: After 24 h of incubation, L929 cells were treated with UiO-66 and incubated for an additional 24 h. Cells were then harvested and transferred to Transwell chambers at a density of 1 × 10^4^ cells/well. Serum-free DMEM was added to the upper chamber, while DMEM containing 10% fetal bovine serum was added to the lower chamber. After 12 h of incubation, the cells were fixed and stained with crystal violet (Beyotime) for 1 h. Migrated cells were imaged using a Nikon TI-S microscope (Nikon, Tokyo, Japan).
Scratch Assay: A uniform linear scratch was made across the confluent monolayer using a sterile pipette tip. Detached cells were removed by rinsing with PBS three times, and the gap area was clearly exposed. For the treatment group, UiO-66 was added at a concentration of 100 µg/mL in serum-free DMEM 24 h after seeding. Images were taken at 0, 4, 8 and 12 h using a Nikon TI-S microscope (Nikon, Tokyo, Japan). Cell migration was quantified by measuring the area of wound closure at each time point using ImageJ software.
Hemolysis assay
Fresh blood was collected from C57 mice and diluted with normal saline at a volume ratio of 4:5. UiO-66 samples were pre-warmed to 37°C for 30 min. The hemolysis working solution was also prepared and incubated at 37°C for 15 min prior to use. In a 24-well plate, 30 μL of the diluted blood was added to each well, followed by treatment with the corresponding samples. The plate was incubated at 37°C for 60 min. After incubation, 1 mL of the reaction mixture was collected from each well and centrifuged at 3000 rpm for 5 min. Then, 15 μL of the supernatant was mixed with 250 μL of the hemolysis working solution, and the absorbance at 545 nm was measured using a microplate reader.
The hemolysis ratio was calculated using the following equation:
where A_sample is the absorbance of the test sample, A_NC is the absorbance of the negative control (normal saline) and A_PC is the absorbance of the positive control (distilled water).
Tube formation assay
A Matrigel-based tube formation assay was conducted to assess the pro-angiogenic potential of UiO-66. HUVECs were seeded at a density of 2 × 10^4^ cells per well in 96-well plates pre-coated with growth factor-reduced Matrigel (Corning, USA). Cells were incubated at 37°C for 4 h in complete endothelial growth medium, with the experimental group treated with UiO-66 and the control group left untreated. Tubular structures were imaged using an inverted light microscope, and the total tube length (mm/field) and number of branch points were quantitatively analyzed using ImageJ software equipped with the Angiogenesis Analyzer plugin.
Gene expression analysis related to wound healing
The expression levels of wound repair-associated genes were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). HUVECs and L929 cells were seeded into 6-well plates at a density of 3 × 10^5^ cells per well and cultured for 24 h. The experimental group was then treated with UiO-66 at a concentration of 100 μg/mL in culture medium and co-incubated for an additional 24 h. Total RNA was extracted from the treated cells using the Total RNA Kit I (Omega Bio-tek, USA), and RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized from the extracted RNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgene, France). qRT-PCR was performed using a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, USA), and gene expression levels were quantified using the comparative Ct (^ΔΔ^Ct) method. The primer sequences employed for quantitative PCR analysis are provided in Supplementary Table S1.
Western blot analysis
VEGF and COL-1 expression in endothelial cells and fibroblasts were evaluated by western blot. Briefly, HUVECs and L929 cells were seeded in 6-well plates and allowed to adhere overnight, then treated with UiO-66 at the indicated concentrations (100 μg/mL) for 24 h under standard culture conditions. After treatment, cells were washed twice with ice-cold PBS and lysed on ice in RIPA buffer supplemented with a protease inhibitor cocktail. The lysates were clarified by centrifugation (12 000 × g, 10 min, 4°C), and protein concentrations were determined using a BCA assay kit. Equal amounts of total protein (20–30 μg per lane) were mixed with 5× SDS loading buffer, boiled at 95°C for 5 min, separated by SDS-PAGE (10% resolving gel with 5% stacking gel) and electrotransferred onto PVDF membranes (0.22 μm). Membranes were blocked with 5% BSA in TBST for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies diluted in TBST containing 1% BSA, including anti-VEGF (1:1000), anti-COL-1 (1:1000) and anti-β-actin (1:3000) as a loading control. After three washes with TBST (3 × 10 min), membranes were incubated with HRP-conjugated secondary antibody (1:3000 in TBST–1% BSA) for 1 h at room temperature, followed by three additional TBST washes (3 × 10 min). Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate on a digital imaging system. The expression levels of target proteins were normalized to β-actin. All western blot experiments were performed in at least three independent biological replicates.
Establishment of diabetic mouse model
All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangdong Provincial People’s Hospital (KY2023-821-01). Male C57BL/6 mice aged 4–6 weeks were used in this study. Prior to modeling, the mice were fasted for 4 h. Streptozotocin (STZ) was freshly prepared at a concentration of 10 mg/mL in a citrate buffer (pH 4.5), which was composed of 21.9 mg/mL citric acid and 29.4 mg/mL trisodium citrate mixed at a 1:1.32 ratio in normal saline. The STZ solution was administered intraperitoneally at a dose of 65 mg/kg based on the mouse body weight. Two hours after injection, mice were provided with 2.5% glucose solution to prevent hypoglycemia.
STZ injections were performed once daily for three consecutive days. Starting three days after the final injection, fasting blood glucose levels were measured every three days. Mice were considered successfully diabetic if their blood glucose levels exceeded 13.9 mmol/L in three consecutive measurements.
In vivo antibacterial and wound healing study
All mice used in the experiments were the diabetic model mice established as described above. Prior to wound modeling, the mice were anesthetized and the dorsal hair was shaved. Full-thickness circular wounds (8 mm in diameter) were created on the back using a sterile biopsy punch. A diabetic infection model was established by injecting 10 μL of S. aureus suspension (10^6^ CFU/mL) directly into the wound site. Following infection, mice were randomly assigned to one of the following treatment groups: control (PBS), US (ultrasound only), UiO-66 and UiO-66 + US. Each group received the corresponding treatment, with US irradiation applied at 1.5 W/cm^2^ and 1 MHz for 5 min. For the UiO-66 and UiO-66 + US groups, 20 μL of UiO-66 suspension (100 μg/mL) was administered, while the control and US groups received 20 μL of PBS. Each group initially included 12 diabetic mice, and the number of animals decreased over time due to scheduled tissue collection (e.g. Day 4 sampling), resulting in 3 mice remaining per group for the final evaluation on Day 10.
