Materials

All raw materials were directly used without further purification. TmCl₃·6H₂O (≥99.99%), YCl₃·6H₂O (≥99.99%), YbCl₃·6H₂O (≥99.99%), 1-octadecene (ODE, ≥90%), oleic acid (OA, ≥85%), sodium fluoride (NaF, ≥99.99%), cetyltrimethylammonium bromide (CTAB, ≥99%), tetraethyl orthosilicate (TEOS, ≥99.99%), 1,2-bis(triethoxysilyl)-ethane (BTEE, ≥96%), sodium salicylate (NaSal, ≥99.5%), triethanolamine (TEA, ≥99.5%), Cyanamide (≥95%), sodium carbonate(Na₂CO₃, ≥99.99%), sodium perchlorate (NaClO₄, ≥99%), Hydrated ruthenium chloride (RuCl₃·nH₂O, ≥99.98%), N,N-Dimethylformamide (DMF, ≥99.9%), 2,2′-Bipyridine (bpy, ≥99%), 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA, ≥90%), isopropanol (IPA, ≥99.9%), potassium bromate (KBrO_3, ≥ 99.99%), ammonium oxalate ((NH₄)₂C₂O₄, ≥99.99%) and 4-Hydroxy-TEMPO (TEMPOL, ≥98%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd Lyticase (L4025, ≥200 units mg⁻¹ solid) was from Sigma Aldrich. Phosphate-buffered saline (PBS), normal saline (0.9% NaCl, sterile), dimethyl sulfoxide (DMSO, ≥99.9%), propidiumiodide (PI, ≥95%), 4′,6-diamidino-2-phenylindole, dimethyl (DAPI, C1002), reactive oxygen species assay kit (DCFH-DA, S0034S), and MTT cell proliferation and cytotoxicity assay kit (C0009M) were obtained from Beyotime Biotechnology (Beijing, China). ConA-FITC (MP6321) was obtained from MKBio Co., Ltd (Shanghai, China). Sabouraud Dextrose Broth (SDB, 021096) medium and Nutrient agar (NA, 021097) were purchased from HuanKai Microbial. Cyclophosphamide (CTX, 97%) and collagenase IV (C8160) were purchased from Solarbio (Beijing). Cell culture dishes/plates and centrifuge tubes were purchased from NEST Biotechnology Co., Ltd (Wuxi, China).

Bacterial strains and cell lines

Candida albicans (SC5314) was obtained from American Type Culture Collection Center. The proliferation of Candida albicans was in SDB liquid medium. The Human umbilical vein endothelial cell line (HUVEC, SCSP-5285) and human immortalized keratinocyte cell line (HaCaT, SCSP-5091) were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences. The human cells were cultured in DMEM (Gibco BRL) containing 10% fetal bovine serum (FBS, Gibco BRL) at 37 °C in 5% CO₂/95% air.

Synthesis of NaYF₄:Yb/Tm upconversion nanoparticles

YCl₃·6H₂O (0.78 mmol, 236.6 mg), YbCl₃·6H₂O (0.215 mmol, 83.4 mg), TmCl₃·6H₂O (0.005 mmol, 1.9 mg) were added in a 100 mL three-necked round-bottom flask and mixed with OA (31 mmol) and ODE (47 mmol). The mixture was stirred and heated to 150 °C for 40 min, forming a clear solution. After cooling to room temperature, a methanol solution (12.5 mL) containing NaOH (2.5 mmol, 100 mg) and NH₄F (4 mmol, 148.2 mg) was injected. The mixture was stirred for 45 min, followed by vacuum heating at 130 °C for 10 min. Subsequently, the solution was heated to 290 °C under an N₂ atmosphere and maintained at this temperature for 90 min. After natural cooling to room temperature, 20 mL ethanol was added to precipitate the nanoparticles and centrifuged in 3500 × g for 10 min. The contents were washed with cyclohexane (5 mL) and ethanol (5 mL) two times, respectively. Then re-dispersed in 10 mL of cyclohexane (≈15 mg mL⁻¹).

Synthesis of silica coated upconversion nanoparticle (UCNP@mSiO₂)

CTAB (100 mg) was dissolved in 20 mL of deionized water, then cyclohexane solution containing NaYF₄: Yb/Tm UCNPs (2 mL, ≈30 mg) was injected under vigorous stirring. When a transparent solution was obtained, the solution was transferred to a 100 mL round-bottom flask and mixed with water (40 mL), ethanol (6 mL), NaSal (40 mg), and TEA (50 μL). Then, BTEE (60 μL), TEOS (40 μL) were added dropwise, and the mixture was stirred overnight. The product was collected by centrifugation in 12,000 × g for 15 min, washed with 10 mL ethanol, recollected using the same centrifugation process and dispersed in 10 mL (≈1 mg mL^-1) ethanol.

