Targeting multidrug-resistant Candida: Cu_xTe nanoflowers synergistically facilitate fungal clearance via cuproptosis death and host immune activation

Abstract

Candida species are common infectious pathogens that can cause invasive infections with potentially life-threatening consequences. Current treatments for invasive Candida infections are primarily azoles and echinocandins. However, emerging antifungal drug resistance compromises therapeutic efficacy. Herein, a stable Cu_xTe nanoflower was designed to combat fungal infections via cuproptosis death and immune activation. Firstly, Cu and Te precursors were added to an auxiliary reducing agent and solvent, and Cu_xTe nanoflowers with a multibranched dendritic structure were synthesized via a temperature-controlled solution-phase method. Cu_xTe nanoflowers effectively inhibit fungal growth, damage fungal cell membranes and walls, and show significant fungicidal activity even against the multidrug-resistant fungus Candida auris. Furthermore, they significantly disrupt fungal biofilms and inhibit their growth and maturation. Cu²⁺ ions released in the weakly acidic fungal microenvironment penetrate fungal cells, inducing copper overload. Transcriptional and cytological analyses revealed that Cu_xTe treatment disrupted the mitochondrial membrane potential, altered membrane permeability, triggered insoluble Dlat protein aggregation, blocked the TCA cycle, impaired mitochondrial function, and ultimately induced fungal cuproptosis death. Simultaneously, they promote proinflammatory macrophage polarization, exerting synergistic antifungal effects. In summary, this therapeutic strategy integrates synergistic dual-pathway mechanisms: fungal cuproptosis death and immune activation, providing a new approach for treating antifungal infections.

Highlights

• •Multi-branched Cu_xTe nanoflowers were synthesized via wet chemistry, showing exceptional metalloenzyme-like activity.
• •They synergistically disrupt fungal structures including biofilms and inhibit growth in vitro.
• •They induce both fungal cuproptosis-like and host immune activation for better clearance.
• •They accelerate healing of fungal-infected wounds in mice with high biocompatibility.

1. Introduction

Candida infection is a common and complex clinical challenge, imposing significant health burdens and medical complications worldwide due to the increasing prevalence of immunocompromised conditions [1,2]. Candida species are ubiquitous, with Candida albicans predominating, accounting for approximately 80% of infections [3]. Moreover, infections caused by Candida tropicalis, Candida glabrata, Candida krusei, and other non-albicans species are increasing annually [4]. In recent years, “multidrug-resistant fungal” Candida auris infection has become more prevalent, and the traditional antifungal agents against this fungus are not effective [5]. Superficial candidiasis is the most common fungal disease in humans, affecting approximately 25% of the global population [6]. Furthermore, invasive Candida infections can lead to life-threatening conditions such as sepsis and pericarditis [7]. In face of increasingly serious fungal infections, the available antifungal agents are limited. In the clinic, only four classes of antifungal agents exist: azoles (fluconazole, voriconazole), polyenes (amphotericin B), echinocandins (caspofungin, micafungin, anidulafungin), and flucytosine[[8], [9], [10]]. Fungal infections pose significant treatment challenges, characterized by limited efficacy and a scarcity of antifungal agents; this contributes to the high mortality rates associated with invasive fungal infections [11,12]. New fungal infection management strategies are urgently needed (see Scheme 1).

Scheme 1.

Schematic diagram of the synthesis of Cu_xTe nanoclusters and effect of Cu_xTe nanoclusters on antibacterial activity and wound healing.

Unlike the peptidoglycan monolayers of prokaryotes, fungal cell walls are composed of β-glucans, chitin, mannoproteins, and glycoproteins, forming a dense and cross-linked multilayer structure that acts as a natural physicochemical barrier [13,14]. Prolonged exposure to antimicrobial agents increases the expression of efflux pump systems in fungal membranes, thereby increasing the development of fungal resistance [15]. Moreover, fungi readily form biofilms on medical devices and human tissues [16]. Biofilm-associated drug resistance can be tens to thousands of times greater than that of planktonic cells, significantly compromising the therapeutic efficacy of antifungal agents [17,18]. In addition, owing to the striking structural similarity between fungal cells and human host cells, some antifungal medications (amphotericin B) exhibit cytotoxic effects on host cells [19,20]. Consequently, suppressing biofilm formation and minimizing drug toxicity represent pivotal aims in antifungal therapy.

The antimicrobial applications of metals such as silver, copper, and zinc span millennia [21]. Recently, the integration of metallic antimicrobial properties with nanotechnology has emerged as a viable therapeutic strategy[[22], [23], [24]]. Metal-based nanoparticles (MNPs) have been extensively studied for their potent antibacterial properties, which act via multiple mechanisms distinct from those of conventional antimicrobial agents. These include protein dysfunction, cell membrane disruption, nucleic acid damage, and biosynthetic disorders, regardless of drug resistance[[25], [26], [27]]. In MNPs, metal ions partially share charge with donor groups, reducing their polarity and enhancing their lipophilicity. This facilitates coordination with microbial lipid layers, causing membrane disintegration [28,29]. Positively charged nanoparticles adhere to negatively charged microbial surfaces and release metal ions intracellularly in specific microenvironments, disrupting fungal anabolism [30,31]. However, clinical reports on the in vivo antifungal applications of metal ions remain limited. Copper is widely utilized because of its stability and cost-effectiveness. Copper-based nanoparticles (Cu-NPs) play increasingly important roles in biochemistry, pharmacology and medicine[[32], [33], [34]]. Sedigheh et al. synthesized Fe₃O₄@SiO₂/Schiff-base/Cu (II) complexes that exhibit significant antifungal activity. Cu-NPs disrupt fungal membranes and walls via protein denaturation, whereas internalized copper ions induce ROS production, causing damage to fungal DNA, ribosomes, and mitochondria [35]. Similarly, Liu et al. synthesized PCW nanoparticles that adhere to fungal walls and degrade it, releasing Cu²⁺ into cells, resulting in intracellular copper overload, and mediating mitochondrial dysfunction [36]. The microenvironment responsive ion release capacity of copper nanomaterials represents a promising antifungal strategy.

Copper, as an essential trace element for cells, plays an irreplaceable structural and catalytic role in biosynthesis [37]. Copper is a very important antifungal agent [38]. Recently, cuproptosis death has been identified as a new cell death mechanism in which excessive Cu²⁺ ions induce cuproptosis death by directly inhibiting the activity of lipoic acid synthase and binding to the lipoylation sites on dihydrolipoamide S-acetyltransferase (Dlat), leading to the formation of insoluble protein aggregates [39]. Moreover, Cu²⁺ disrupts Fe‒S clusters, leading to protein‒toxic stress responses, perturbed energy metabolism, and eventual cell death [39]. Clinically, targeting cuproptosis death has emerged as a promising strategy for treating various cancers and diseases [40,41]. However, the mechanism underlying cuproptosis fungal death has not been described in detail in the context of antifungal applications. The manifestation of cuproptosis death is contingent upon mitochondrial respiration, a process shared between fungi and mammalian cells due to their structural similarities [42]. The tricarboxylic acid (TCA) cycle is a pivotal metabolic pathway in both fungal and animal cells, and presents a possibility for cuproptosis death in fungi.

Here, we propose a strategy for the wet chemical synthesis of copper telluride (Cu_xTe) nanoflowers for antifungal applications, employing copper chloride (CuCl₂) and telluric acid (H₆TeO₆) precursors with a reducing agent. Cu²⁺-based nanoflowers are expected to treat fungal infections in vivo by triggering fungal cuproptosis death and immune activation. In this method, the liquid-phase reaction system of CuCl₂ and H₆TeO₆ precisely controls the reduction power of the auxiliary reducing agent/solvent acrylic acid (AA) and benzyl alcohol (BP), and polyvinyl pyrrolidone (PVP) achieves directional growth of the nanostructure through a surface modification process, facilitating the self-assembly of 2D nanosheets into hierarchical 3D nanoflower morphologies [[43], [44], [45]]. In a specific microenvironment for fungal growth, Cu_xTe nanoflowers can adhere to the fungal surface and release Cu²⁺. Excessive amounts of Cu²⁺ penetrate the cell interior to exert antifungal activity. Owing to the formation of nanoflowers, Cu_xTe maintains excellent antifungal properties even under protein-rich conditions. Transcriptome sequencing analysis, quantitative detection kit and mitochondrial function tests were used to explore the antifungal mechanism of the Cu_xTe nanoflowers. The results showed that Cu_xTe nanoflowers could trigger fungal cuproptosis death through intracellular Cu²⁺ overload. In addition, Cu_xTe nanoflowers can induce macrophages to polarize in a proinflammatory direction, enhance the immune system, and cooperate with Cu_xTe to treat fungal infected wounds. Its excellent superoxide dismutase activity plays an antioxidant role, protects normal cells from excessive free radical and exogenous hydrogen peroxide damage, and has the ability to promote wound healing. The antifungal properties and wound healing-promoting abilities of the Cu_xTe nanoflowers in vivo were verified in a C. albicans-induced skin infection model. Cu²⁺-based nanoflowers provide a strategy for treating fungal infections through fungal Cuproptosis death and immune activation. Great promise in combating drug-resistant fungal infections and accelerating wound healing.

2. Materials and methods

2.1. Cu_xTe nanomaterial synthesis

This work details the wet-chemical synthesis of copper telluride (Cu_xTe) nanomaterials using copper chloride (CuCl₂) and telluric acid (H₆TeO₆) as precursors, with polyvinylpyrrolidone (PVP), ascorbic acid (AA), and benzyl alcohol (BP) serving as reducing and stabilizing agents. The experimental procedure comprises three sequential stages: initially, copper and tellurium reactants undergo uniform dispersion via 30-min ultrasonication; subsequently, a 3-h thermal reaction proceeds at 160 °C in an oil bath, during which PVP suppresses excessive grain growth through pyrrolidone-metal ion coordination, whereas BP and AA control nucleation and growth processes via their moderate reducing properties, thereby enabling the formation of morphologically uniform Cu_xTe nanocrystals that facilitate controlled crystal development with specific architectures while preventing aggregation. These components synergistically promote the directional binding of Cu²⁺ with TeO₆²⁻ to generate Cu_xTe crystal nuclei, with the final product undergoing three alternating ethanol/acetone wash cycles to remove unreacted organics and ionic residues. This approach achieves efficient and controllable synthesis of Cu_xTe nanostructures through surfactant-mediated morphological regulation coupled with thermodynamic optimization, with the specific formulation for three-dimensional structures being 4.1 mg of CuCl₂ + 5.8 mg of H₆TeO₆ + 50 mg of PVP +18 mg of AA + 10 ml of BP.