To monitor the wound healing process over time, photographs of the wound sites were taken on Day 0, 1, 4, 7 and 10. Wound area was quantified using ImageJ software, and wound closure percentage was calculated according to the following formula: Wound closure rate (%) = [(wound area on Day 0—wound area on Day x)/wound area on Day 0] × 100%, where x represents Day 1, 4, 7 or 10.
To evaluate wound closure, inflammation resolution and epidermal regeneration, skin tissues were collected on Day 4 and 10 post-treatment for histological analysis. Samples were fixed in 4% paraformaldehyde for 24 h at room temperature, followed by graded ethanol dehydration and paraffin embedding. Serial paraffin sections (5 μm thickness) were prepared using a microtome and subjected to hematoxylin and eosin (H&E) staining, Giemsa staining and immunofluorescence/immunohistochemical staining. Type I collagen (COL-1) staining was performed to assess extracellular matrix deposition and early tissue remodeling within the wound bed. All slides were scanned using a Pannoramic MIDI whole-slide scanner (3DHISTECH, Hungary). For quantitative analysis, five randomly selected, nonoverlapping fields were analyzed per section and three sections were evaluated per sample. Image quantification was performed using ImageJ by an investigator blinded to group allocation to minimize bias.
Statistical analysis
All data are presented as mean ± standard deviation (SD). Prior to statistical testing, datasets were assessed for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test. For normally distributed data with equal variances, one-way ANOVA followed by Tukey’s post hoc multiple-comparison test was applied. For non-normal data or data with unequal variances, the Kruskal–Wallis test followed by Dunn’s post hoc correction was used. Exact p-values are provided in the figure legends or indicated directly in the graphs. A P value <0.05 was considered statistically significant (**P *< 0.05, ***P *< 0.01, ****P *< 0.001, *****P *< 0.0001). All statistical analyses were performed using GraphPad Prism 8. Quantification of histological and immunostaining images was conducted from five randomly selected, nonoverlapping fields per section and three sections per sample and the analysis was performed in a blinded manner.
Results and discussion
Synthesis and characterization of UiO-66
UiO-66 nanoparticles were synthesized via a solvothermal strategy and systematically characterized to confirm their morphology, composition and structural integrity (Figure 2). As shown in TEM images (Figure 2A), UiO-66 exhibited uniform polyhedral particles with well-defined shapes. High-resolution TEM further displayed distinct lattice fringes, indicative of the excellent crystallinity of the material. Energy-dispersive X-ray spectroscopy (EDS) elemental mapping demonstrated homogeneous distribution of C, Zr and O elements throughout the particles, consistent with the expected chemical composition of UiO-66. The X-ray diffraction (XRD) pattern (Figure 2B) exhibited sharp and intense peaks at approximately 7.4°, 8.5°, 17.1° and 25.7°, which are in excellent agreement with the reference pattern of UiO-66 [35]. The presence of multiple sharp reflections in the low-angle region further confirmed the successful formation of a highly ordered crystalline framework.
*(A) TEM Images of UiO-66 nanoparticles. Inset: HRTEM and EDS images. Scale bars: 50 nm (left), 20 nm (middle) and 30 nm (right). (B) XRD patterns of UiO-66 compared with the simulated standard pattern. (C) FTIR spectrum. (D) XPS survey spectrum. (E) High-resolution Zr 3d XPS spectrum. (F) Particle size distribution by DLS. (G) Zeta potential distribution. (H) Release of Zr4+ from UiO-66 under different conditions. (I) Release of Zr4+ from UiO-66 during non-ultrasound and ultrasound irradiation. (J) The sonodynamic activity of UiO-66 under different conditions. **P < 0.001.
FTIR spectroscopy (Figure 2C) showed distinct bands at ∼1580 cm^−1^ and ∼1390 cm^−1^, corresponding to the asymmetric and symmetric stretching vibrations of coordinated carboxylate groups, respectively, suggesting the successful coordination between the carboxylic acid groups of the organic linker and Zr^4+^ nodes. Additionally, strong bands in the 750–500 cm^−1^ region were assigned to Zr–O and Zr–O–C vibrations, further validating the formation of the metal–ligand coordination structure [36]. To gain further insights into the elemental composition and chemical states, XPS analysis was performed. The survey spectrum (Figure 2D) confirmed the presence of O 1 s (∼531 eV), C 1 s (∼284.8 eV) and Zr 3d (∼183 eV) peaks, in line with the molecular framework comprising Zr_6_ clusters and 1,4-benzenedicarboxylate linkers. In addition, several minor unassigned peaks were observed, likely arising from Zr-related Auger signals or residual surface species. Nonetheless, the overall spectrum exhibits a clean background with no significant extraneous signals, indicating high sample purity. High-resolution XPS analysis of the Zr 3d region (Figure 2E) showed a well-defined spin–orbit doublet with peaks at ∼182.2 eV (Zr 3d_5/2_) and ∼184.6 eV (Zr 3d_3/2_), confirming that Zr exists predominantly in the +4 oxidation state and is stably coordinated within the UiO-66 framework.
Dynamic light scattering (DLS) measurements (Figure 2F) revealed a narrow, monomodal size distribution centered on 150 nm, indicating good particle uniformity and dispersion. Furthermore, zeta potential analysis (Figure 2G) showed a negative surface charge of approximately –40 mV, suggesting excellent colloidal stability in aqueous media, which is crucial for subsequent applications in water-based systems.