Synthesis of hollow silica coated upconversion nanoparticle (UCNP@hmSiO₂)

UCNP@mSiO₂ (≈20 mg) was dispersed in a solution containing CTAB (120 mg), ethanol (12 mL), water (38 mL), and TEA (30 μL). After ultrasonic dispersion for 1 hour and stirring at room temperature for 1 hour, a mixture of TEOS (100 μL) and BTEE (100 μL) was quickly added to the suspension. After stirring at 25 °C for 16 h, a double-layer silica modified UCNP was obtained. Then, the obtained nanoparticles were dispersed in 450 mL of water and subjected to 12 h of hydrothermal treatment at 100 °C. After removing the surfactant through 60 mL HCl/ethanol (1:10) extraction, UCNP@hmSiO₂ (≈10 mg) nanoparticles were collected by centrifugation in 12,000 × g for 30 min.

Synthesis of carbon nitride coated upconversion nanoparticle (UCNP@g-C₃N₄)

UCNP@hmSiO₂ (≈20 mg) was dispersed in a 4 mL aqueous solution containing 2 g of cyanamide, after stirring at room temperature for 24 h. The product was centrifuged in 12,000 × g for 30 min and transferred to a Porcelain boat. Then the mixture was heated to 550 °C at a rate of 5 °C min⁻¹ under N₂ atmosphere for 4 h. The UCNP@g-C₃N₄ was obtained after reacted in 12 mL Na₂CO₃ solution (280 mg, 0.2 mol L⁻¹) at 60 °C for 16 h.

Synthesis of ruthenium(II) complex

The Ru complex was synthesized according to our previously report⁵⁴. Cis-[Ru(bpy)₂Cl₂]·2H₂O (262 mg, 0.5 mmol) was dissolved in 20 mL methanol aqueous solution (methanol/water = 4/1, v/v), and 2-(3-carboxy-4-hydroxyphenyl)imidazo[4,5-f]phenanthroline (177.5 mg, 0.5 mmol) was added under N₂ atmosphere, refluxing for 12 h at 80 °C in darkness. After the reaction completion, the solvent was evaporated. Then, 5 mL saturated sodium perchlorate solution was added to the mixture to give a participate. After stirring for 0.5 h, red solid was obtained by filtering and washed with 20 mL distilled water and ether, respectively (343 mg, yield 70%). ¹H NMR (400 MHz, DMSO-d₆) δ: 9.36 (d, 1H), 9.11 (d, 1H), 8.94 (s, 1H), 8.89 (d, 2H), 8.85 (d, 2H), 8.17-8.31 (m, 3H), 8.11 (t, 2H), 8.02 (d, 2H), 7.94–7.84 (m, 4H), 7.67–7.56 (m, 4H),7.37 (t, 2H), 6.88 (d, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 171.45, 163.65, 157.22, 157.02, 151.94, 151.83, 149.94, 145.21, 138.39, 138.25, 132.87, 132.68, 132.47, 131.09, 130.74, 129.14, 128.32, 128.19, 126.42, 124.83, 123.65, 119.63, 118.96, 116.84, 116.36. HRMS (ESI) calculated for C₄₃H₃₆N₈O₃Ru [M-2ClO₄]²⁺: 385.0664; found: 385.0663.

Synthesis of ruthenium complex loaded carbon nitride coated upconversion nanoparticle (UCNP@g-C₃N₄-Ru)

UCNP@g-C₃N₄ (5 mg) was carefully grinded with an agate mortar for 1 h and dispersed in 5 mL water, then 0.5 mL ethanol solution of Ru complex (2 mg) was added. After vigorously stirring overnight, the final product was collected by centrifugation in 6000 × g for 10 min.

Loading amount of Ru complex

The prepared UCNP@g-C₃N₄-Ru were dispersed in 5 mL DMSO and treated under ultrasound for 1 h and soaked for another 24 h. The nanoparticles were centrifuged in 6000 × g for 10 min, and the supernatant was collected. The amount of Ru complex was determined by characteristic UV absorbance at 460 nm.

Detection of ROS generation

The generation of singlet oxygen (¹O₂) was detected by ABDA. When compared the singlet oxygen generation capacity of UCNP@CR and Ru complex, a 460 nm blue LED is used. DMPO was used as trapping agent when collecting ESR spectroscopy.