2.2. Cu_xTe morphology and characterization

Microstructural morphology was analyzed via transmission electron microscopy (TEM) with elemental mapping to characterize the spatial distribution patterns of the constituent elements; crystal structure identification and phase composition analysis were performed via powder X-ray diffraction (XRD); chemical states were calibrated, and surface elemental species were determined through X-ray photoelectron spectroscopy (XPS); the atomic-scale elemental distribution was resolved via high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), which simultaneously visualized the morphological and internal structural variations to complement compositional analysis; high-resolution TEM (HRTEM) coupled with selected-area electron diffraction (SAED) was used to probe the atomic lattice fringes to determine the interplanar spacings and crystallographic parameters; high-resolution XPS spectra were acquired to verify the elemental speciation and quantify the valence states of copper and tellurium; and zeta potential measurements were conducted via hyphenated dynamic light scattering-electrophoretic light scattering (DLS-ELS) techniques via a zeta potential analyzer.

2.3. Peroxidase-like catalytic activity

The horseradish peroxidase-like (POD) activity of the material was systematically evaluated via a chromogenic reaction system under the following experimental conditions: in an acetate buffer solution (pH 4.0–5.5), with 3,3′,5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) as chromogenic substrates, and hydrogen peroxide (H₂O₂) as the cosubstrate. After a specific reaction duration, peroxidase-like activity was determined by measuring the characteristic absorption peak intensities of oxidized TMB (OxTMB) at 652 nm and oxidized OPD at 492 nm via ultraviolet-visible spectrophotometry.

2.4. DPPH radical scavenging assay

Using copper telluride as the research subject, serial concentration solutions (0, 6.25, 12.5, 25, 50, and 100 μg/ml) were prepared via dispersion in deionized water and subjected to equal-volume mixing with 0.2 mmol/L DPPH-ethanol solution, followed by a 30-min reaction at room temperature protected from light. Absorbance measurements were conducted at 517 nm via ultraviolet‒visible spectrophotometry with absolute ethanol as the blank control, where the free radical scavenging rate (%) was calculated as [1 - (A_Sample – A_Control)/A_Blank] × 100%.

2.5. Glutathione depletion assay

The DTNB (5,5′-dithiobis(2-nitrobenzoic acid), Ellman's reagent) method was employed to characterize the glutathione (GSH) oxidation process induced by the Cu_xTe nanomaterials. Copper telluride was coincubated with GSH (1 mM) in phosphate-buffered saline (PBS, pH 7.4) under light-protected conditions at 25 °C for 15 min, followed by centrifugation (12,000 rpm, 5 min). The supernatant was subsequently analyzed by measuring the absorbance at 412 nm, and the residual GSH concentration was quantified through a standard calibration curve. All the experimental procedures were conducted under light-free conditions to prevent photolytic interference from DTNB.

2.6. ABTS ⁺ scavenging assay

The ABTS⁺∙ radical detection system was established via a preoxidation protocol, wherein ABTS (7.4 mM) and K₂S₂O₈ (2.6 mM) were mixed in equal volumes and reacted under light-protected conditions for 16 h to generate a radical stock solution, which was subsequently diluted 50-fold with deionized water or absolute ethanol prior to use. Sample solutions (0.6 mL, concentration range: 3.125–100 μg/ml prepared by serial dilution) were mixed with 1.6 mL of ABTS⁺∙ working solution, and a mixture of deionized water and working solution was used as the negative control. Following a 10 min light-protected reaction, the absorbance was measured, and the scavenging rate was calculated as [(A _negative – A _Control)/A _Control] × 100%.

2.7. Fungal growth curve analysis

Single colonies (1-2) from a fresh YPD agar plate were inoculated into 25 mL of YPD liquid media (containing 1% yeast extract, 2% peptone, and 2% glucose). For the experimental groups, ultrasonicated dispersions of copper telluride in deionized water (power: 80 W; cycle: 5 s on/1 s off; total duration: 5 min; maintained at 4 °C in an ice bath) were added to achieve final concentrations of 0 μg/mL (negative control), 25 μg/mL, and 50 μg/mL. The positive control group received fluconazole (final concentration: 8 μg/mL). All the groups were incubated in a shaking incubator at 30 °C and 220 rpm. Starting from the initiation of culture (t = 0), 0.2 mL aliquots of the yeast suspension were aseptically collected every 2 h, and the optical density at 600 nm (OD₆₀₀) was measured via a microplate reader for 22 consecutive hours. Each experimental condition was independently repeated in triplicate, and the data are presented as the means ± standard deviations (SDs). Sigmoidal growth curves were generated through curve fitting.

2.8. Agar plate antimicrobial testing

To further evaluate the antimicrobial efficacy of Cu_xTe against Candida species, an antibacterial activity assay was performed on solid agar plates. Single colonies (1-2) from fresh fungal cultures were inoculated into liquid media and grown to the logarithmic growth phase; 0.5 mL of each culture was washed three times with phosphate-buffered saline (PBS) and resuspended in PBS. Five concentrations of Cu_xTe suspensions (0–100 μg/mL) in PBS were prepared. Equal volumes of the fungal suspension and Cu_xTe suspensions were mixed and incubated at 30 °C with shaking at 220 rpm for 4 h. After triple washing with PBS, the mixtures were diluted 2000-fold, and 100 μL aliquots were spread onto YPD agar plates. Following overnight incubation at 30 °C, the plates were photographed. Colony counting and inhibition rate calculations were performed via Photoshop software, with statistical significance determined via one-way ANOVA with t-tests (p < 0.05); all experiments were conducted in triplicate.

2.9. Fungal biofilm formation assay

The inhibitory effects of the Cu_xTe nanomaterials on Candida biofilm formation and preformed biofilms were assessed via crystal violet staining. For the prebiofilm inhibitory assay, logarithmic-phase fungal suspensions (1 × 10⁶ CFU/mL) were mixed with equal volumes of copper telluride solutions (0–100 μg/mL, ultrasonicated dispersion); for the postbiofilm inhibitory assay, Cu_xTe was added after biofilm maturation. Following 24 h of dynamic culture at 30 °C with shanking at 80 rpm, the biofilms were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, stained with 0.1% crystal violet for 10–15 min at room temperature, and solubilized with 33% acetic acid for 10–15 min before the absorbance was measured at 595 nm via a microplate reader. The drug-free group served as the negative control (100% biofilm formation). The inhibition rate was calculated as [(mean OD₅₉₅ of negative control - OD₅₉₅ of treated well)/(mean OD₅₉₅ of negative control - mean OD₅₉₅ of blank control)] × 100%, with statistical significance determined by one-way ANOVA with t-tests (p < 0.05); all experiments were performed in triplicate.

2.10. SEM analysis of fungal morphology

To directly assess the extent of the cellular damage inflicted by copper telluride on Candida species, fungal suspensions were diluted to 1 × 10⁵ CFU/mL and incubated with various concentrations of copper telluride nanoflowers in liquid media for 6 h. Following centrifugation, the samples were immediately washed three times with PBS. After the supernatant was discarded, the pellets were fixed with 2.5% glutaraldehyde solution at 4 °C for 12 h. After fixation, the samples were centrifuged again, the supernatant was discarded, and the samples were washed three times with PBS. Sequential dehydration was then performed in 30%, 50%, 70%, 80%, 90%, and 100% ethanol aqueous solutions (20 min per step), followed by three additional dehydration cycles in 100% ethanol. The samples were subsequently immersed in a 1:1 isoamyl acetate/ethanol mixture for 20 min, dehydrated in pure isoamyl acetate for 20 min, and dried using a critical point dryer. Finally, the samples were subjected to scanning electron microscopy (Gemini SEM 300) to directly visualize structural alterations to the cell walls and membranes of Candida species.

2.11. Fungal live/dead staining

To evaluate the viability of Candida species following copper telluride treatment under liquid culture conditions, fungal suspensions (10⁵ CFU/mL) were cocultured with gradient concentrations of copper telluride in 12-well plates and incubated statically overnight at 30 °C. After full interaction with Cu_xTe, 30 μL of SYTO-9 fluorescent dye (50 μM) and 20 μL of propidium iodide (PI) dye (50 μM) were added to each well, followed by incubation in the dark at 30 °C for 40–50 min. The samples were washed 2–3 times with PBS before fluorescence microscopy imaging, where SYTO-9 was excited by blue light (emission: green) and PI by green light (emission: red).

2.12. Fungal transcriptome sequencing

C. albicans samples treated with PBS or copper telluride nanoparticles (three biological replicates per group) were submitted to Tsingke Biotechnology Co., Ltd. for RNA-seq analysis. Total RNA was extracted from each treatment group via TRIzol reagent, and RNA integrity was verified via an Agilent 2100 Bioanalyzer (RIN ≥7.0). Sequencing was performed on the Illumina NovaSeq 6000 platform (paired-end 150 bp), yielding ≥10 Gb of clean data per sample on average. The raw FASTQ files were subjected to quality control via in-house Perl scripts (Q30 ≥ 90%), and low-quality reads (Phred score <20) were discarded. Clean reads were aligned to the C. albicans SC5314 reference genome (Assembly 22, GenBank accession GCA_000182965.3) via Bowtie2 v2.2.3 with the parameters --end-to-end --sensitive. Gene expression counts were quantified with HTSeq v0.6.1, retaining genes with counts per million (CPM) > 1 in ≥50% of the samples. Differential expression analysis was performed with DESeq2 v1.18.0 (|log₂ (fold change) | >1 and Benjamini‒Hochberg adjusted p < 0.05). Functional enrichment included GO term analysis via GOseq v1.34.1 (adjusted p < 0.05), KEGG pathway analysis (version 2021.09), and predefined gene set enrichment assessment via GSEA v4.1.0 (Broad Institute; FDR <0.25).

