Ultrasound-activated piezo-hot carriers trigger tandem catalysis coordinating cuproptosis-like bacterial death against implant infections – Nature Communications

Chemicals

Barium carbonate (99%), titanium dioxide (99%), calcium carbonate (99.9%), zirconium dioxide (99.99%), cupric sulfate (99%), and nano HA were from Sinopharm Chemical Reagent Co., Ltd. (China). Dopamine hydrochloride (DA-HCl), tri(hydroxymethyl) amino methane hydrochloride (Tris-HCl, 1 M), methylene blue (MB), vancomycin, Calcein, and ARS were from Sigma Chemical Co. (USA). SOSG was from Invitrogen (USA). The Bacterial Live/Dead Bac Light viability kit was from Thermo Fisher (USA). Calcein/PI Cell Viability/Cytotoxicity Assay Kit, CCK-8 kit, and ARS dye were purchased from Beyotime (China).

Synthesis of CpBT nanoreactors

The mBT was prepared in the following steps: Weigh the elemental components according to the chemical formula Ba_0.90Ca_0.10Ti_0.91Zr_0.09. Ball-mill the weighed components in nylon jars for 24 hours. Calcined the obtained powders at 1260 ^oC for 3 hours. Sand-milled the calcined powders at 2000 revolutions per minute for 4 hours to obtain mBT. Then, 1 mg mL^-1 mBT was dispersed in the Tris-HCl solution (10 mM, pH = 8.5) and sonicated for 40 min. DA-HCl was added and sonicated for another 10 min and stirred for 2 hours. After centrifuging and washing with DI water for three times, pBT nanoparticles were obtained. Then, 10 uM cupric sulfate solution was added to pBT and stirred for 2 hours, and then were collected by centrifugation and washed with DI water for three times to obtain CpBT nanoreactor.

Preparation of PH-CpBT scaffold

3D printing technology utilizing fused deposition modeling was employed to fabricate PEKK scaffolds. The design of the structures was accomplished using Materialise 3-Matic software, resulting in bone scaffolds measuring 3 mm in diameter and 4 mm in height for in vivo experiments, as well as 10 mm in diameter and 1 mm in height for in vitro experiments involving cells and bacteria. Medical-grade PEKK filaments were extruded into the deposition bin of the 3D printer, enabling layer-by-layer preparation of the scaffolds according to pre-determined shapes at a temperature of 200 ^oC. The PEKK scaffold was stirred in Tris-HCl solution (10 mM, pH = 8.5) containing 3 mg ml^-1 DA-HCl for 24 hours to obtain PEKK@pDA (Pp). Then Pp scaffold was immersed in HA solution (2 mg ml^-1) for 12 hours to get Pp@HA (PH). The scaffolds of PH-pBT and PH-CpBT were constructed by soaking PH scaffolds in different solutions (2 mg ml^-1) for 12 hours.

Characteristics

The crystal structures were unveiled through XRD utilizing Cu Kα radiation (λ = 1.5406 Å) (Empyrean, Malvern Panalytical, UK). The SEM (SUPRA 55, Carl Zeiss AG, Germany) was used to examine the surface morphologies. High-resolution portraits and the related lattice fringes were captured by HRTEM (Talos F200i, FEI, USA). Raman spectra were examined by Raman spectroscopy with an excitation source of 532 nm (Invia Reflex, Renishaw, UK). The chemical state and valence band were obtained by XPS (K-Alpha + , Thermo Fisher, USA). The switching spectroscopy piezo-response force microscopy loops were collected by a commercial atomic force microscope (MFP−3D, Asylum Research, UK). The temperature-dependent dielectric constant (ε_r–T) was obtained via an LCR meter (TH2816A, Tonghui, China). The 5982 Universal testing machine (Instron, USA) was used to perform the compression test. The contact angle of the surfaces of scaffolds was measured by A JY-82C contact angle apparatus (Dingsheng Testing Equipment Co. Ltd., China). Then, the concentration of Cu was obtained by an inductively coupled plasma-optical emission spectrometer (ICP-OES, model 5100, Agilent, USA). Cu²⁺ ions released from PH-CpBT scaffolds were analyzed by an ICP spectrometer (ICP-MS, 7850, Agilent, USA). Specifically, PH-CpBT scaffolds were immersed in a 0.9 % NaCl solution at 37 ± 1 °C with the surface-area to -volume ratio was 3 cm² mL^-1 according to the international standard ISO 10993-12. Triplicate samples were used to obtain an average value with standard deviation.