Electrochemical analysis

The linear sweep voltammogram (LSV) curves of the nanohybrid at different rotation speed was collected in O₂-saturated phosphate buffer (0.1 M, pH = 7) by a three-electrode system (Working electrode: glassy carbon electrodeg; Counter electrode: gold electrode; Reference electrode: saturated calomel electrode) using IGS-6030 electrochemical station (Guangzhou ingsens sensor technology Co., Ltd). The suspension was prepared by adding 2 mg UCNP@g-C₃N₄ in 0.5 mL ethanol solution containing 0.1% Nafion. The suspension was dropped on the working electrode and dried. The electron transfer number (n) was calculated by Koutecky-Levich (K-L) equation⁵⁵.

In the equation, i and i_k are the tested current and the kinetics current, respectively. And ω is the angular rotation rate of the electrode, n is the electron transfer number, F is the Faraday constant (96485 C mol⁻¹), A is the surface area of working electrode (0.196 cm²), D is the diffusion coefficient of O₂ (2.7 × 10⁻⁵cm²s⁻¹), C is the bulk concentration of O₂ in solution (1.3 × 10^-6mol cm⁻³), v is the kinetic viscosity of water (0.01 cm²s⁻¹).

Characterization

The structure of nanoparticles were observed using transmission electron microscope (JEM-1400 Plus and Thermo Scientific Talos F200i). The diameter distribution and zeta-potential of the nanoparticles (NPs) were determined by dynamic light scattering (DLS) using ZS Nano S (Malvern). UV-Vis diffuse reflectance spectra was collected by UV-2700. The powder X-ray diffraction (XRD) patterns were collected by ULTIMA IV (Rigaku) with Cu Kα radiation (λ  =  0.15418 nm). ¹H NMR and ¹³C NMR spectra were recorded on a Avance 400 III 400 MHz (Bruker) in d-dmso solution. X-Ray photoelectron spectroscopy (XPS) were collected by K-Alpha (Thermo Scientific) with Al Kα radiation (hv = 1486.7 eV), and all binding energies were referenced to the adventitious carbon C1s at 284.8 eV. The fourier transform infrared (FT-IR) spectral was measured by INVENIO R (Bruker). The UV-Vis absorption spectra and photoluminescence (PL) were obtained by Lambda UV365 (Perkin Elmer) and RF-6000 (SHIMADZU), respectively. The electron paramagnetic resonance (EPR) spectroscopy was tested using ELEXSYS-II E500 CW (Bruker). Upconversion emission spectra were measured on Aurora 4000 spectrometer under with fiber-coupled MDL-H-980 (CNI, China) as excitation sources. The portable sonicator was purchased from Shenzhen WELLD Co., Ltd.

Anti-fungal effect of nanoparticles in suspension

After culturing a single colony of Candida albicans in 50 mL SDB medium for 16 h at 30 °C, a density of 2 × 10⁶ colony forming units (CFU) mL⁻¹ was obtained after dilution. The suspension of fungi was incubated with different concentration NPs (0, 12.5, 25, 50, 100 µg mL⁻¹) for 4 h and treated with different condition (Light: 980 nm, 0.5 W cm⁻², 60 min; Ultrasound: 1.0 W cm⁻², 5 min). For the ultrasound (US) or light (L) group, the stimulation time is 0–15 min and 0–60 min, respectively. The anti-fungal effect of US + L was treated with 5 min ultrasound and 15 min light. After treatment the suspension was diluted 10-fold serially and spread 100 μL of dilutions on agar plate. The viability of bacteria was calculated by counting the number of colony forming unit after incubation at 30 °C for 16 h. The morphology of treated Candida albicans was observed by scanning electron microscope (Zeiss Sigma 300) after fixed with 2.5% glutaraldehyde and gradient dehydration.

Biofilm elimination performance

An in vitro biofilm model was used for evaluating treatment Candida albicans biofilm. First, 300 μL culture medium (RPMI1640/SDB = 4:1,v/v) suspension containing Candida albicans 5 × 10⁴ CFU mL^-1 was added to each hole in a 48 well plate. After culture for 16 h, remove the culture medium and replace with new 1640 culture medium, continue to culture for 12 h. Then carefully remove the culture medium, and rinse the surface with PBS solution. The biofilm was further incubated with 50 μg mL⁻¹ nanoparticles for 4 h and treat with different exogenous stimulation (Light: 980 nm, 0.5 W cm⁻², 30 min; Ultrasound: 0.5 W cm⁻², 150 s). In vivo experiment, infected wound of different groups were soaked with 15 μL PBS, the collected suspension was mixed with 190 μL PBS and spread on SDB plate. The number of colony-forming unit were calculated after 16 h.