To further assess the physicochemical robustness of UiO-66 under physiologically relevant conditions, we evaluated its structural stability in different media and during ultrasound irradiation. As shown in Figure 2H, UiO-66 exhibited extremely low Zr^4+^ release in PBS, simulated acidic wound microenvironment and serum, with cumulative leaching remaining below 0.5% in all cases. The slightly elevated release observed in acidic and serum conditions is consistent with the expected influence of pH and protein-rich environments, yet the overall leaching level remains minimal, confirming the inherent stability of the framework. We next investigated the effect of ultrasound irradiation on material stability (Figure 2I). Although ultrasonication moderately increased Zr^4+^ release, even three consecutive ultrasound cycles resulted in only ∼0.08% leaching, indicating that ultrasound activation does not compromise the integrity of the UiO-66 structure. These observations align with the well-established chemical durability of UiO-66, which originates from the strong Zr–O coordination bonds and high connectivity of the Zr_6_ cluster nodes. Importantly, the functional performance of UiO-66 remained stable under these conditions. As shown in Figure 2J, its sonodynamic ROS-generating capability displayed no significant variation across PBS, acidic medium or serum and simulated acidic wound environments did not negatively affect biocompatibility. Together, these results highlight that UiO-66 possesses exceptional structural and functional stability under diverse physiological and sonodynamic conditions, supporting its suitability for further biological and therapeutic applications.
Sonodynamic ability of UiO-66 and the behind mechanism
To elucidate the mechanism of ROS generation by UiO-66 under ultrasound stimulation, both experimental evidence and electronic structure analysis were comprehensively examined. As shown in Figure 3A, MB degradation was employed as an indicator of •OH production. The absorbance at ∼664 nm significantly decreased in the UiO-66+US group, whereas negligible changes were observed in the control (Ctrl), UiO-66 alone and ultrasound alone (US) groups. This sharp decline suggests that efficient •OH generation occurs only when UiO-66 is activated by ultrasound. In parallel, the generation of was quantified using the WST-8 assay. As shown in Figure 3B, the characteristic absorbance of the WST-8 reduction product (formazan) at ∼450–480 nm was markedly enhanced in the UiO-66+US group, whereas the other groups exhibited only weak signals. This result confirms that ultrasound stimulation enables UiO-66 to effectively produce . Further strong evidence for ROS generation was obtained through electron spin resonance (ESR) spectroscopy. Using DMPO as the trapping probe for •OH, the UiO-66+US group displayed a pronounced quartet signal (Supplementary Figure S1), whereas only weak or negligible signals appeared in the remaining groups, confirming robust •OH formation under ultrasound excitation. Similarly, using BMPO as the trapping agent for , the UiO-66+US group exhibited characteristic multipeak ESR spectra with substantially stronger intensity compared to UiO-66 alone, US alone and the Ctrl group (Supplementary Figure S2). These ESR signatures provide direct molecular evidence supporting the ultrasound-triggered generation of both •OH and . Taken together, these results indicate that ultrasound excitation facilitates charge separation within the UiO-66 framework, enabling both reduction of dissolved O_2_ to and oxidation of OH^-^ to •OH. This dual pathway establishes a robust sonodynamic ROS generation mechanism.
ROS generation performance and electronic structure analysis of UiO-66. (A) Methylene blue degradation to evaluate •OH production. (B) WST-8 assay for O2•– detection. (C) UV–Vis absorption spectrum. (D) Tauc plot for bandgap estimation. (E) Ultraviolet photoelectron spectroscopy (UPS) for valence band maximum. (F) Schematic illustration of ROS generation mechanism under ultrasound.
To further elucidate the structural basis for the ROS-generating capability of UiO-66, its optical properties and electronic band structure were systematically investigated. As shown in Figure 3C, the UV–Visible absorption spectrum of UiO-66 exhibits strong absorption in the ultraviolet region, with negligible absorption in the visible range. The absorption edge is located at approximately 330–340 nm, indicating that UiO-66 is essentially transparent to visible light and can be classified as a wide-bandgap insulator or semiconductor. This characteristic suggests that only high-energy excitation—such as ultrasound-induced sonoluminescence—can provide sufficient energy to trigger electronic transitions and subsequent ROS generation.
Based on the absorption data, the optical bandgap of UiO-66 was estimated to be approximately 3.99 eV using a Tauc plot derived from (αhv)^2^ vs hv fitting (Figure 3D), confirming its wide-bandgap nature. This optical feature explains why UiO-66 is inert under ambient light but can exhibit ROS-generating activity under physical stimulation such as ultrasound. To probe the band structure in greater detail, ultraviolet photoelectron spectroscopy (UPS) analysis was conducted. As shown in Figure 3E, the valence band maximum (VBM) is located at ∼3.64 eV (relative to the vacuum level). Combined with the Tauc-estimated bandgap of 3.99 eV, the conduction band minimum (CBM) was calculated to be approximately –0.35 eV. This electronic structure further supports the notion that external stimulation is required to initiate charge excitation in UiO-66, accounting for its low activity under visible light but robust ROS generation when activated by ultrasound.
Together, the UV–Vis absorption spectrum (Figure 3C), Tauc-derived bandgap (Figure 3D) and UPS-determined VBM (Figure 3E) provide a comprehensive picture of the electronic structure of UiO-66, which underpins the ROS generation mechanism depicted in Figure 3F. Under ultrasound irradiation, localized cavitation events generate high-energy sonoluminescence that excites electrons from the valence band to the conduction band. The excited electrons (e^-^) can reduce dissolved oxygen (O_2_) to , as the CBM (–0.35 eV) lies above the redox potential of the O_2_/ couple (–0.33 eV vs NHE). Simultaneously, the photogenerated holes (h^+^) in the valence band can oxidize hydroxide ions (OH^-^) to generate •OH, since the VBM (3.64 eV) exceeds the OH^-^/•OH redox potential (1.99 eV). Therefore, the ultrasound-activated band structure of UiO-66 facilitates the generation of both and •OH through coordinated redox pathways, highlighting its excellent sonodynamic properties.
To further assess the durability of UiO-66’s sonodynamic performance, we examined whether its ROS-generating capability could be maintained during prolonged incubation and repeated ultrasound activation. As shown in Supplementary Figure S3, UiO-66 preserved stable ROS production after immersion in PBS for 1 day and even up to 7 days, indicating that extended storage does not impair its intrinsic catalytic activity. Moreover, the ROS generation rate remained essentially unchanged after three consecutive ultrasound irradiation cycles (Supplementary Figure S4), demonstrating that repeated acoustic stimulation does not diminish its sonodynamic efficiency. These results collectively confirm that UiO-66 possesses robust and long-lasting sonodynamic activity, which is essential for achieving sustained antibacterial effects in practical applications.