2.13. Hemolysis assessment

Fresh rabbit erythrocytes were diluted to 2% in PBS buffer, mixed with various concentrations of copper telluride (Cu_xTe) solutions and incubated at 37 °C for 4 h. After centrifugation at 7000 rpm for 3 min, the supernatants were collected. Diluted blood mixed with ultrapure water and PBS served as positive and negative controls, respectively; both control groups underwent identical mixing and incubation conditions prior to supernatant collection. The absorbance of all the supernatants was measured at 540 nm via a microplate reader to calculate hemolysis rates according to the following formula: hemolysis (%) = [(sample absorbance – blank control absorbance)/(positive control absorbance – blank control absorbance)] × 100%.

2.14. Fungal ROS detection

Candida species in the logarithmic growth phase were washed twice with PBS and diluted to 10⁷ CFU/mL in PBS. The negative control group consisted of a 1 mL C. albicans suspension mixed with 1 mL of PBS; for the experimental groups, a 1 mL Candida albicans suspension was mixed with copper telluride (Cu_xTe) diluted in PBS at gradient concentrations (100, 50, 25, 12.5, and 6.25 μg/mL) and cocultured for 4 h. The DCFH-DA fluorescent probe (final concentration: 10 μM) was subsequently added, followed by incubation in the dark at 30 °C for 30 min. After three washes with PBS, the samples were resuspended in 500 μL prechilled PBS and subjected to fluorescence microscopy observation. Following probe loading, changes in fluorescence intensity (excitation/emission wavelengths: 488–495 nm/520–530 nm) were measured to assess intracellular ROS levels.

2.15. Intracellular Cu²⁺ quantification (ICP-OES)

Single colonies (1--2) freshly picked from agar plates were inoculated into liquid YPD media and cultured until they reached an OD₆₀₀ = 1.0--1.2; the cells were then washed three times with PBS and resuspended. The suspension was mixed 1:1 with copper telluride (Cu_xTe) solutions to achieve final concentrations of 0, 25, and 50 μg/mL and incubated for 4 h. After three washes with deionized water, the cells were resuspended in 1 mL of deionized water and lysed via ultrasonication; the lysates were subsequently centrifuged at 12,000 rpm for 10 min, after which the supernatants were collected. The samples were prepared via microwave-assisted nitric acid digestion: concentrated nitric acid was cautiously added dropwise to the supernatants (1:3 v/v) to prevent violent reactions, followed by 30-min of equilibration. The mixtures were transferred to sealed digestion vessels and subjected to stepwise heating: 80 °C for 10 min; 120 °C for 10 min; 180 °C for 20 min; and then cooled to 50 °C for 10 min until solutions became colorless and clear. The digested samples were transferred to glass beakers, evaporated to near-dryness (∼0.1 mL residual liquid) on a 120 °C hot plate, and then diluted to 1 mL with 2% HNO₃ for subsequent copper ion quantification via inductively coupled plasma‒optical emission spectrometry (ICP‒OES).

2.16. qPCR for gene expression

Total RNA extraction was performed as follows: C. albicans cells in the logarithmic growth phase were collected after 4 h of copper telluride treatment and washed three times with PBS, and 0.1 g of fungal cells was ground in liquid nitrogen with 1 mL of total RNA extractor. The mixture was incubated at room temperature for 10 min before 0.2 mL of chloroform was added, the mixture was vigorously vortexed for 15 s, and the mixture was incubated at room temperature for 3 min. After centrifugation at 12,000 rpm (4 °C) for 10 min, the aqueous upper phase was transferred and mixed with an equal volume of isopropanol, followed by a 20-min incubation at room temperature. After subsequent centrifugation at 12,000 rpm (4 °C) for 10 min, the RNA was pelleted the supernatant was discarded, and the pellet was washed with 1 mL 70% ethanol. After centrifugation at 12,000 rpm (4 °C) for 3 min, the supernatant was discarded again, and the pellet was air-dried at room temperature for 5–10 min. The RNA was dissolved in 50 μL of RNase-free ddH₂O, thoroughly mixed, and quantified by measuring the absorbance at 260 nm and 280 nm, with the OD₂₆₀/OD₂₈₀ ratios maintained between 1.8 and 2.0. Reverse transcription was performed under the following conditions: Step 1, 42 °C for 15 min; Step 2, 85 °C for 5 s; and Step 3, 4 °C for 5 min. The qPCR protocol included predenaturation at 94 °C for 30 s; denaturation at 94 °C for 5 s; annealing at 55 °C for 15 s; extension at 72 °C for 10 s; and 39 cycles.

2.17. Western blot analysis of protein expression

Total fungal protein extraction was performed via the alkaline lysis method: C. albicans in the logarithmic growth phase were collected after 4 h of copper telluride treatment, washed three times with prechilled PBS, and lysed in 350 μL prechilled alkaline lysis buffer (1 N NaOH: 488 μL, β-mercaptoethanol: 21 μL, ddH₂O: 892 μL) with thorough pipetting; the mixture was incubated on ice for 15 min before 150 μL of prechilled 30% TCA was added, followed by vortexing for 2 min. After centrifugation at 12,000 rpm (4 °C) for 3 min, the supernatant was discarded. The pellet was resuspended in 700 μL of prechilled acetone with pipette mixing, centrifuged at 12,000 rpm for 3 min, and then inverted on absorbent paper for 3 min to dry and remove the acetone. Proteins were resuspended in 100 μL of urea buffer by pipetting, denatured in a metal bath at 100 °C for 10 min, and centrifuged at 12,000 rpm (room temperature) for 5 min; the supernatant was transferred to a new tube and mixed 1:1 with 20% glycerol bromophenol blue buffer. After thorough mixing, the samples were stored at −20 °C for subsequent Western blot analysis of protein expression levels.

2.18. Mitochondrial TEM imaging

This experiment employed a dual-fixation method to prepare TEM samples of C. albicans biofilms: fungal suspensions were washed three times with deionized water and then fixed overnight at 4 °C in PIPES buffer (pH 6.8) containing 2.5% glutaraldehyde and 4% paraformaldehyde; samples were subsequently rinsed three times with 0.1 M PBS (15 min per wash), followed by light-protected fixation in 3% potassium permanganate at 4 °C for 3 h; after three additional deionized water rinses (15 min each until clarification), gradient acetone dehydration was performed (30%, 50%, 70%, 85%, 95%, 100%; 5 min per step at 4 °C); specimens were then infiltrated with Spurr's resin, embedded, polymerized, and sectioned to 70 nm thickness via a Leica UC7 ultramicrotome; and unstained sections were directly observed via transmission electron microscopy.

2.19. JC-1/Rh123 mitochondrial staining

C. albicans cells in the logarithmic growth phase were collected after 4 h of copper telluride treatment, washed three times with PBS, and pretreated for 30 min with 1 mg/mL lyticase buffer (1 M sorbitol + 10 mM HEPES, pH 7.5). After two additional PBS washes, the fungal suspension was uniformly mixed with JC-1 (final concentration of 10 μM) and Rh123 (final concentration of 5 μM), followed by light-protected incubation for 30 min. The samples were then washed three times with prechilled PBS, resuspended in PBS, and observed under a fluorescence microscope to record fluorescence intensity images. Quantitative analysis of red/green fluorescence intensity ratios was performed for graphical representation.

2.20. Mitochondrial complex I/II activity assay

This experiment measured mitochondrial respiratory chain complex I activity on the basis of its catalysis of NADH dehydrogenation to generate NAD+, where NADH has a characteristic absorption peak at 340 nm,; thus, complex I activity was characterized by the determination of the NADH oxidation rate. Complex II activity was assessed through the reduction of 2,6-dichloroindophenol (exhibiting a characteristic absorption peak at 605 nm) by reduced coenzyme Q generated via complex II catalysis, quantified by the decomposition rate of 2,6-dichloroindophenol. BoxBio (Beijing) kits AKOP005U-1 and AKOP006C-1 were used to determine fungal mitochondrial respiratory chain complexe I and II activities, respectively.

2.21. Fungal MDA content measurement

Malondialdehyde (MDA) reacts with thiobarbituric acid (TBA) under acidic and high-temperature conditions to form a brownish-red trimethene (3,5,5-trimethyloxazole-2,4-dione), which has a maximum absorption peak at 532 nm; thus, the lipid peroxidation level can be estimated by measuring the absorbance at this wavelength. This experiment utilized the BoxBio (Beijing) commercial kit AKFA013M to determine the fungal MDA content (indicating lipid peroxidation levels).

2.22. Lipid peroxidation (C11-BODIPY staining)

C. albicans cells in the logarithmic growth phase were collected after 4 h of copper telluride treatment, washed three times with PBS, treated with 0.1% EDTA for 10 min, washed twice with PBS, resuspended in 1 mL of PBS, mixed with 1 μL of C11-BODIPY (2 mM), and incubated under light-protected conditions for 60 min. Following three additional PBS washes, the fungal suspension was resuspended in PBS for subsequent analysis, and the fluorescence intensity was detected in the red channel (excitation/emission: 581 nm/591 nm) and green channel (excitation/emission: 488 nm/510 nm), while images were captured for quantitative analysis and graphical representation of red-to-green fluorescence intensity ratios.

2.23. Fungal gene knockout (C. albicans)

This study generated knockout mutants of the FDX1 (YAH1) and LIAS (LAB5) genes in the C. albicans strain SC5314 via CRISPR-Cas9 technology; the knockout plasmid PV1524 (conferring nourseothricin resistance) was employed, for which guide RNA sequences were designed via the Benchling platform following retrieval of gene sequences from NCBI; homologous recombination repair templates were constructed by seamlessly cloning ∼1000 bp upstream and downstream flanking regions of each target gene; competent SC5314 cells were transformed via the PEG/LiAc method, involving coincubation of the cells, homologous repair template, single-stranded salmon sperm DNA, and linearized PV1524 plasmid in 40% PEG4000/TE/LiAc solution at 30 °C with 220 rpm shaking overnight, followed by a 42 °C heat shock for 1 h; and transformed cells were recovered in nonselective YPD medium for 4‒5 h before being plated on nourseothricin-containing agar plates and incubated at 30 °C for 2‒3 days; successful gene knockouts were confirmed via PCR, agarose gel electrophoresis, and Western blot analysis.