Detection of ROS in vitro

The ROS generated from the samples when exposed to US stimulation were tested using MB, SOSG, and ESR. To assess •OH production, different samples were mixed with a solution containing MB and subjected to US stimulation (1 MHz, 1.0 W cm^-2, 50% duty cycle) with or without H₂O₂ (50 μM). The changes in absorption of MB at 664 nm before and after US stimulation were recorded using the ultraviolet and visible spectrophotometer (UV-5200, METASH, China). Similarly, the presence of singlet oxygen was confirmed by the fluorescence intensity of SOSG at 525 nm using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). The identification of ROS species was performed using an ESR spectrometer (JES-FA200, JEOL, Japan). For trapping the singlet oxygen, we utilized 2,2,6,6-tetramethylpiperidine (TEMP) at a concentration of 50 mM, while for detecting •OH, we employed 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) at a concentration of 0.1 mM.

Detection of Cu⁺ of CpBT

Neocuproine (Aladdin, China) was selected as an indicator for detecting in-situ Cu⁺ production. Briefly, 1.04 mg of neocuproine was dissolved in 5 mL of ethyl alcohol and then diluted five times with ultrapure water. Subsequently, the buffer solution at pH 6.5 was prepared by dissolving KH₂PO₄ and NaOH in ultrapure water. CpBT dispersion at a concentration of 400 μg mL^-1 was prepared by dissolving itself in ultrapure water, and then the final working solution, consisting of 0.75 mL of buffer solution, 1.0 mL of CpBT solution, and 0.8 mL of neocuproine solution, was irradiated under sonication for 0-12 min (1 MHz, 1.0 W cm^-2, 50% duty cycle). Finally, the absorption of the reaction solution was measured by a UV-vis absorbance spectrometer at 452 nm.

Simulation details

The dimer, trimers, and tetramers structures of pDA employed in the current study were extracted from the most stable geometry among all the structures generated using a brute-force algorithmic generator³⁷. The layered aggregates consistent of the stacked dopamine (DA) and dihydroxyindole (DHI) via π-π interactions³¹. The pDA-Cu²⁺/Cu⁺ composites were constructed by chelating Cu²⁺/Cu⁺ ions near the dihydroxyl site^38,39. Unless otherwise specified, the Becke three-parameter Lee-Yang-Parr (B3LYP) functional was employed to optimize the molecular structure and generate the wavefunction, together with the def2-TZVP basis set. The BLYP functional combined with the 6−31+g (d, p) basis set was employed to optimize the structures of the layered aggregates and analyze the wavefunction. To account for weak interactions in the layered aggregates systems, the dispersion correction DFT-D3 with Becke-Johnson damping (D3BJ) was implemented⁴⁰. The Pipek-Mezey method⁴¹ was chosen to localize occupied molecular orbitals (MOs) and differentiate σ and π characters. For investigating the delocalization channel of π electrons, the LOL-π variant of the localized orbital locator (LOL) function was utilized^27,28. All quantum chemistry calculations were performed using the Gaussian16 program⁴², while wavefunction analysis was conducted using the Multiwfn 3.8 (dev) code⁴³. Isosurface graphs were generated using the VMD program for improved visual representation. To investigate the quantum transport properties of multilayer stacked pDA, we designed a vertical transport architecture device with Au electrodes being the source and drain contacts, and pDA was acted as the transport channel region. The simulations were performed using the first-principles methods, with the combination of the NEGF-DFT²⁹, conducted in the first-principles quantum transport software package Nanodcal⁴⁴. The local density approximation (LDA) was embraced to portray the exchange and correlation function, and atomic cores were defined by the standard norm-conserving nonlocal pseudo potentials⁴⁵. A double-ϛ polarized (DZP) atomic orbital basis⁴⁶ was conducted for Au metal electrode and pDA to expand all physical quantities with a kinetic energy cutoff of 4500 eV. Furthermore, a k-point mesh of 3 × 3 × 1 and 17 × 17 × 1 was applied to sample the first Brillouin zone for integrations in the reciprocal space of the scattering region (pDA) and Au electrode, respectively. In addition, self-consistent calculations were converged until each component of the density matrix have declined to 10⁻⁵ Hartree. Employing the Landau-Ginsburg-Devonshire phenomenological model, the Landau free energy was expressed as △G = α₁(P₁² + P₂² + P₃²) + α₁₁(P₁⁴ + P₂⁴ + P₃⁴) + α₁₂(P₁²P₂² + P₂²P₃² + P₁²P₃²) + α₁₁₁(P₁⁶ + P₂⁶ + P₃⁶) + α₁₁₂[P₁⁴(P₂² + P₃²) + P₂⁴(P₁² + P₃²) + P₃⁴(P₁² + P₂²)] + α₁₂₃P₁²P₂²P₃², where α₁, α₁₁, α₁₂, α₁₁₁, α₁₁₂ and α₁₂₃ represented Landau energy coefficients. Finite element method calculations were carried out with COMSOL Multiphysics 5.4 with a model of a piezoelectric device based on a steady-state study.