Real time anti-fungal effect monitoring

The culture medium (RPMI1640/SDB = 4:1, v/v) containing Candida albicans at a density of 1 × 10⁴ CFU mL⁻¹ was incubated in 96-well plate for 12 h. After incubation with/without UCNP@g-C₃N₄-Ru (50 μg mL⁻¹) for 4 h, the fungi was treated by NIR, Ultrasound, NIR+Ultrasound (Light: 980 nm, 0.5 W cm⁻², 30 min; Ultrasound: 0.5 W cm⁻², 150 s) and further incubated for another 36 h. The fungal growth was evaluated by real-time monitoring of absorbance at 600 nm (OD₆₀₀) by microplate reader (INFINITE M NANO, TECAN) every 6 h.

Dead staining of fungi

The Candida albicans suspension of 1 × 10⁷ CFU mL⁻¹ or cultured biofilm was incubated with 50 μg mL⁻¹ UCNP@g-C₃N₄-Ru for 4 h. After NIR (980 nm, 0.5 W cm⁻², 15 min), Ultrasound (1 MHz, 1.0 W cm⁻², 5 min) or NIR+Ultrasound treatment, the corresponding fungi suspension or biofilm was washed with 200 μL PBS three times, stained with PI and DAPI for 10 min at 30 °C. The fluorescent images were captured using confocal fluorescence microscope (A1 + , Nikon).

Detection of reactive oxygen species levels in fungi

Fungi suspension and biofilm were obtained using the same condition as mentioned above. The intracellular ROS of fungi were detected by fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 mM). The fungi were incubated with/without NPs (50 μg mL⁻¹) for 4 h, then transfer the medium containing DCFH-DA and incubated for 20 min in dark. After giving the stimulation (Light: 980 nm, 0.5 W cm⁻², 15 min; Ultrasound: 1.0 W cm⁻², 5 min), the fungi were washed and observed using fluorescence microscope.

Adhesion and uptake of nanoparticles

The yeast and pseudohyphae of Candida albicans was growing on the slides of cells. Typically, 500 μL Candida albicans in RPMI1640/SDB culture medium (2 × 10⁴ CFU mL⁻¹) was seeded per well in 24 well plate for 12 h at 30 °C. Then, the culture medium was replaced with the medium containing Ru complex or NPs. After 4 h incubation, the fungi were washed and observed using confocal fluorescence microscope and high-resolution fluorescence microscope (N-SIM/N-STORM/TIRF, Nikon). The cell wall (1,3)-β-D glucan related impact on the anti-fungal effect and adhesion of the NPs were tested using the lyticase pretreated Candida albicans or lyticase pretreated NPs. The adhesion of NPs over time were obtained through flow cytometry.

Contribution of different reactive species

IPA (0.5 mM), TEMPOL (0.5 mM), KBrO₃ (0.5 mM), and (NH₄)₂C₂O₄ (0.5 mM) were used as scavengers to investigate the effects of generated •OH, •O₂⁻, e-, h+ in the fungi inactivation process, respectively.

Computational details

To derive reliable initial structures for the candidate complexes, a systematic conformational search was conducted utilizing crest 3.0⁵⁶. Subsequently, geometry optimizations were carried out using GFN2-xTB method with verytight keyword⁵⁷. The conformation with the lowest energy was then selected as the initial structure for subsequent calculations. All density functional theory (DFT) computations were executed using the Gaussian 16 rev. C.01 suite of programs⁵⁸. Geometry optimizations and frequency analyses for all compounds were performed at the B3LYP-GD3(BJ)/def2-SVP level^59,60,61, ensuring the attainment of stable structures devoid of imaginary frequencies (the atomic coordinates of the optimized computational models are listed in Supplementary Data).

Biosafety evaluation

The stability of UCNP@CR in FBS was evaluated by particle size change measured by Malvern Laser Particle Size Analyzer. The effect of ultrasound on viability of HUVEC and HaCaT was carried under 1.0 W cm⁻¹ (1 MHz) from 0 to 10 min. For NPs’ toxicity evaluation, HUVEC was incubated with different concentrations of UCNP@CR (0, 6.25, 12.5, 25, 50, 100 μg mL⁻¹). After 24 h of co-incubation, the viability of cells were assayed by MTT. The cytotoxicity of UCNP@CR was further evaluated using HaCaT cells after 24 h and 72 h incubation. The cell migration tests and hemolysis tests were carried under the concentration of 100 μg mL⁻¹. Further we administered UCNP@CR (100 μL, 1 mg mL⁻¹) via tail vein injection into male mice and the fluorescence of the Ru complex in the heart, liver, spleen, lungs, and kidneys were tracked at 1, 3, 6, 12, and 24 h. On the 7 day, the main blood parameters and indicators of liver and kidney function were tested (n = 3 mice), the major organs (heart, liver, spleen, lung, and kidney) of mice were harvested at 7 days for histological analysis.