2.24. Flow cytometry analysis

Raw 264.7 cells retrieved from liquid nitrogen were revived and passaged to the third generation and then seeded in 12-well plates. After reaching 80‒90% confluency, the cells were treated with Cu_xTe nanoparticles dispersed in 2% FBS-supplemented DMEM at concentrations of 0, 25, and 50 μg/mL, with the experimental groups designated as follows: control, 25 μg/mL, 50 μg/mL, and LPS (1000 ng/mL). After 24 h of incubation, the cells were harvested, washed once with ice-cold PBS, counted to approximately 10⁷ cells, and resuspended in 100 μL of PBS. The cells were then incubated with TruStain FcX™ PLUS (anti-mouse CD16/32) Antibody (1:100 dilution) on ice for 10 min, followed by staining with APC anti-mouse CD86 Antibody and PE anti-mouse CD206 Antibody (both at 1:100 dilution) under light-protected conditions at 4 °C for 30 min. Subsequently, the cells were washed with ice-cold PBS, resuspended in 200 μL of buffer, filtered through 70-μm cell strainers, and immediately subjected to flow cytometry analysis.

2.25. In vivo antimicrobial evaluation

This study established a cutaneous wound healing model using eight 9-week-old female BALB/c mice divided into three groups: a PBS-treated control, Cu_xTe-treated experimental, and a fluconazole-treated positive control (4 μg/mL); on day −1, circular full-thickness wounds (8 mm diameter) were created on the dorsum of all the mice and inoculated with a 10⁷ CFU/mL C. albicans (SC5314) suspension until air-dried; the mice with confirmed infection received designated treatments on days 1 and 3, with wound healing documented photographically on days −1, 1, 3, 6, 9, and 12; the wound healing percentage was calculated via the following formula: (Original wound area - Wound area at time point)/Original wound area × 100%, while body weight was monitored throughout; on day 12, the wound fungal burden was quantified via plating, followed by the collection of orbital blood and organs (heart, liver, spleen, lungs, and kidneys); and complete blood count and biochemical analyses were performed on blood samples, while H&E staining was conducted for visceral organs; wound tissues that underwent H&E and Masson staining, with additional immunohistochemical staining for IL-6 and VEGFA expression. The experimental protocol has been approved by the Anhui Medical University Laboratory Animal Ethics Committee (Approval No. LLSC20251618).

3. Results

3.1. Synthesis and characterization of copper telluride nanoflowers

Copper-based nanoparticles hold great promise for biomedical applications. The excessive growth of copper-based nanoparticles was controlled by the addition of additional reducing agents and stabilizers. Cu-based telluride nanomaterials with three-dimensional nanoflower structures were successfully fabricated on the basis of wet chemical reaction pathways. Specifically, copper chloride (CuCl₂) and telluric acid (H₆TeO₆) were first added to a mixed solvent of acrylic acid (AA), benzyl alcohol (BP) and polyvinyl pyrrolidone (PVP), sonicated for 30 min, and then fully reacted in an oil bath at 160 °C for 3 h (Fig. 1A). AA and BP, as auxiliary reducing agents, can increase the stability of the reduction potential, and their hydrophobic groups participate in the regulation of the interface energy. PVP forms coordination bonds with copper ions through carbonyl groups in molecular chains, selectively adsorbs on specific crystal surfaces, reduces surface energy, and finally guides Cu_xTe 2D nanosheets to self-assemble into 3D Cu_xTe nanoflowers. This multi-branched structure endows it with a high specific surface area and abundant active sites, and also increases the surface charge density, thereby promoting targeted enrichment on the surface of fungi [46,47]. The transmission electron microscopy (TEM) images in Fig. 1B–S1 and S2 show that Cu_xTe has a flower-shaped nanostructure morphology, with tentacles radiating from the c enter outward, increasing the surface area and reactivity, and improving physical and chemical properties of the material. The particle size of the nanoflowers is approximately 200 nm. Each nanoflower structure exists mainly in the aggregation state, and the overall appearance has no obvious rules. CuTe lattice fringes were observed only in some regions in the HRTEM images, and the lattice fringe spacing was calculated to be approximately 0.24 nm (Fig. S3). The SAED pattern can further confirm the crystal structure of the material, and the selected area electron diffraction pattern shows dispersion diffraction rings, which further proves the polycrystalline/low crystalline characteristics of the material (Fig. S4). X-ray diffraction (XRD) patterns of the Cu_xTe nanoflowers show that their diffraction peaks do not match those of standard Cu₂Te (PDF #04-007-6583), CuTe (PDF #04-008-8274), indicating that they have a complex crystal structure that is not a single phase (Fig. 1C). The dark field and element mapping images of Cu_xTe taken in Fig. 1D shows the overall morphology of the photographed part, which shows element mapping images of Cuand Te. Both the Cu and Te signals have strong signals, and the distributions of the two elements are consistent with the dark field image, indicating that the spatial distributions of the Cu and Te signals are highly coincident and consistent with the morphology profile, indicating the successful synthesis of the Cu_xTe nanomaterials. These results confirmed that the elements coexist uniformly in the nanoclusters. Movere, Cu and Te were present, which was further confirmed by the combined elemental mapping images of Te and Te.

Fig. 1.

Synthesis and characterization of copper telluride nanoflowers. (A) Schematic diagram of synthesis and preparation process of Cu_xTe nanoflowers. (B) TEM images of Cu_xTe. (C) The particle size distribution of Cu_xTe. (D) HAADF-STEM image and elemental mappings. (E)Energy Dispersive Spectrometer analysis of each element in Cu_xTe. (F) Cu_xTe was measured for X-ray diffraction at different incident beam angles. (G) The total counts of Cu_xTe appearing at different zata potential. (H-J) The XPS survey of Cu_xTe, as well as the XPS spectra of Cu 2p and Te 3d.

In addition, the elemental chemical states of Cu and Te in Cu_xTe were studied via XPS. The XPS spectra of the Cu 2p peak indicate that the valence state of Cu is +2. The binding energies of Cu are 932.00 eV and 951.76 eV, corresponding to the 2p3/2 and 2p1/2 orbitals of Cu²⁺, respectively. In addition, the electron binding energy positions correspond to satellite peaks at 932.00, 934.34, 951.76, and 954.02 eV (Fig. 1E). Fig. 1F shows the Cu LMM spectrum of the Cu_xTe nanomaterials, showing the oxidation state of the Cu in Cu_xTe. In Fig. 1G, the XPS spectrum of Te 3d shows characteristic peaks of Te²⁻ at 576.81 eV (3d5/2) and 587.17 eV (3d3/2), indicating that Te has a valence state of −2. XPS full spectrum determination of Cu_xTe, it provides the overall information of all elements in the peak positions of the Cu, Te, C, N and O in the sample, including the types and relative contents of the elements (Fig. 1H). In terms of the valence states of several elements, Cu and Te are likely to combine at a ratio of 1:1, so their chemical formula may be Cu_xTe, which indicates that PVP has successfully modified the Cu_xTe nanomaterials. The Cu_xTe nanoflowers have an average zeta potential of 12.3 mV (Fig. 1I), indicating that the Cu_xTe surface is positively charged. These findings suggest that Cu_xTe can interact with fungal surfaces via electrostatic attraction. Fig. 1J shows a number diagram of the hydrated particle size of the Cu_xTe nanomaterials. In three parallel measurements, the maximum range of hydrated particle sizes is approximately 100–300 nm, which indicates that the hydrated particle size of most Cu_xTe nanoparticles is between 150 and 250 nm. In this work, a wet chemical method was adopted to synthesize Cu_xTe nanomaterials. XRD and XPS measurements revealed that the Cu_xTe nanomaterials were successfully synthesized at room temperature. The multiphase composite structure endows the material with high specific surface area characteristics, the three-dimensional flower-like structure morphology increases the surface-active site density, and the positive surface charge characteristics and the negativity of the fungal cell membrane generate strong electrostatic adsorption, which provides a structural basis for antifungal applications.

3.2. Copper telluride exhibits excellent metalloenzyme-like activity

Cu_xTe nanoflowers were synthesized and their metalloid enzyme activity and ability to regulate free radicals were evaluated systematically. In previous studies, copper-based nanoparticles have been shown to mimic a variety of enzyme activities because of their abundant d electron configurations and variable oxidation states. Owing to its high peroxidase-like catalytic efficiency and high biocompatibility under acidic conditions, it has been studied as an anti-infective therapeutic material. In addition, peroxidase can initiate Fenton like reactions involving reactive oxygen species (ROS) via the use of exogenous hydrogen peroxide as a catalyst. TMB and OPD are commonly used as substrates to determine peroxidase activity in samples. Cu_xTe nanoflowers were uniformly dispersed in deionized water via ultrasonic treatment (Fig. S5). Under acidic conditions, Cu_xTe nanoflowers can catalyze the decomposition of hydrogen peroxide to ROS, and then oxidize TMB to oxTMB; its absorbance at 652 nm significantly increases. As shown in Fig. 2A, the Cu_xTe nanoflowers react with TMB under acidic conditions, and the solution is blue; with the addition of H₂O₂, the color intensifies, and the absorbance at 625 nm increases. Moreover, the blue color of the reaction system gradually increased with increasing H₂O₂ concentration (Fig. 2B). Similarly, at the same H₂O₂ concentration, the color of the solution increased with increasing Cu_xTe nanoflower concentration, and the absorbance at 625 nm increased in a concentration-dependent manner (Fig. 2C). In a similar OPD catalytic system, after the same treatment, the OPD probe showed a similar phenomenon, the absorbance at 492 nm increased linearly with increasing material concentration, and the reaction rate was comparable to that of TMB system (Fig. 2D, E, 2F). Cu_xTe nanoflowers exhibit significantly higher peroxidase activity at lower concentrations than do Cu_xTe nanospheres and nanowires prepared via similar synthetic processes, which is likely due to their high surface area/volume ratio, polysynaptic structure and efficient electron transfer properties. These results indicate that the Cu_xTe nanoflowers have peroxidase-like activity under acidic conditions. The duration and diffusion of ROS limit the role of reducing substances in the fungal microenvironment to some extent. However, the reduction in these reducing substances may disrupt the internal balance of the fungal environment and ultimately accelerate fungal death. Interestingly, Cu_xTe nanoflowers can consume GSH. The GSH consumption ability of the Cu_xTe nanoflowers was determined by using 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) as a probe. After the incubation of the Cu_xTe nanoflowers with GSH, the absorbance of GSH bound to DTNB decreased at 412 nm. We measured the absorbance at 412 nm after a certain concentration of GSH was added, and the concentration of the Cu_xTe nanoflowers was increased for some time. The results showed that the higher the material concentration was, the faster the GSH consumption rate (absorbance decreased to 72%), indicating that the Cu_xTe nanoflowers have good GSH peroxidase like (GSH-Px) activity (Fig. 2G). Therefore, Cu_xTe nanoflowers, as multifunctional enzymes with similar properties to POD and GSH-Px, have great antibacterial potential.