Culturing of bacteria

Gram-positive S. aureus (ATCC 25923) and Gram-negative E. coli (ATCC 25922) were cultured in a sterile Luria-Bertani (LB) medium (10 g L⁻¹ of back to-tryptone,10 g L⁻¹ of NaCl, and 5 g L⁻¹ of bacto-yeast extract). The bacterial counts were obtained from the spread plate of different samples.

Antibacterial assessment in vitro

The effectiveness of Pp, PH, PH-pBT, and PH-CpBT against S. aureus and E. coli with and without US stimulation was assessed using the spread plate method. The different scaffolds were exposed to bacterial suspensions (2×10⁷ CFU mL^-1) in 48-well plates for specified durations and subjected to US stimulation (1 W cm^-2) for 9 mins or left untreated. Bacterial growth was cultured on agar plates at 37 °C for 18 h using the spread plate method to quantify colony-forming units (CFUs). To simulate the high H₂O₂ environment in vitro, PH-CpBT+H₂O₂ was supplemented with 50 μM H₂O₂. Furthermore, under identical conditions, antibacterial experiments were conducted to compare copper chelator (TTM, 20 μM, Aladdin, China), copper release from the scaffold, the coating of mBT and copper ions (PH-mBT/Cu), and similar materials (commercial TiO₂, ZnO, MoS₂, and BT, Aladdin, China). The adherent bacteria on PEKK were fixed, dried, dehydrated, and coated with gold. SEM (ZEISS Gemini 300, Germany) was employed to examine bacterial morphology and integrity. Bacteria treated with US and PH-CpBT+H₂O₂ + US were collected, cryo-centrifuged at high speed for 2 mins, embedded into blocks, and sliced into 50 nm thick sections using an ultrathin microtome (EM UC7, Leica, Germany). Subsequently, TEM images were captured after staining the samples with uranyl acetate and lead citrate, and placing on copper grids. After 5 days of bacterial cultivation on various scaffolds, a live/dead staining assay was conducted employing the Live/Dead BacLight viability kit. The data were collected by the confocal laser scanning microscopy (CLSM, N-SIM S, Nikon, Japan). Moreover, intracellular ROS levels were measured using the ROS assay kit (Beyotime, China) four hours after coculturing with different scaffolds, and the outcomes were also visualized using CLSM. The assessment of bacterial DNA damage induced by various samples utilized the Bacterial DNA Kit (Beyotime, China). After treatment, bacteria underwent collection through centrifugation at 5000× g for 5 mins at 4 °C. Following the manufacturer’s instructions, the Bacterial DNA kit was employed for the purification of intact DNA strands from the bacteria. Subsequently, complete DNA fragments were subjected to quantitative analysis using a UV-vis spectrophotometer. This comprehensive methodology aimed to discern and quantify the extent of DNA damage caused by the diverse samples. The supernatant obtained by centrifugation was detected by BCA Protein Quantitation Assay (Beyotime, China), and the bacterial leakage protein was quantified by a microplate reader.