In addition, considering that glutathione (GSH) is a major intracellular antioxidant capable of scavenging ROS, we further examined whether a GSH-rich environment would impair the sonodynamic activity of UiO-66. As shown in Supplementary Figure S5, UiO-66 and UiO-66+US exhibited negligible GSH depletion under all tested conditions, indicating that the nanoparticles themselves do not directly consume GSH. To further clarify the influence of GSH on ROS production, we compared the ROS-generating capability of UiO-66 in the presence and absence of GSH (Supplementary Figure S6). Although GSH partially quenched the generated ROS—as expected for a biological reductant—the UiO-66+US group still maintained a clear and detectable ROS signal. These findings demonstrate that the ultrasound-activated ROS-generation process of UiO-66 remains functional even in GSH-rich environments, and that the observed decrease in ROS intensity is attributable to the scavenging effect of GSH rather than suppression of UiO-66’s intrinsic sonodynamic activity. Collectively, these results indicate that UiO-66 retains its ROS-generating capability under physiologically relevant reductive conditions, further supporting its robustness for antibacterial applications.
In vitro antibacterial activity of UiO-66 combined with ultrasound
To systematically evaluate the in vitro antibacterial performance of UiO-66 combined with ultrasound against S. aureus, we first optimized the sonodynamic treatment parameters. A series of time-course experiments (1–5 min) were conducted, and 5 min was identified as the optimal duration for maximal antibacterial activity (Supplementary Figure S7a and b). We then compared ultrasound intensities of 0.8, 1.0, 1.2, 1.5 and 2.0 W/cm^2^ (Supplementary Figure S7c and d) and the condition “5 min at 1.5 W/cm^2^” was selected for all subsequent assays. In addition to optimizing irradiation parameters, we further investigated the minimum inhibitory concentration (MIC) and the duration of antibacterial activity. As shown in Supplementary Figure S8, the antibacterial efficacy increased progressively with concentration, and 100 μg/mL was identified as the MIC, at which nearly all colonies were eliminated under ultrasound activation. Notably, this concentration also exhibited a sustained antibacterial effect, as the bacterial viability remained almost completely suppressed even after 24 h of post-treatment incubation. Under these optimized conditions, we conducted a series of antibacterial assays to verify the synergistic effect of ultrasound-activated UiO-66. As shown in Figure 4A, dense bacterial colonies were observed in the Ctrl, US and UiO-66 groups, whereas the colony number was markedly reduced in the UiO-66+US group, indicating excellent antibacterial activity. Corresponding CFU quantification (Figure 4B) demonstrated a 94.60 ± 1.33% reduction in S. aureus viability compared to the control, confirming the enhanced bactericidal effect of UiO-66 upon ultrasound activation.
*In vitro antibacterial performance of UiO-66 combined with ultrasound against S. aureus. (A) Representative spread plate images of S. aureus after different treatments. (B) Quantification of bacterial viability by antibacterial rate (mean ± SD). (C) Live/dead fluorescence staining of bacteria. Scale bars: 100 μm. (D) SEM images of S. aureus morphology in Ctrl and UiO-66+US groups. Scale bars: 1 μm. (E) Intracellular ROS detection using fluorescent probe. Scale bars: 100 μm. ***P < 0.0001.
Live/dead fluorescence staining was further used to assess bacterial viability at the cellular level (Figure 4C). In the Ctrl and UiO-66 groups, most S. aureus cells displayed intact membranes with strong green fluorescence, indicating high survival rates. A small amount of red fluorescence was observed in the US group, likely due to limited ROS generation caused by ultrasound-induced cavitation, resulting in mild antibacterial activity [37]. In contrast, the UiO-66+US group showed extensive red fluorescence, indicating severe membrane disruption and widespread bacterial death. These findings demonstrate that UiO-66, under sonodynamic activation, can significantly enhance its antibacterial potency against S. aureus.
Furthermore, to validate the broader antibacterial applicability of UiO-66, additional plating assays were performed using E. coli and methicillin-resistant S. aureus (MRSA). As shown in Supplementary Figures S9 and S10, both E. coli and MRSA exhibited almost complete eradication under UiO-66+US treatment, with antibacterial rates approaching 100%, whereas the Ctrl, US and UiO-66 groups showed dense colony formation. These results further demonstrate that ultrasound-activated UiO-66 possesses robust and broad-spectrum antibacterial efficacy against both Gram-negative and antibiotic-resistant bacteria.
To investigate the morphological changes associated with sonodynamic treatment, SEM imaging was performed on S. aureus in the Ctrl and UiO-66+US groups (Figure 4D). Bacteria in the Ctrl group maintained intact, smooth and well-defined cellular structures, indicative of normal physiological morphology. In contrast, S. aureus exposed to UiO-66+US displayed pronounced membrane rupture, cytoplasmic leakage and severe structural collapse, reflecting substantial mechanical disruption and oxidative injury likely caused by ROS-mediated damage. Notably, because both the US-alone and UiO-66-alone groups exhibited minimal antibacterial activity and showed no appreciable morphological alterations in preliminary observations, SEM imaging focused on the Ctrl and UiO-66+US groups to more clearly demonstrate the distinctive structural damage induced by the synergistic sonodynamic treatment.
Furthermore, a ROS fluorescence probe was used to detect intracellular ROS levels in S. aureus after different treatments (Figure 4E). Strong ROS signals were detected only in the UiO-66+US group, with minimal signals in the US group and negligible levels in the Ctrl and UiO-66 groups. These results confirm that ultrasound-activated UiO-66 efficiently generates ROS, which induces oxidative stress and membrane disruption in S. aureus, ultimately achieving a potent synergistic antibacterial effect.