Fig. 2.

Multienzyme activity of the Cu_xTe nanoclusters. (A) UV–vis spectra of solutions including TMB alone, H₂O₂, TMB, TMB + H₂O₂, Cu_xTe + TMB, and Cu_xTe + TMB + H₂O₂ after 30 min of reaction. (B) POD-like activity; (C) POD-like activity of Cu_xTe nanoclusters using a TMB probe; (D) UV–vis spectra of solutions including OPD alone, H₂O₂, OPD, OPD + H₂O₂, Cu_xTe + OPD, and Cu_xTe + OPD + H₂O₂ after 30 min of reaction. (E) POD-like activity; (F) POD-like activity of Cu_xTe nanoclusters using an OPD probe. (G) Concentration-dependent GSH scavenging activities of Cu_xTe nanoflowers using the DTNB probe. DPPH (H) and ABTS (I) scavenging ratios of the Cu_xTe nanoclusters.

After controlling fungal infections, excess OH toxicity can have side effects on healthy tissues. SOD in the body also plays an important role in free radical scavenging. This enzyme consumes oxygen to form hydrogen peroxide and oxygen, thereby reducing the degree of tissue damage caused by excess free radicals. Cu-based nanomaterials, as the catalytic centers of SOD, are also considered to have good SOD properties. ABTS and DPPH radical scavenging experiments were performed to evaluate the ability of the Cu_xTe nanoflowers to scavenge nitrogen radicals in neutral environments. When ABTS is oxidized, ABTS + radicals are formed with absorbance maxima at 414, 645, 734 and 815 nm. The antioxidants added to the ABTS working solutions interact with ABTS+ and cause discoloration of the ABTS working solutions. The absorbance of ABTS⁺ in solution at its maximum absorption wavelength (734 nm) was measured via an ultraviolet spectrophotometer to assess the free radical scavenging capacity of the material. The addition of Cu_xTe nanoflowers to the prepared ABTS solution resulted in a decrease in the absorbance at 734 nm. There is a positive correlation between the concentration of Cu_xTe nanoflowers and their ability to scavenge ABTS⁺. The results show that the Cu_xTe nanoflowers can effectively scavenge ABTS ⁺ ions (Fig. 2H). Similar results were obtained for the reaction of DPPH with the Cu_xTe nanoflowers. After DPPH reacts with the Cu_xTe nanoflowers, the absorbance of DPPH at 517 nm decreases, resulting in decolorization of the solution system (Fig. 2I). These findings confirm the potent free radical scavenging activity of the Cu_xTe nanoflowers against nitrogen-centered radicals. In summary, Cu_xTe nanoflowers exhibit unique two-way redox regulatory functions: ROS generation via peroxidase/oxidase-mimetic activity, and free radical scavenging, which disrupts cellular redox homeostasis. In antifungal applications, ROS production directly disrupts fungal cell membrane integrity, whereas GSH depletion weakens intracellular antioxidant defenses, leading to the accumulation of oxidative stress. Moreover, the efficient scavenging ability of the material for ABTS· and DPPH free radicals can effectively reduce the oxidative damage to host tissues and create an anti-inflammatory microenvironment for wound repair. Compared with the one-way sterilization mode of traditional antibacterial materials (such as silver nanoparticles), this dual mechanism of “synergistic sterilization and host protection” result in a better balance of biological functions. The multiple biological activities of Cu_xTe flowers stem from their unique composition and surface chemistry, and their bidirectional redox regulation provides a new way to develop innovative solutions that are both highly effective antifungals and promote tissue repair.

3.3. In vitro antifungal activity of Cu_xTe

On the basis of the excellent oxidase-like/peroxidase activity, positively charged surface characteristics (favorable for binding to negatively charged fungal cell walls), and potential for physical and mechanical damage of the Cu_xTe nanoflowers, this study speculated that the Cu_xTe nanoflowers might exhibit significant antifungal activity. The fungicidal activity of the Cu_xTe nanoflowers in vitro was evaluated via the experimental standard strain SC5314 (C. albicans) and five clinically resistant fungi, C. auris, C. tropicalis, C. parapsilosis, C. krusei and C. dubliniensis as model fungal strains, and the antifungal activity of the Cu_xTe nanoflowers in vitro was systematically evaluated (All strains of Candida were derived from the North District Laboratory Department of the First Affiliated Hospital of Anhui Medical University.) The minimum inhibitory concentration (MIC) of each strain against clinical antifungal drugs was determined by a drug sensitivity board. The test results showed that all the tested strains exhibited resistance to these three drugs (Figs. S6 and S7). First, the effects of the Cu_xTe nanoflowers on Candida proliferation were evaluated via growth curve measurements. As shown in Fig. 3A and S8, after overnight culture in YPD media containing 0, 25 and 50 μg/mL Cu_xTe nanoflowers, the growth of each Candida genus was significantly inhibited with increasing material concentration, indicating that the growth lag period was prolonged and that the OD value of the fungal mixture in the plateau period was reduced. At a concentration of 50 μg/mL, the fungistatic effect of the Cu_xTe nanoflowers was even stronger than that of the fluconazole positive control. Notably, a highly resistant strain of Candida auris was not sensitive to fluconazole at 8 μg/mL but still showed an inhibition rate of nearly 50% at 50 μg/mL Cu_xTe. These results demonstrate that Cu_xTe nanoflowers have broad-spectrum antifungal activity against a variety of Candida species and are effective against clinically common drug-resistant strains. To verify these results, the in vitro antifungal properties of Cu_xTe were evaluated via standard plate counting methods. Fig. 3B and S9 shows solid agar plate images of the killing effect of Cu_xTe nanoflowers on Candida at different concentrations. Agar plate images after treatment with different concentrations of Cu_xTe revealed that the number of plate colonies in the Cu_xTe nanoflower treatment group was significantly lower than that in the PBS control group. With increasing Cu_xTe concentration from 3.125 μg/mL to 50 μg/mL, the number of fungal colonies decreased in a concentration-dependent manner. Specifically, at concentrations of 50 and 25 μg/mL, the number of individual Candida colonies was much lower than that in the negative control group. It is worth noting that the antifungal efficacy of nanoflower-shaped Cu_xTe is significantly superior to that of linear and spherical counterparts (Fig. S10). This experiment confirmed that the Cu_xTe nanoflowers themselves possess strong intrinsic fungicidal activit, even without the aid of external conditions such as exogenous H₂O₂ or light irradiation. To evaluate fungal activity in the coculture system of Cu_xTe nanoflowers and fungi, live/dead cell staining (SYTO-9/PI) was used. The PBS control fungi presented green fluorescence (live cells) and no red fluorescence (dead cells). The Cu_xTe treatment group presented a red fluorescence signal for dead bacteri, and the red fluorescence intensity (proportion of dead bacteria) increased significantly with increasing Cu_xTe concentration, whereas the green fluorescence intensity (viable bacteria) decreased correspondingly. At a concentration of 50 μg/mL, the proportion of viable fungal cells was approximately 10% (Fig. 3C and S11). This result is consistent with the plate bacteriostasis experiment, which once again confirms the potent antifungal activity of the Cu_xTe nanoflowers.

Fig. 3.

In vitro fungicidal activity of Cu_xTe. (A) Growth curves of Candida species in Cu_xTe. (B) Photograph of fungal colonies. Fungal viability of C. albicans, C. auris, C. tropicalis, C. parapsilosis, C. krusei and C. dubliniensis (n = 3). (C) Fluorescence images of dead (red) and live (green) fungi following various treatment methods: Candida species.

In addition, scanning electron microscopy (SEM) was used to observe the morphological changes in Candida plants treated with Cu_xTe nanoflowers, and damage to the fungal cell membrane/cell wall was observed more directly. Fig. 4A and S12 shows that the fungal cell wall of the control group was smooth and complete, and the morphology was full. After incubation with 25 μg/mL Cu_xTe, the fungal cell wall showed slight shrinkage or collapse and an abnormal morphology. When the concentration was increased to 50 μg/mL, the fungal surface severely collapsed, and even cell wall rupture and content leakage occurred. These findings indicate that Cu_xTe nanoflowers can significantly disrupt Candida cell wall/membrane integrity. These results are consistent with those of previous studies showing that Te ions can damage the structure of biofilms. In view of the critical role of fungal biofilms (composed of cells and their extracellular polymers EPS) in adhesion, aggregation, resistance to drugs and host defense, the resulting treatment difficulties. This study evaluated the effects of Cu_xTe nanoflowers on Candida biofilms via crystal violet staining. To investigate the antibacterial activity of the Cu_xTe nanoflowers at the initial stage of biofilm formation and at the mature stage, two strategies of Cu_xTe nanoflower treatment were carried out during biofilm formation and after fungal biofilm maturity. In the experiment in which biofilm formation was inhibited, the biofilms in the PBS control group were thick, intact and dark purple. With increasing Cu_xTe concentration, the integrity of the biofilm was destroyed, pores appeared, and the staining became lighter and thinner. At concentrations of 25 and 50 μg/mL, the grayish white biofilm almost disappeared and the staining was light purple; the absorbance at 570 nm was significantly lower than that of the control group, indicating that biofilm formation was hindered. Cu_xTe at 50 μg/mL had an effect comparable to or stronger than that of the fluconazole positive control; notably, fluconazole was ineffective against C. auris biofilms, while Cu_xTe still significantly inhibited their formation (Fig. 4B and S13). In the experiment in which mature, biofilms were destroyed, the fluconazole treatment group was not significantly different from the control group, indicating that mature biofilm could effectively resist traditional drugs. In contrast, Cu_xTe nanoflowers significantly damaged the mature biofilms of Candida species, especially at 50 μg/mL, causing 80% damage to the biofilms of C. albicans and C. krusei and more than 40% damage to the remaining strains (Fig. 4C and S14). Overall, the Cu_xTe nanoflowers exhibited excellent antifungal/anti-biofilm activity against multiple drug-resistant Candida species and their biofilms. It is more effective than traditional antifungal agents such as fluconazole, especially against resistant strains and biofilm bacteria. This study provides valuable information for the development of new drugs against drug-resistant fungal infections. Cu_xTe nanoflowers exhibit antifungal effects due to their multiple mechanisms of action and do not easily induce drug resistance, indicating good application prospects.