Treatment of biofilm infected wound in immunosuppressed mice

All experiments involving animals were approved by the Animal Ethics Committee of Guangdong Laidi Biomedical Research Institute Co., Ltd (Approval Number: 2024035-2). Male Balb/c mice (6–8 weeks old) were purchased from the Guangzhou Ruige Biotechnology Co., Ltd, and raised in a specific pathogen-free (SPF) animal laboratory. The purchased male Balb/c mice were adjustable fed for one week to adapt the environment. Mice were housed at 20–22 °C (30–70% humidity) with a 12 h light-dark cycle. On day -4 and day -1, CTX (100 mg kg⁻¹) was intraperitoneally injected into the mice. On day 0, the mice were anesthetized with isoflurane inhalation, and their dorsal fur was shaved. Two 6 mm diameter open wound was created on the back of the mice using a skin punch, and 10 μL Candida albicans suspension (2 × 10⁸ CFU mL⁻¹) was inoculated onto the wound. The wound was covered with a 3 M film for 24 h and showed evident yellowish exudate. (A small portion of the exudate was collected and stained with cotton blue for observation. The successful establishment of the model was further confirmed by histopathological examination of the tissue sections).

On day 1, 20 mice were divided into 4 groups randomly (Control, Light, Ultrasound, Light+Ultrasound). The two wounds on one mouse were divided into a PBS group and UCNP@CR group (20 μL, 100 μg mL⁻¹). Four hours after drug administration, the mice were kept in darkness and subjected to the corresponding treatment. Light treatment condition involved irradiation with a 980 nm laser at a power density of 500 mW cm⁻² for 15 min. The ultrasound treatment condition involved applying coupling agent to the ultrasound probe at room temperature, covering the probe with a transparent 3 M film, and treating the wound site with ultrasound 0.3 W cm⁻² for a total of 60 s (three sessions, each session 20 s). Images of the wound site were captured on days 0, 1, 3, 5, 7, 9, and 11 (days post-infection). On day 3, the exudate from the wound site was collected for colony counting.

Histopathology study and immunofluorescence imaging

The surrounding tissue of uninfected wound treated with and without ultrasound for 24 h were harvested and stained by hematoxylin-eosin (H&E). The tissue slice of infected wounds were harvested on Day 11 for analysis of hematoxylin-eosin staining (H&E) and periodic acid-schiff (PAS) staining. Further, the harvested tissues slices were incubated with the antibodies of anti-TNF-α (1:200, Cat#BS-10802R, Bioss), anti-IL-6 (1:200, Cat#GB11117, Servicebio), anti-IL-10 (1:200, Cat#BA1201-1, BIOSTER), anti-Ly6g (1:200, Cat#GB11229, Servicebio), anti-MPO (1:500, Cat#ab208670, Abcam), anti-F4/80 (1:500, Cat#GB113373, Servicebio), anti-CD206 (1:500, Cat#GB113497, Servicebio), anti-CD86 (1:200, Cat#19589T, CST) and further staining with DAPI. Slices were imaged by using a digital pathological section scanner (Olympus VS200).

Immune cell analysis

Place the wound tissue in 200 μL of DMEM medium containing 5% FBS (fetal bovine serum) and mince the wound tissue. Subsequently, supplement with 400 μL of DMEM and 200 μL of 10 mg ml⁻¹ collagenase IV, and incubated at 37 °C for 1–1.5 h. After terminating the digestion, the suspension were filter through a 70 μm mesh. The prepared suspension were centrifuged at 500 × g for 10 min at 4 °C and perform red blood cell lysis on ice for 4 min. Finally, the cells were collected by centrifugation and stained with DAPI and antibody listed: APC-Cy7 anti-CD45 (Cat#103116, Clone#30-F11, BioLegend), PE/Dazzle 594 anti-Ly6c (Cat#128043, Clone#HK1.4, BioLegend), BV650 anti-CD11b (Cat#101239, Clone#M1/70, BioLegend), BV605 anti-F4/80 (Cat#743281, Clone#T45-2342, BD OptiBuild), APC anti-CD206 (Cat#141707, Clone#C068C2, BioLegend), PE anti-CD80(Cat#104707, Clone#16-10A1, BioLegend), Alexa Fluor 700 anti-CD11c (Cat#117319, Clone#N418, BioLegend), FITC anti-MHC-II (Cat#11-5321-82, Clone#M5/114.15.2, eBioscience). All antibodies were diluted 200 times before used.

Reporting summary

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