Underlying antibacterial mechanism of ultrasound-activated UiO-66
To further elucidate the molecular mechanisms underlying the antibacterial effect of ultrasound-activated UiO-66 against S. aureus, we conducted comprehensive RNA transcriptome sequencing. As shown in the volcano plot (Figure 5A), significant transcriptional differences were observed between the UiO-66+US-treated group and the untreated control. A total of 1275 differentially expressed genes (DEGs) were identified, including 615 upregulated and 660 downregulated genes, indicating a widespread transcriptional response triggered by the UiO-66+US intervention. Principal component analysis (Supplementary Figure S11a) revealed a marked segregation between the control and UiO-66+US groups, reflecting substantial alterations in the global transcriptional landscape. Pearson correlation analysis (Supplementary Figure S11b) demonstrated excellent reproducibility among biological replicates within each group and markedly reduced correlations between groups. In agreement, hierarchical clustering of gene expression profiles based on P-values (Supplementary Figure S11c) further accentuated the distinct transcriptional signatures induced by UiO-66+US, underscoring its profound regulatory influence on S. aureus gene expression.
Antibacterial mechanism of UiO-66 activated by ultrasound. (A) Volcano plot showing DEGs in S. aureus following UiO-66+US treatment. (B) KEGG pathway enrichment of DEGs, indicating widespread disruption of bacterial metabolic and biosynthetic processes. (C, D) GO enrichment analysis highlighting ROS-induced interference in energy metabolism, membrane structure and protein homeostasis.
Furthermore, KEGG pathway enrichment analysis was conducted to identify the top 20 pathways most affected by DEGs. As shown in Figure 5B, a series of pathways closely associated with bacterial core metabolism and membrane biosynthesis were significantly enriched, suggesting that ROS may exert potent bactericidal effects through multidimensional metabolic disruption. Specifically, the enrichment of the phosphotransferase system (PTS) and galactose metabolism pathways indicates impaired carbon source uptake and utilization [38, 39], thereby restricting energy production and precursor supply for biosynthesis. Alterations in glycolysis/gluconeogenesis and 2-oxocarboxylic acid metabolism further imply that ROS may oxidatively damage key metabolic enzymes, leading to ATP depletion and metabolic breakdown [40]. In addition, the enrichment of fatty acid degradation and branched-chain amino acid biosynthesis pathways (including valine, leucine and isoleucine) reveals severe interference with membrane lipid remodeling and protein synthesis [41]. Notably, the significant enrichment of the C5-branched dibasic acid metabolism pathway suggests that structurally unique intermediates may accumulate abnormally or undergo dysregulated degradation under oxidative stress, further disturbing amino acid homeostasis and compromising membrane stability [42]. Collectively, these findings indicate that the UiO-66+US treatment induces bacterial inactivation through multilevel disruption of core energy metabolism, biosynthetic processes and structural integrity.
In parallel, GO enrichment analysis was conducted to further clarify the functional implications of the DEGs (Figure 5C and D). GO annotations across molecular function (MF), biological process (BP) and cellular component (CC) categories revealed patterns highly consistent with the KEGG pathway results. Among the up-regulated genes, the term “cellular biogenic amine metabolic process” was significantly enriched, suggesting enhanced amine metabolism in S. aureus. This may reflect ROS-induced accumulation of toxic amine derivatives such as histamine and indoleamines [43]. Additionally, enrichment in “indole-containing compound metabolic process” and “indolalkylamine metabolic process” indicates disruption of intracellular metabolic balance and regulatory networks. On the CC level, many upregulated genes were associated with membrane structures, implying that the bacterial membrane is a direct target of ROS attack. The increase in “aldehyde dehydrogenase activity” indicates excessive lipid peroxidation and the accumulation of toxic aldehydes [44], while the enrichment of “unfolded protein binding” points to widespread protein misfolding—hallmarks of severe oxidative stress [45]. In contrast, down-regulated DEGs were primarily involved in core carbon metabolism, energy production and redox-related membrane complexes. Specifically, significant suppression of the “glycolytic process,” “pyruvate metabolic process,” and “nucleoside triphosphate metabolic process” suggests that ROS impairs energy-generating pathways, resulting in ATP depletion [46–48]. The downregulation of the “proton-transporting ATP synthase complex”, a central component of the bacterial “energy factory,” further implies that S. aureus undergoes metabolic exhaustion [49]. In addition, the decreased expression of “cytochrome-c oxidase activity” and “nitrate reductase activity” suggests that electron transport chain function is disrupted, halting respiratory ATP synthesis [50, 51]. From the CC perspective, significant changes in “cell periphery,” “plasma membrane part,” and “integral component of membrane” indicate ROS-induced structural damage to the membrane, likely resulting in leakage and cell death [52, 53]. Collectively, GO enrichment analysis reinforces the mechanisms revealed by KEGG, demonstrating that ultrasound-activated UiO-66 exerts antibacterial effects through ROS-mediated damage to metabolic pathways, protein function and membrane integrity—ultimately leading to systemic collapse and rapid bacterial inactivation.
Taken together, these transcriptomic alterations are fully consistent with the well-established molecular pathways through which ROS exert antibacterial activity. Previous studies have demonstrated that ROS rapidly oxidize central metabolic enzymes—especially Fe–S cluster–containing proteins—leading to the collapse of glycolysis, the TCA cycle and amino-acid biosynthesis, as summarized by Imlay [54]. Likewise, ROS-induced lipid peroxidation and oxidation of membrane-associated proteins disrupt membrane integrity and electrochemical homeostasis, triggering broad transcriptional remodeling of cell-envelope and energy-regulation pathways, a mechanism highlighted by Ezraty et al. [55]. Moreover, the overall transcriptional signature observed in our study strongly parallels the cumulative oxidative injury model described by Hong et al. [56], in which simultaneous oxidation of lipids, proteins and DNA leads to global suppression of metabolic and membrane-associated genes. These well-established mechanistic frameworks provide strong molecular support for the RNA-seq patterns observed here, reinforcing that oxidative damage is the primary driver of bacterial inactivation in the UiO-66+US treatment group.