Fig. 4.

Antifungal performance of Cu_xTe nanoclusters in vitro. (A) Candida species imaged via SEM after different treatments. (B) Biofilm images and inhibition rates of Candida at different Cu_xTe concentrations during biofilm formation. (C) Candida biofilm images and inhibition rates were obtained at different concentrations when the Cu_xTe was used to treat mature fungal biofilm.

3.4. Cu_xTe nanoflowers inhibit the expression of genes related to mitochondrial energy metabolism in fungi

Cu²⁺ plays a key role in cell physiology and homeostasis and is also an essential element for fungal growth and reproduction. Fungi maintain intracellular Cu²⁺ homeostasis through a sophisticated regulatory system, and excessive copper can induce their death. To further investigate the antifungal mechanism of the Cu_xTe nanoflowers, transcriptome sequencing was performed on C. albicans incubated with the Cu_xTe nanoflowers. A total of 543 differentially expressed genes (DEGs) were identified in the Cu_xTe nanoflower treatment group compared with the control group, of which 164 genes were significantly downregulated and 376 genes were significantly upregulated (Fig. 5B). In addition, through Gene Ontology (GO) annotation enrichment analysis, compared with those in the control group, the DEGs in the Cu_xTe nanoflower treatment group were enriched mainly in the gene expression, ribosome metabolism and transcription and translation metabolic pathways (Fig. 5C). Changes in the functional pathways of C. albicans treated with Cu_xTe nanoflowers were studied via Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The 25 most important pathways in the KEGG enrichment analysis are shown in bubble plot format. Compared with the control, Cu_xTe nanoflowers significantly affected C. albicans pathways associated with protein processing, peroxidase, the TCA cycle, pyruvate metabolism, and starch and sucrose metabolism (Fig. 5D). Importantly, the enrichment scores for the TCA cycle pathway for the Cu_xTe and control groups were padj<0.1, indicating that the superior fungicidal performance of Cu_xTe is highly correlated with the TCA cycle (Fig. 5D). GO analysis also revealed significant effects of the Cu_xTe nanoflowers on fungal transmembrane transport and mitochondria-related pathways (Fig. 5C). Copper death has been reported to be dependent on mitochondrial respiration, where excess copper promotes the acylation of pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-KDH), thereby affecting the TCA cycle. Therefore, Cu_xTe may exert antibacterial effects by triggering fungal cuproptosis death. The classification and thermal map of DEGs after Cu_xTe nanoflower treatment on the basis of gene function are shown in Fig. 5E. Specifically, genes related to Cu²⁺ uptake, such as CTR1, were significantly downregulated in the Cu_xTe-treated group, whereas genes related to Cu²⁺ transport and copper resistance (ATX1, CCC2 and YCF1) were significantly upregulated, We think that this differential expression pattern reflects the adaptive regulatory response of fungi to Cu_xTe NFs. When copper ions accumulate intracellularly, fungi activate negative feedback regulation as a protective measure, reducing copper ion influx and promoting copper ion efflux [48,49]. Indicating that intracellular copper overload occurs in C.albicans. In addition, PDH-related genes, such as PDX1, PDA1 and PDB1, were significantly downregulated after Cu_xTe treatment, further confirming that Cu_xTe can significantly affect the fungal TCA cycle.

Fig. 5.

Transcriptomic analysis of SC5314 killing by Cu_xTe nanoclusters. (A) Schematic diagram of the Cu_xTe nanocluster antifungal mechanism in SC5314. (B) Volcano map of the distribution of DEGs. The red and green points correspond to 2.0-fold changes in the two groups. (C) GO analysis of the functions of the DEGs. (D) KEGG enrichment analysis scatter plot. (E) Hierarchical clustering analysis of the two groups.

According to the transcriptomic analysis described above, the fungicidal effect of Cu_xTe nanoflowers is highly correlated with the TCA cycle, suggesting a trigger for fungal cuproptosis death. In addition, the expression of genes associated with Fe-S cluster proteins (ISA1 and ISA2), which are important markers of copper death was significantly reduced. Notably, genes associated with fungal cell wall synthesis were also downregulated, suggesting that Cu_xTe nanoflowers can disrupt cell wall integrity (Fig. 5E). These results indicate that Cu_xTe nanoflowers exert fungicidal effects by triggering fungal cuproptosis death. Cu_xTe blocks fungal metabolism by interfering with mitosis, inducing DNA damage and inhibiting biofilm formation under the synergistic effects of copper accumulation and cell wall damage. Comprehensive analysis of the transcriptome sequencing results revealed the mechanism of action of PCW nanoflowers on fungal cells, as shown in Fig. 5A. In summary, Cu_xTe can adhere to the surface of fungi and increase the local Cu²⁺ concentration of the fungal cell wall, resulting in Cu²⁺ overload in fungi. PDH activity subsequently decreased, the TCA cycle was restricted, Fe-S protein levels decreased, MDA accumulation was significant, and cuproptosis death occurred in fungal cells treated with Cu_xTe nanoflowers.

3.5. Cu_xTe induced cuproptosis death in fungi

Previous studies have shown that mitochondrial dysfunction adversely affects basic cellular processes such as energy metabolism and substance transport, thus interfering with cell survival and metabolic homeostasis. Copper death mediates cell death mainly by affecting mitochondrial function. On the basis of previous GO and KEGG enrichment analyses, the PDH complex and key genes of the TCA cycle were significantly downregulated in C. albicans treated with Cu_xTe nanoflowers. Given that mitochondria are the energy factories of cells, downregulation of these key genes suggests impaired mitochondrial activity, suggesting that Cu_xTe may trigger cuproptosis death by inducing fungal copper overload. To verify the above hypothesis, we first detected reactive oxygen species (ROS) levels in fungi treated with Cu_xTe nanoflowers via a DCFH-DA probe. As shown in Fig. 6A and S15, the intracellular ROS levels of fungi in the Cu_xTe-treated groups were significantly greater than those in the control groups, and the ROS levels were positively correlated with the Cu_xTe concentration. In addition, the lipid peroxide malondialdehyde (MDA) accumulated in fungal cells treated with Cu_xTe nanoflowers. C11-BODIPY 581/591 fluorescence staining and MDA quantification revealed that the MDA levels in the Cu_xTe treatment group were significantly increased (Fig. 6B and C). The results revealed that a high level of oxidative stress occurred in fungi and that the DNA, biosynthesis and mitochondrial functions of fungi might be obviously damaged. Mitochondrial dysfunction is a key marker of copper mortality. We measured the mitochondrial membrane potential and ATP production levels of fungi via fluorescence staining for JC-1 and Rhodamine 123 (Rh123). The results revealed that the fungal membrane potential of the Cu_xTe nanoflower treatment group was obviously disrupted (Fig. 6D) and that the ATP production level of mitochondria was significantly reduced (Fig. 6E), indicating that Cu_xTe nanoflowers can affect the normal energy metabolism of mitochondria. In addition, we measured the activity of mitochondrial respiratory chain complexes I and II in fungi. As shown in Fig. 6F, the activities of mitochondrial respiratory chain complexes I and II were significantly reduced after fungi were treated with the Cu_xTe nanoflowers, which was consistent with the results of the transcriptome enrichment analysis. Moreover, the results of biological transmission electron microscopy revealed that the mitochondria of fungi treated with Cu_xTe lost their normal elliptical shape and that the crista structure disappeared (Fig. 6G). These findings confirmed that Cu_xTe nanoflowers could destroy the mitochondrial function of fungi. Cell wall damage and cytoplasmic leakage were observed in fungal cells treated with Cu_xTe nanoflowers (Fig. S16). Following exposure to CuTe nanoflowers, fungal cells presented nucleic acid and protein leakage, which increased in a time-dependent manner (Fig. S17 and S18). Additionally, intracellular potassium release was increased (Fig. S19). Inductively coupled plasma-mass spectrometry (ICP‒MS) results revealed that the copper content in C. albicans treated with Cu_xTe was significantly greater than that in the control group (Fig. 6H), suggesting that the bacteriostatic effect may be due mainly to intracellular copper ions overload. To investigate the selective ion release ability of copper telluride, we measured its copper ion release under different pH conditions. The results show that under weakly acidic conditions (pH 5-6), copper telluride exhibits a significantly enhanced ability to release copper ions. This finding indicates that within the weakly acidic microenvironment conducive to fungal growth, copper telluride can selectively release more copper ions, thereby inducing fungal cuproptosis (Fig. S20). In conclusion, we hypothesized that Cu_xTe nanoflowers adhere to the fungal surface through their positively charged nature and locally release Cu²⁺, which penetrates into the cell, resulting in intracellular copper overload and subsequently causes fungal mitochondrial dysfunction and triggers intense oxidative stress in fungal cells, resulting in fungal death.

Fig. 6.

Molecular mechanisms of Cu_xTe-induced cuproptosis death in C. albicans. (A) Species-specific ROS generation in Candida spp. assessed by DCFH-DA staining. (B) Lipid peroxidation levels measured via C11-BODIPY 581/591 (fluorescence images, upper; oxidation index, lower). (C) Malondialdehyde (MDA) accumulation. (D) Mitochondrial depolarization visualized by JC-1 fluorescence (representative images, left; intensity quantification, right). (E) Mitochondrial functionality via Rh123 staining (representative panels, top; signal quantification, bottom). (F) Respiratory chain complex I and II enzymatic activities. (G) Ultrastructural alterations under Cu_xTe treatment (TEM). (H) Intracellular copper concentration (ICP‒OES).