Transcriptome analysis

S. aureus was cultivated with PH-CpBT with and without US stimulation. The bacteria were collected by cryo-centrifugation during the logarithmic phase and immediately frozen in liquid nitrogen. Three sets of biological replicates were carried out under identical conditions: the control group (US-1, US-2, US−3), the experimental group 1 (PH-CpBT+H₂O₂-1, PH-CpBT+H₂O₂-2, PH-CpBT+H₂O₂−3), and the experimental group 2 (PH-CpBT+H₂O₂ + US-1, CpBT+H₂O₂ + US-2, CpBT+H₂O₂ + US−3). Total RNA isolation and cDNA library construction were executed according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq platform at Majorbio Bio-pharm Technology Co., Ltd. The expression quantification results were subjected to analysis using DESeq2 (Version 1.24.0) software to discern DEGs with a screening threshold of |log2FC | ≥ 1 and a p-value < 0.05. We utilized Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) databases to elucidate the biological implications of DEGs and explain gene functional differences between samples. Furthermore, we conducted KEGG pathway analysis by KOBAS. The significance of KEGG pathways was evaluated through a Fisher’s exact test.

Metabonomics analysis

The sample preparation for the metabolomic analysis was performed as described in transcriptome analysis, and metabolomics analysis was performed under the same conditions for each group of six biological replicates. The experimental procedure was as follows: preparation of the central carbon metabolite standard solution; pre-processing of the standard curve, treatment of the bacterial cell precipitate samples, and qualitative and quantitative detection of target compounds in the samples using LC-ESI-MS/MS (UHPLC-Qtrap) with the ExionLC AD system; Chromatographic analysis was performed using the Waters HSS T3 column (2.1 ×150 mm, 1.8 μm); Mass spectrometry analysis was conducted using the SCIEX QTRAP 6500+ in both positive and negative modes. Finally, the Sciex OS quantitative software was utilized for the automatic identification and integration of ion fragments to analyze the data.

Biocompatibility assessment in vitro

Mouse osteoblastic MC3T3-E1 cells (GNM15) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The HUVECs (CP-H082) were purchased from Procell Life Science & Technology Co., LTD. (Wuhan, China). MC3T3-E1 cells cultured with α minimum essential medium (α-MEM, Gibcom, USA), including 10% fetal bovine serum (FBS, Gibcom, USA) and 1% penicillin-streptomycin solution. The cultures were maintained in a humidified atmosphere incubator with 5% CO₂ at 37 °C for in vitro cytocompatibility and osteogenic differentiation studies.

Cell proliferation and spreading assay

Cell viability and proliferation were quantified through a CCK-8 assay. Various scaffolds were co-cultured with cells, and the optical density (OD) at 450 nm was gauged on days of 1, 3, and 5 using a microplate reader. Moreover, cells were seeded onto different scaffolds for 3 days, and their viability was gauged using a Calcein/PI Cell Viability/Cytotoxicity Assay Kit, followed by observation using laser scanning CLSM. MC3T3-E1 cells were seeded onto different scaffolds in a 24-well plate for 24 h, and the cells were stained with fluorescein isothiocyanate-phalloidin and 4,6-diamidino-2-phenylindole, then the arrangement of F-actin and the cell nuclei were then observed using CLSM.

Osteogenic differentiation and angiogenesis in vitro

MC3T3-E1 cells were seeded on the surface of different scaffolds. After 24 h, the culture medium α-MEM was replaced with an osteoinductive medium containing 10 mM β-glycerophosphate, 50 µg mL^-1 ascorbic acid, and 10 nM dexamethasone (all from Sigma, USA) to prompt the osteogenic commitment of MC3T3-E1 osteoblasts. This osteoinductive medium was renewed every 3 days. On days 7 and 14, the activity of ALP was assessed using a 5-bromo-4-chloro−3-indolyl phosphate/nitro-blue tetrazolium (BCIP/NBT) ALP color development kit (Beyotime, China). For the evaluation of calcified extracellular matrix, cells were fixed and treated with ARS dye on days 14 and 21. Then subsequent to the removal of excess dye, images were captured using a scanner. HUVECs were cultured for in vitro angiogenesis investigation. The suitable cells and samples were introduced into the μ-Slide 15 well 3D (Ibidi, Germany) and allowed to incubate for 6 hours. Afterward, the cells were stained with Calcein and subsequently imaged using a fluorescence microscope.