Biocompatibility of UiO-66
Good biocompatibility is a fundamental prerequisite for ensuring that UiO-66 can effectively perform its intended functions. In this study, we systematically evaluated the biocompatibility of UiO-66 through both cytocompatibility and hemocompatibility assays to ensure that this nanomaterial does not pose harm to human cells while exhibiting ultrasound-responsive antibacterial properties. As shown in the live/dead staining results of HUVECs and L929 cells treated with UiO-66 (Figure 6A and B), the majority of the cells exhibited green fluorescence (Calcein-AM), with only a small fraction showing red fluorescence (PI staining), indicating intact cell membranes and minimal disruption of metabolic activity. Further, CCK-8 assays (Figure 6C and D) confirmed these findings, showing that cell viability remained above 95% across concentrations ranging from 50 to 400 μg/mL, indicating that UiO-66 induces negligible cytotoxicity under physiological conditions and does not affect cell proliferation or vitality. Moreover, UiO-66 maintained excellent biocompatibility even under simulated acidic wound conditions, as demonstrated in Supplementary Figure S12.
(A, B) Live/dead staining of HUVECs and L929 cells. Scale bars: 100 μm. (C, D) CCK-8 assay for cell viability. (E) Hemolysis assay showing low hemolysis.
Furthermore, hemolysis testing was conducted to evaluate the compatibility of UiO-66 with red blood cells, which is a critical factor for any material intended for systemic administration or subcutaneous implantation, especially in the context of wound healing in diabetic patients. The material must not only be biocompatible but also nontoxic to blood components. As shown in Figure 6E, the hemolysis ratios for all tested concentrations of UiO-66 were well below the clinically accepted threshold of 5%, indicating that the material does not compromise the stability of erythrocyte membranes or induce significant hemolysis. These results provide strong evidence for the safety of UiO-66 in blood-contacting biomedical applications.
Angiogenic potential of UiO-66
To evaluate the pro-angiogenic potential of UiO-66, a series of functional assays were conducted using HUVECs as the in vitro model. As shown in Figure 7A, the scratch wound healing assay revealed that UiO-66 treatment significantly accelerated endothelial cell migration compared to the control group. Quantitative analysis of the cell-covered area (Figure 7B) further confirmed this observation, with significantly higher migration rates observed at 8 and 12 h post-treatment (*P *< 0.05 and *P *< 0.01, respectively). These findings indicate that UiO-66 effectively enhances endothelial motility and supports wound closure, which are essential steps in angiogenesis and tissue repair.
*(A, B) Wound healing assay showing enhanced cell migration after UiO-66 treatment. Scale bars: 100 μm. (C) qPCR analysis of angiogenesis-related genes (HIF-1α, PDGF, KDR). (D) VEGF protein expression in HUVECs with or without UiO-66 treatment. β-actin served as the internal reference. (E, F) Tube formation assay showing increased total segment length in UiO-66-treated cells. *P < 0.05, **P < 0.01, ***P < 0.0001.
To further elucidate the intrinsic regulatory effects of UiO-66 on endothelial cell function, qPCR was performed to evaluate the expression of key pro-angiogenic genes in HUVECs following exposure to UiO-66. The results revealed a significant upregulation of HIF-1α, PDGF and KDR (*P *< 0.01 or *P *< 0.0001; Figure 7C), indicating that UiO-66 can induce a transcriptional program favorable for angiogenesis. Specifically, the increased expression of HIF-1α suggests that UiO-66 may modulate redox homeostasis or mimic hypoxia-like signaling, thereby priming endothelial cells for angiogenic activation. The enhanced expression of PDGF implies strengthened autocrine and paracrine signaling, which contributes to endothelial proliferation and neovessel stabilization. Moreover, the upregulation of KDR indicates an increased sensitivity of endothelial cells to VEGF cues, promoting cell migration, proliferation and tube formation. Importantly, western blot analysis further confirmed that UiO-66 stimulation led to a noticeable increase in VEGF protein expression (Figure 7D), demonstrating that the transcriptional activation of angiogenic genes is translated into functional protein-level responses. This concordance between gene and protein expression provides direct mechanistic evidence that UiO-66 not only primes endothelial cells at the transcriptional level but also enhances key pro-angiogenic signaling outputs essential for neovascularization. Collectively, these findings demonstrate that UiO-66 can actively engage the endothelial angiogenic gene network, laying a mechanistic foundation for its potential application in vascular regeneration therapies.
Critically, the ability of endothelial cells to form capillary-like structures is a fundamental step in neovascularization and tissue regeneration. As shown in Figure 7E, HUVECs treated with UiO-66 formed significantly more organized and extensive tubular networks compared to the control group, which displayed only sparse and poorly connected structures. Quantitative analysis using the Angiogenesis Analyzer plugin in ImageJ confirmed that UiO-66 treatment markedly increased both the number of junction points (Supplementary Figure S13) and the total length of tubular segments (Figure 7F), indicating enhanced endothelial morphogenesis and vascular network formation.
These findings provide strong evidence that UiO-66 effectively promotes tube formation in endothelial cells, supporting its intrinsic pro-angiogenic potential. Robust tube formation is a prerequisite for the development of functional vasculature, which is essential for oxygen and nutrient delivery in ischemic or chronic wound environments—particularly relevant in pathological conditions such as diabetes, where vascular regeneration is impaired.
Pro-regenerative potential of UiO-66
To evaluate the potential of UiO-66 in promoting wound healing, we further investigated its intrinsic bioactivity in modulating fibroblast behavior. Scratch wound assays revealed that treatment with UiO-66 significantly accelerated the migration of L929 fibroblasts toward the wound center (Figure 8A), while Transwell assays independently confirmed the enhanced migratory capacity at the single-cell level (Figure 8B and Supplementary Figure S14).
*(A) Scratch wound assay of L929 fibroblasts treated with UiO-66. Scale bars: 100 μm. (B) Transwell migration assay. Scale bars: 100 μm. (C) qPCR analysis of Col1a, Col3a and TGF-β expression. (D) COL-1 protein expression in L929 cells with or without UiO-66 treatment. β-actin served as the internal reference. P < 0.05.