To explore the molecular mechanism of copper death, the expression of key genes related to copper transport (CCC2, CRP1, and CTR1) was verified by qPCR under Cu_xTe treatment. As shown in Fig. 7A, genes associated with Cu²⁺ uptake, such as CTR1, were significantly downregulated in the Cu_xTe-treated group, whereas genes associated with Cu²⁺ transport and copper resistance (CCC2 and CRP1) were significantly upregulated, indicating that intracellular copper overload in C. albicans may lead to fungal copper overload death. Ferredoxin 1 (Fdx1), lipoic acid synthase (Lias), dihydrothionamide acetyltransferase (Dlat) and heat shock protein 70 (Hsp70) have been identified as key proteins involved in copper cell death, and their expression signatures change when copper death is triggered. Fdx1 participates in the mitochondrial electron transport chain (ETC) as an iron‒sulfur cluster protein responsible for the transfer of electrons from complex I/II to ubiquinone (CoQ). Hsp70 has cytoprotective and stress response regulatory functions, whereas Lias is involved in metabolism, the TCA cycle and the copper toxicity response. Both play key roles in the copper-induced death of C. albicans. As shown in Fig. 7B, compared with those in the control group, the expression levels of the FDX1 and LIAS genes were significantly lower, and the expression of the stress protein HSP70 and HSP60 was significantly greater in the Cu_xTe-treated group. Western blot analysis revealed that Lias and Fdx1 protein expression decreased and the Hsp70 and Hsp60 protein expression increased in the Cu_xTe treatment group (Fig. 7C and S21). The results showed that Cu_xTe nanoflowers increased the level of mitochondrial stress, inhibited the expression of key proteins of the TCA cycle and impaired mitochondrial function. Dlat is the core component of the pyruvate dehydrogenase complex (PDH), and its thiocaprylation domain is responsible for transferring acetyl groups to coenzyme A to form acetyl coenzyme A in the TCA cycle. During copper death, high concentrations of Cu²⁺ bind specifically to the thiocapillary side chain of Dlat, inducing it to form insoluble oligomers, resulting in impaired mitochondrial function. Oligomerization of the Dlat protein is a key marker of copper death. Western blot analysis revealed that the Dlat protein in the Cu_xTe-treated groups presented multiple protein bands with higher molecular weights above the original protein size, indicating Dlat protein aggregation (Fig. 7D). FDX1 and LIAS genes play important roles in fungal growth and metabolism, so we constructed FDX1 and LIAS gene knockout strains via the CRISPR-Cas9 system and conducted drug sensitivity experiments to verify the antibacterial mechanism of Cu_xTe nanoflowers (Fig. S22). As shown in Fig. 7E, FDX1 and LIAS knockout strains presented significantly increased sensitivity to Cu_xTe, and low concentrations of Cu_xTe also significantly inhibited fungal growth. The above studies, revealed that Cu_xTe nanoflowers exert antifungal effects through the local release of Cu²⁺, resulting in intracellular copper overload. To further verify whether fungal death is caused by copper ion penetration, we treated the Cu_xTe nanoflowers with the copper ion chelator tetrathiomolybdate (TTM). The results showed that TTM alleviated the ability of Cu_xTe to kill fungi, while the addition of exogenous copper ions slightly enhanced the antifungal ability of Cu_xTe, and TTM itself had no effect on fungal growth (Fig. 7F). Moreover, the qPCR results revealed that TTM alleviated the downregulation of key copper death genes by Cu_xTe (Fig. 7G). These results confirmed that Cu_xTe nanoflowers mediate fungus-triggered cuproptosis death via cascade reactions. The positive charge of the surface of Cu_xTe strongly adhere to fungal cells and can release Cu²⁺ ions locally. The Cu²⁺ accumulated in fungal cells leads to copper overload. Excessive Cu² inhibits PDH activity, blocks the TCA cycle and leads to the disintegration of Fe‒S cluster proteins. In addition, Cu²⁺ directly binds to the lipoic acid groups on Dlat, inducing insoluble aggregation of lipoylated proteins and mitochondrial proteotoxic stress. Relevant studies show the main difference between copper-induced death (cuproptosis) and cuproptosis-like death: cuproptosis is a copper ion-dependent programmed cell death (PRCD). Its core mechanism is that copper ions specifically bind to lipoylated proteins in the tricarboxylic acid cycle (TCA cycle), causing protein aggregation and metabolic disorders. Cuproptosis-like death refers to toxic cell death from non-specific oxidative damage caused by copper [39,50]. Since we observed clear cuproptosis characteristics in fungi, we define this mechanism as cuproptosis.

Fig. 7.

Molecular mechanisms of Cu_xTe-induced cuproptosis death in C. albicans. (A) Expression profiling of core cuproptosis death regulators. (B) Copper transporter gene modulation in the SC5214 strain post-Cu_xTe exposure (qPCR). (C) Immunoblotting of cuproptosis death executers via densitometric analysis. (D) DLAT protein expression (western blot). (E) Cu_xTe sensitivity profiles of the wild-type, FDX1Δ/Δ, and LIASΔ/Δ mutants. (F) Antifungal susceptibility in copper-chelated cultures. (G) Chelator-mediated regulation of copper homeostasis genes (qPCR).

3.6. Cu_xTe nanoflowers have good biosafety and can induce macrophage polarization to exert synergistic antifungal effects

Good biocompatibility and safety are prerequisites for clinical application. To address this concern, the biological safety of Cu_xTe nanoflowers was investigated at the cellular level. In this study, HUVECs (human umbilical vein endothelial cells), L929 cells (mouse fibroblast epithelial cells) and Raw264.7 cells (mouse macrophages) were selected as the research objects, and the cytotoxicity of Cu_xTe nanoflowers was detected via a CCK-8 cytotoxicity test. The results showed that at the effective antibacterial concentration, the cells were in good condition and the cell survival rate was above 80% (Fig. 8A). In addition, in the hemolysis experiment with rabbit erythrocytes, the hemolysis rate was less than 5% at the effective antibacterial concentration (50 μg/mL) (Fig. 8B). The results confirmed that the Cu_xTe nanoflowers maintained good biosafety at an effective antifungal concentration (50 μg/ml), meeting the therapeutic window requirements. Fungal infection wounds become a clinical problem because they cannot heal according to normal physiological processes. Copper is an essential trace element in wound healing, and is involved in inflammatory regulation, cell proliferation, angiogenesis and extracellular matrix remodeling. On the basis of the excellent enzyme-like catalytic activity (peroxidase-like enzyme/oxidase activity) and free radical scavenging ability of the Cu_xTe nanoflowers in this study, a systematic evaluation of their potential in wound repair was performed. To explore the mechanism by which Cu_xTe nanoflowers promote healing, we further treated HUVECs stimulated with β-glucans with Cu_xTe. The results revealed that β-glucans treatment group presented a strong ROS fluorescence signal, whereas the Cu_xTe-treated HUVECs presented significantly reduced fluorescence intensity, and the scavenging efficiency was concentration dependent. Quantitative analysis further confirmed that Cu_xTe could efficiently scavenge the ROS induced by β-glucans (Fig. 8C). In addition, HUVECs and L929 cells were treated with Cu_xTe, and growth factor expression levels were measured. The qPCR results revealed significant upregulation of the proangiogenic genes Vegfa, Hif-1α, and Angiogenin (Fig. 8D and S23), and the Western blot results revealed a simultaneous increase in the protein expression of cell growth factors (Fig. 8E). In conclusion, Cu_xTe nanoflowers can reduce ROS levels in wound tissues and activate vascular endothelial cells and fibroblast epithelial cells to release growth factors to accelerate angiogenesis and tissue remodeling.

Fig. 8.

Evaluation of cytotoxicity and anti-inflammatory activity in vitro. (A) Cytocompatibility evaluation of HUVECs, L929 cells, and Raw264.7 cells (CCK-8 assay). (B) Hemolytic activity assessment with erythrocyte morphology (representative micrographs). (C) Intracellular ROS visualization (representative fluorescence fields). (D) Inflammatory protein expression (western blot). (E) Cytokine transcriptional regulation in HUVEC cells (qPCR).

Previous studies have demonstrated the fundamental role of copper ions in immunostimulation. Fungal biofilms impede the ability of immune cells to kill fungi, and immune escape caused by biofilm-infected microenvironments promotes fungal retention in the host. Disruption of this immunosuppressive state is therefore crucial for preventing the persistence of chronic infections. However, macrophages play a key role in the immune system by recognizing and killing fungi. We treated the RAW264.7 cells with Cu_xTe nanoflowers and evaluated their ability to promote their differentiation. Flow cytometry was used to assess the polarization level of macrophages by analyzing the expression levels of Cd86 and Cd206 (Fig. 9A). The results revealed that the RAW264.7 (CD86⁺) ratio after incubation with Cu_xTe was significantly greater than that of the control group (Fig. 9B). qPCR results subsequently revealed that Cu_xTe treatment significantly upregulated the Raw264.7 M1-type marker Cd86 and proinflammatory factor genes (Il-1β, Il-6, Tnf-α, and inos), whereas the M2-type markers Cd206 and Arg-1 remained unchanged (Fig. 9C). Cu_xTe nanoflowers significantly increased protein levels of IL-6 and TNF-α in RAW264.7 cells (Fig. S24). Moreover, the Il-6 level in Raw264.7 cells treated with Cu_xTe was detected via immunofluorescence staining and Western blotting. As shown in Fig. 9D, Cu_xTe nanoflowers significantly increased IL-6 secretion and protein expression in Raw264.7 cells. These findings clearly indicate that Cu_xTe nanoflowers can drive M0 macrophages to polarize to the M1 phenotype, increase their phagocytic fungicidal ability and release proinflammatory mediators. This immune activation and direct antibacterial activity of Cu_xTe form a synergistic effect, jointly clearing pathogens and initiating inflammation‒repair cascade reactions, providing dual protection for wound healing.

Fig. 9.

Evaluation of cytotoxicity and anti-inflammatory activity in vitro. (A) Macrophage immunophenotyping strategy (flow cytometry gating). (B) Quantified surface marker expression (flow cytometric intensity). (C) Macrophage polarization dynamics: M1/M2 markers and cytokine transcriptomics (qPCR). (D) IL-6 distribution patterns (immunofluorescence imaging). (E) Proteomic profiling of treated Raw264.7 cells (immunoblotting).