In vivo experiments

Male SD rats (200–220 g, about two-months old) were bought from the Beijing Huafukang Bioscience Cojnc. Animal experimentation in this study received ethical approval from the Laboratory Animal Ethics Committee of West China Hospital, Sichuan University (IACUC number 20221216022). All rats were raised in 25 ± 3 °C (temperature), 60-70% (humidity), and 12 h light/dark cycle conditions for two weeks before the experiments. SD rats (n = 8 per group) were divided randomly into seven groups: Pp, PH, PH-pBT, PH-mBT/Cu, PH-CpBT(-), PH-CpBT, and Van.. Except for PH-CpBT(-) and Van., other groups underwent ultrasound intervention. To create an implant-related contamination model, the engineered PEKK implants (⦶ 3 mm × 4 mm) were immersed in S. aureus (2 × 10⁷ CFU ml^-1) solution at 37 °C for 4 hours. Subsequently, a rat lateral femoral condyle model for cylindrical bone defect repair was created, and the implants with S. aureus were implanted in the bilateral femoral condyles. After 1st, 4th, and 8th weeks, the rats were sacrificed to assess the antibacterial effect and new bone formation.

Antibacterial activity in vivo

Starting from the first to sixth days after surgery, rats were exposed to US stimulation (1 MHz, 1.0 W cm^-2, 50% duty cycle) for 8 mins under gas anesthesia induction. On the seventh day post-surgery, the rats were sacrificed, and the implants were removed and placed in sample collection tubes containing PBS to collect bacteria. Dilutions were cultured on agar plates to assess implant infection. Moreover, bone tissue around the scaffolds was gathered for histological analysis, including H&E and Giemsa staining. These sections were observed and captured using an inverted microscope (Olympus BX53, Japan).

The volume of new bone and bone growth rate

To evaluate the volume of new bone and trabecular thickness within the implants, femoral condyles were scanned using the Quantum GX Micro CT (PerkinElmer, USA). The 3D reconstruction of the CT images was generated by Imaris 9.9 (BitPlane, Oxford Instruments). Following reconstruction, parameters such as BV, Tb. Th, % BS/BV, % BV/TV, Tb. N, and BMD were quantified using Skyscan NRecon software. For the assessment of new bone growth rate, ARS (30 mg kg^-1) and Calcein (20 mg kg^-1) were intraperitoneally injected into the rats at 4th and 6th week after implantation. Infected rats were euthanized at 8th week post-surgery, and their implants and surrounding bone were fixed, sliced, and examined using CLSM.

The microstructure of new bone ingrowth: histomorphometry, immunohistochemistry, and immunofluorescence

Histological sections were generated parallel to the long axis of the implants around the undecalcified femoral condyle before decalcification. Sections were ground to 100 μm thickness, and slides were polished and stained with H&E and Goldner. Additionally, other bone samples without implants were fixed, decalcified, dehydrated, and embedded in paraffin. The tissues were cut into sections, being prepared for IHC staining (TNF-α, 1:200, Abcam ab1793) and 4-color immunofluorescence staining with the following primary antibodies (Abcam, UK): CD31 (1:500, ab182981), RUNX2 (1:200, ab236639), BMP-2 (1:200, ab214821), and DAPI (1:1000, ab285390) at 4 °C overnight. Finally, 4-color immunofluorescence staining was performed and visualized with Vectra Polaris (PerkinElmer, USA).

Statistics analysis

Data were presented as mean values ± standard deviations (SD); error bars = SD. Statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison with statistical significance assigned at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-45619-y