At the molecular level, quantitative PCR analysis demonstrated that UiO-66 markedly upregulated the expression of Col1a, Col3a and TGF-β (Figure 8C), suggesting the activation of transcriptional programs associated with extracellular matrix (ECM) remodeling. Specifically, Col1a encodes the alpha chain of type I collagen, the primary structural component of the ECM, which provides tensile strength and mechanical integrity to the tissue, while serving as a scaffold for cell adhesion and migration [57]. Col3a, encoding type III collagen, is frequently co-expressed with Col1a and predominates in flexible connective tissues such as skin and vasculature. It plays a critical role during the early stages of wound repair by forming a compliant ECM network that facilitates fibroblast and endothelial cell migration and alignment [58]. The coordinated upregulation of both collagen isoforms indicates that UiO-66 may promote tissue regeneration by modulating ECM architecture and plasticity. Importantly, western blot analysis further confirmed that UiO-66 stimulation led to a pronounced increase in COL-1 protein levels in L929 fibroblasts (Figure 8D), demonstrating that the transcriptional enhancement of collagen-related genes is indeed translated into functional ECM protein production. This concordance between mRNA and protein expression provides direct molecular evidence that UiO-66 actively promotes collagen synthesis, thereby supporting its role in facilitating fibroblast-mediated matrix deposition and tissue repair.
Taken together, our findings demonstrate that UiO-66 not only serves as a sonosensitizer for ROS-mediated antibacterial therapy, but also actively contributes to tissue repair by promoting angiogenesis, fibroblast recruitment and matrix remodeling. This integrated pro-regenerative profile renders UiO-66 a promising candidate for the treatment of complex wounds, particularly diabetic or infected wounds where simultaneous antimicrobial activity and tissue regeneration are critically required.
Wound healing in bacterially infected diabetic model
Following the above in vitro experiments and mechanistic analyses, an in vivo diabetic wound infection model was established using mice with full-thickness skin defects to further investigate the therapeutic potential of UiO-66 for treating infected wounds under diabetic conditions. The experimental protocol for the animal study is depicted in Figure 9A. Given that infected diabetic wounds typically exhibit persistent inflammation during the first several days and begin to enter the early regenerative phase around Day 7–10, a 10-day endpoint was selected to concurrently evaluate antibacterial efficacy and initial tissue-repair progression while minimizing the confounding effects of late-stage wound contraction.
In vivo evaluation of UiO-66 combined with ultrasound in a diabetic infected wound model. (A) Schematic of animal experiment design. (B) Representative wound images and heatmap analysis at indicated time points. (C) Quantification of wound closure rate over time. (D) Bacterial colony formation on agar plates from wound tissue homogenates at Day 4. (E) Giemsa staining on Day 4 showing bacterial presence (arrows); scale bars: 100 μm. (F) Immunohistochemical staining of iNOS at Day 4, scale bars: 100 μm. (G) H&E staining of wound sections at Day 4. Scale bars: 100 μm.
As shown in Figure 9B, on Day 0 post-injury, all groups exhibited characteristic signs of wound infection, including exudation, erythema and tissue degradation, indicating successful establishment of the infected diabetic wound model. Over time, the therapeutic effects of each treatment became increasingly evident. In the control group, wounds remained swollen and covered with thick scabs throughout the 10-day period, with evident signs of unresolved inflammation and poor closure, highlighting the impaired healing process under hyperglycemic and infectious conditions. In the US group, slight improvements in wound appearance were observed, potentially attributable to the mechanical vibration and mild thermal effects induced by ultrasound, which may transiently improve local circulation and reduce bacterial load. However, substantial scabbing and delayed closure were still observed on Day 4 and 7, suggesting limited antibacterial and regenerative efficacy. The UiO-66-treated group showed moderate reductions in inflammation and a visible acceleration of tissue regeneration. Although signs of infection persisted during the early stages, granulation tissue formation and wound edge contraction became prominent by Day 7, indicating a certain degree of pro-healing activity. Most notably, the UiO-66+US group demonstrated the most effective wound resolution. As early as Day 1, reduced exudate and attenuated infection were observed compared to other groups. By Day 4, a dry, intact scab had formed, providing effective wound coverage and protection. By Day 10, the wounds in this group were nearly completely healed, with restored skin tone and minimal residual scarring. Figure 9C presents the quantitative analysis of wound closure across different treatment groups, further illustrating that the UiO-66+US group exhibited significantly enhanced wound healing capacity as early as Day 4.
On Day 4 post-treatment, bacterial plating and histological analyses were performed on wound tissues. The plating results (Figure 9D) showed a significant reduction in bacterial load in the UiO-66+US group, the antibacterial rate reached 92.02 ± 2.65% (Supplementary Figure S15). Consistent with the antibacterial outcomes, Giemsa staining (Figure 9E) showed a markedly reduced bacterial burden in the UiO-66+US group, as indicated by the sparse distribution of purple-stained cocci and the absence of dense bacterial aggregates that were prominent in the control, US and UiO-66 groups. This substantial reduction in bacterial colonization confirms the enhanced in vivo antibacterial efficacy of the combined treatment.
Additional histological assessments further corroborated the resolution of infection and inflammatory responses. iNOS immunohistochemistry (Figure 9F), a classical indicator of M1-type pro-inflammatory activation, revealed intense iNOS-positive staining throughout the wound bed in the control and US groups, reflecting persistent inflammatory stress. In contrast, the UiO-66+US group exhibited markedly diminished iNOS expression, indicating effective suppression of inflammation. The UiO-66 group showed an intermediate phenotype with partial reduction of iNOS levels. H&E staining (Figure 9G) provided further structural evidence of tissue recovery. The control and US groups exhibited extensive inflammatory cell infiltration, disrupted epithelial continuity and disorganized extracellular matrix architecture, characteristic of unresolved infection. The UiO-66 group displayed reduced inflammatory infiltration and early signs of granulation tissue formation. In striking contrast, the UiO-66+US group presented minimal inflammatory cell presence, restored epithelial integrity and well-organized collagen and stromal structures, closely resembling the early regenerative stage. These findings collectively demonstrate that the combined UiO-66+US treatment not only suppresses bacterial burden but also accelerates inflammation resolution and promotes coordinated tissue repair.