3.7. In vivo therapeutic effects of Cu_xTe against wound fungal infections

Nanomaterials have shown important value in the treatment of infected wounds, and previous studies have highlighted both the healing and antifungal potential of copper-based nanomaterials. According to our previous studies, Cu_xTe nanoflowers can effectively destroy multidrug-resistant fungi and promote wound healing in vivo by inducing fungal copper death and disrupting biofilms in vitro. In addition, Cu_xTe exhibited excellent biocompatibility. We hypothesized that Cu_xTe could be used for the systemic treatment of wounds infected with C. albicans. The effect of Cu_xTe on wound healing was studied in an established infected wound model. As part of the experiments using Cu_xTe, a 9 mm wound model infected with C. albicans was used in BALB/c mice. A schematic of the in vivo wound model is shown in Fig. 10A. A full-thickness wound model was established in mice to determine the healing effects of PBS, Cu_xTe, and FLC (positive control group) in vivo. Wound healing was dynamically monitored within 12 days after treatment, images of the healing process were acquired, and the kinetics of wound closure were recorded (Fig. 10B). Fig. 10C shows the wound healing rates at various time points over 12 days for each group. The results revealed that the scab rate of the Cu_xTe group was significantly greater than that of the PBS group and that the scar area was significantly smaller. The wound surface in the Cu_xTe group was basically epithelialized, while the scar in the fluconazole group was small, and the scar in the PBS group was still large. Quantitative analysis with ImageJ software revealed that the wound closure rate of the Cu_xTe group was significantly greater than that of the control group at all time points (P < 0.05) in terms of the wound healing of the mice in each group during the treatment period. There was no significant difference (P > 0.05) in the body weight of mice in each group during the treatment period (Fig. 10D). The culture results of wound secretions from mice on day 1 and 6 further confirmed that only a small number of colonies were seen in CuTe group (Fig. E), which was significantly lower than that in PBS group and fluconazole group (P < 0.01), indicating that CuTe group had strong anti-infection ability. (Fig. 10E).

Fig. 10.

In vivo efficacy assessment of Cu_xTe nanoclusters in infected wound healing. (A) Schematic of the murine wound infection model. (B) Representative wound images across treatment groups at the indicated timepoints (days −1, 1, 3, 6, 9, and 12). (C) Quantitative wound area reduction (control vs. fluconazole vs. Cu_xTe; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (D) Longitudinal body weight monitoring (n = 3 per group). (E) Fungal burden quantification: (Top) Day 1 and day 6 wounds homogenate cultures; (Bottom) Colony enumeration.

We collected wound tissues and performed HE and Masson staining and histological analysis to determine the ability of the Cu_xTe nanoflowers to promote wound healing (Fig. 11A). H&E staining revealed that the Cu_xTe group had well-differentiated epithelial cells, the epidermal layer significantly thickened and good continuity was observed, whereas the PBS group and fluconazole group still presented a tissue fracture zone, the epidermis. regeneration was weak. The remodeling phase is important for collagen deposition and remodeling to promote tensile strength and regeneration. Therefore, the amount of collagen produced in each group of wound tissues was further observed. Compared with the control group, the Cu_xTe group presented significantly greater collagen deposition, more compact arrangement, more collagen production and more obvious differentiation (Fig. 11A). In the Cu_xTe group, many skin attachments and new wounds were observed in the central area of the wounds. Immunofluorescence assays revealed that the levels of the proinflammatory factor IL-6 in tissue samples from PBS- and fluconazole-treated mice were high, whereas the levels of inflammatory factors in Cu_xTe treated mice were significantly decreased. The immunofluorescence intensity of the inflammatory factor IL-6 in the wound tissues of the Cu_xTe-treated mice was significantly lower than that in the other two groups. Furthermore, vascular endothelial growth factor (VEGF) levels increased in tissues (Fig. 11B,C). These findings indicate that in addition to controlling wound infection and promoting wound healing, Cu_xTe nanoflowers can also improve wound inflammation and stimulate angiogenesis at a later stage to accelerate the repair process.

Fig. 11.

In vivo wound healing efficacy and biocompatibility assessment of the Cu_xTe nanoclusters. (A) Histopathological analysis: Representative H&E and Masson's trichrome-stained wound sections on day 12. (C) Fluorescence intensity statistics. (B) Immunofluorescence localization of IL-6 and VEGF in wound tissues. (D) Hematological and biochemical profiling: Complete blood counts and serum biochemistry in treatment groups versus healthy controls (n = 6). (E) Systemic biosafety evaluation: H&E-stained sections of major organs (heart, liver, spleen, lung, and kidney) on day 12.

In addition, we monitored the toxicity of the Cu_xTe nanoflowers in vivo to verify their safety. The blood biochemical parameters of the different groups of mice were assessed on day 7 (Fig. 11D). The Cu_xTe group was not significantly different from the PBS group or FLC group in terms of routine blood tests (RBCs, platelets, WBCs) (P > 0.05). Compared with those in the control group, the levels of liver function indicators (glutamate aminotransferase and glutamate aminotransferase) and renal function indicators (blood urea nitrogen, creatinine, and creatinine) in the Cu_xTe group were not significantly different (P > 0.05). In addition, HE staining of major organs (heart, liver, spleen, lungs and kidneys) in each group did not reveal significant histopathological changes (Fig. 11E). In vivo evaluation of the Cu_xTe nanoflowers confirmed their biocompatibility. In conclusion, the Cu_xTe nanoflowers had excellent antifungal effects both in vitro and in vivo. Antifungal effects are exerted synergistically mainly by triggering fungal copper death and host immune activation. In addition, it can promote the secretion of tissue growth factors and accelerate wound healing. In vivo, Cu_xTe nanoflowers promote infected wound healing through a twofold synergistic mechanism (direct killing of pathogens and regulation of the repair microenvironment). Its efficacy is superior to that of the traditional antifungal drug fluconazole, and it has excellent in vivo safety at therapeutic doses, providing an experimental basis for translational clinical application.

4. Discussion

In summary, Cu_xTe nanoflowers featuring a multibranched structure were successfully synthesized via a wet chemical method. Experiments revealed that Cu_xTe possesses a unique structure and a multimode antifungal mechanism, conferring advantages over traditional copper-based nanomaterials. First, Cu_xTe nanoflowers exhibit multienzyme-like activities that efficiently catalyze the production of reactive oxygen species (ROS) in acidic microenvironments, triggering intense oxidative stress in fungi. While stable under physiologically neutral pH conditions, they effectively release Cu²⁺ions in weakly acidic environments. The pH-responsive ion release behavior of Cu_xTe is similar to that reported for antifungal nanomaterials (PDA@Kana-AgNPs, ZnO) [51,52]. Targeted therapy can be achieved by leveraging the weak acidity characteristic of infection microenvironments. However, the unique branching structure of the Cu_xTe nanoflowers significantly increased the specific surface area. This enhancement not only improves their initial adhesion efficiency to fungal cells but also facilitates the generation of locally high concentrations of Cu²⁺ ions around fungal cells upon decomposition in acidic microenvironments. To the best of our knowledge, the antifungal activity of pyroptosis-like death has not been extensively investigated. This study directly links the antifungal effect of Cu_xTe nanoflowers to the emerging mechanism of cuproptosis death. Cu_xTe treatment induced mitochondrial stress, characterized by inactivation of the pyruvate dehydrogenase complex (PDH), inhibition of the tricarboxylic acid (TCA) cycle, and oligomerization of the Dlat protein [39]. These results indicate that Cu_xTe nanoflowers drive an irreversible cell death program, which is less likely to induce drug resistance, by causing mitochondrial metabolic dysfunction and energy depletion. Additionally, Cu_xTe nanoflowers exhibit dual functional characteristics as “direct antibiotic–immune regulators.” They effectively induce macrophage polarization toward the proinflammatory M1 phenotype, exerting synergistic antibacterial effects and accelerating wound closure.

Recent years have seen great progress in copper-induced cell death mechanisms (cuproptosis and cuproptosis-like pathways) for antimicrobial applications. The key to this progress is the targeted antimicrobial effect of copper-based nanomaterials, which reduces the systemic toxicity and poor targeting of traditional copper preparations. Studies demonstrate that disrupting bacterial copper homeostasis and inducing copper-mediated proteotoxic stress effectively eliminate bacteria and degrade biofilms [53]. Furthermore, researchers have exploited multi-ionic effects to induce cuproptosis—for instance, manganese ions in copper-manganese composites deplete bacterial glutathione, thereby impairing copper chelation and promoting copper binding to lipoylated TCA cycle proteins to trigger cuproptosis-like death [54]. Additionally, integrating photothermal capabilities into copper sulfide nanomaterials and nanomotors compromises bacterial membrane integrity, facilitates copper influx, and generates reactive oxygen species via copper-catalyzed Fenton-like reactions, yielding synergistic antibacterial outcomes [55]. Collectively, these developments mark a shift in cuproptosis-based therapy-from nonspecific copper ion toxicity to targeted delivery systems that disrupt bacterial homeostasis. This new approach provides promising strategies to fight drug-resistant pathogens and biofilm-associated infections.

In conclusion, the Cu_xTe nanoflowers represent an efficient, multi mechanism synergistic antifungal nanoplatform. It not only has excellent direct antifungal activity but also effectively eradicates persistent biofilms through a unique synergistic strategy combining “microenvironment regulation, chemodynamic therapy (CDT), and cuproptosis.” Furthermore, they demonstrate significant antifungal efficacy against “the multidrug-resistant fungus” Candida auris. This study provides a solid theoretical and experimental foundation for the development of a new generation of antifungal therapies based on nonclassical antifungal mechanisms. While the current work primarily utilizes in vitro and skin wound fungal infection models, future studies need to further investigate the long-term in vivo biodistribution and clearance pathways of Cu_xTe nanoflowers. Additionally, exploring their potential for surface modification or combination with other antifungal agents is warranted to accelerate their translational clinical application.

CRediT authorship contribution statement

Yong he: Conceptualization, Data curation, Writing – original draft. Jianguo Niu: Investigation, Methodology. Wenqiang Huang: Formal analysis. Changshan Ju: Data curation. Shengqun Deng: Data curation, Methodology. Juanjuan Cui: Funding acquisition. Xianwen Wang: Resources, Supervision, Writing – review & editing. Mao Zhang: Funding acquisition, Supervision, Writing – review & editing.

Declaration of competing interest

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

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.