Meanwhile, the expression of TGF-β was examined on Day 4 (Figure 10A). As a key regulatory factor in tissue repair, TGF-β plays a crucial role in modulating cell proliferation, differentiation and extracellular matrix production. The results showed that TGF-β expression was significantly upregulated in both the UiO-66 and UiO-66+US groups compared to the control, suggesting activation of regenerative signaling pathways and initiation of the tissue repair process (Figure 10E). The elevated TGF-β expression observed in the US group may be attributed to ultrasound-induced microenvironmental modulation via mechanical and thermal effects [59].
*Histological and immunohistochemical analysis after different treatments. (A) Immunohistochemical staining of TGF-β on Day 4; scale bars: 100 μm. (B) Masson staining for collagen deposition on Day 10; scale bars: 100 μm. (C) Immunohistochemical staining of COL-1 on Day 10; scale bars: 100 μm. (D) Immunofluorescence staining of CD31 on Day 10. Scale bars: 100 μm. (E) Quantitative analysis of TGF-β expression area (%) on Day 4. (F) Quantitative analysis of collagen fiber area (%) on Day 10. (G) Quantitative analysis of COL-1 expression area (%) on Day 10. (H) Quantitative analysis of CD31-positive area (%) on Day 10. **P < 0.01, **P < 0.001.
On Day 10, Masson’s trichrome staining (Figure 10B) was used to assess collagen deposition and tissue structure. The Ctrl group showed loose tissue with sparse blue staining and extensive red areas, indicating delayed healing. The US group exhibited slight improvement, but collagen remained limited. In contrast, the UiO-66 group showed denser and more extensive blue staining, suggesting enhanced regeneration. The UiO-66+US group demonstrated the most abundant collagen and well-organized structure, confirming accelerated matrix maturation and tissue repair with the combined treatment (Figure 10F).
In parallel, immunohistochemical staining of COL-1 was performed to assess collagen synthesis (Figure 10C). The control and US groups exhibited weak COL-1 positivity, indicating limited collagen production. In contrast, the UiO-66 group showed stronger brown staining, suggesting a moderate enhancement in type I collagen expression. Most notably, the UiO-66+US group demonstrated the most intense and uniformly distributed COL-1 signal, indicating that the combined treatment significantly promoted collagen synthesis and extracellular matrix remodeling (Figure 10G). This enhancement likely contributes to improved tissue mechanical integrity and accelerated wound healing.
Furthermore, to evaluate the impact of different treatments on angiogenesis, CD31 immunofluorescence staining was performed on Day 10 (Figure 10D). In the Ctrl and US groups, only weak CD31 signals and sparse neovessel formation were observed, indicating limited angiogenic activity. The UiO-66 group showed a moderate increase in CD31 expression, suggesting a partial enhancement in vascular development. Strikingly, the UiO-66+US group exhibited the most intense CD31 fluorescence, with dense and well-organized capillary networks, highlighting a robust pro-angiogenic response in vivo (Figure 10H). These findings are further supported by our in vitro experiments, in which UiO-66 demonstrated strong tube formation ability, confirming its intrinsic potential to promote angiogenesis. The consistency between in vitro and in vivo results underscores the pivotal role of UiO-66 in stimulating endothelial activity. Importantly, the enhanced neovascularization observed in the UiO-66+US group is likely to improve local perfusion and oxygenation, thereby accelerating tissue regeneration and wound healing. In parallel, the in vivo biosafety of UiO-66 was thoroughly evaluated to ensure its suitability for therapeutic use. Major organs—including the heart, liver, spleen, lung and kidney—were collected on Day 10 for H&E staining. As shown in Supplementary Figure S16, no histopathological abnormalities, inflammatory infiltration or structural damage were detected in any group, demonstrating that UiO-66 exhibits excellent systemic biocompatibility under the current dosing regimen.
Together, these results demonstrate that the combination of UiO-66 and ultrasound not only facilitates bacterial clearance but also modulates the inflammatory microenvironment and accelerates tissue regeneration, offering a promising therapeutic strategy for infected diabetic wounds.
Conclusion
In this study, we introduced a new perspective by demonstrating that pristine, unmodified UiO-66 itself can serve as an effective sonosensitizer for antibacterial therapy and skin regeneration. Our in vitro results confirmed that UiO-66 generates substantial ROS under ultrasound, enabling potent antibiotic-free antibacterial activity. Prokaryotic RNA sequencing further revealed that the bactericidal mechanism involves membrane disruption and broad suppression of metabolic, biosynthetic and energy-related pathways. Recent progress in MOF-based sonodynamic therapy has largely relied on structural engineering or electronic modification—such as Ag-decorated MIL@Ag heterostructures [27], MXene–porphyrin Schottky composites (PCMX) [34] and other metal-incorporated MOF nanocomposites [60]—to enhance charge separation and improve catalytic efficiency. While these systems achieve strong SDT performance, they typically require complex multistep synthesis and introduce additional components whose biological contributions must be carefully evaluated. By contrast, our findings highlight that pristine UiO-66 alone exhibits intrinsic sonodynamic activity and notable pro-regenerative potential, despite lacking any architectural or compositional enhancement. UiO-66 is easy to synthesize, highly stable and biocompatible, yet still capable of promoting angiogenesis, collagen deposition and accelerated wound closure in a diabetic infected-wound model. These results reveal previously underappreciated inherent properties of zirconium-based MOFs and position UiO-66 as a simple yet powerful foundation for future SDT-based wound-healing platforms. We acknowledge that the in vivo study did not include a clinical positive-control treatment, which limits direct comparison with existing therapeutic standards. However, the primary aim of this work was to establish the intrinsic bioactivity of unmodified UiO-66 rather than benchmark it against optimized or clinically deployed agents. Future studies will incorporate standard antibacterial therapies or established sonosensitizers as comparative controls to further evaluate the translational potential of UiO-66. Collectively, this work not only provides a promising antibiotic-free strategy for managing infected diabetic wounds, but also establishes a fundamental rationale for subsequent functionalization of UiO-66 to develop next-generation, high-performance MOF-based therapeutic platforms.
Supplementary Material
rbag012_Supplementary_Data
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