Introduction

Fungal infections have become a global public health issue with high morbidity1. Superficial fungal infections are the most common fungal diseases in humans, affecting ∽25% global population2. Invasive fungal infections can lead to serious diseases and even life-threatening3. For example, fungal keratitis is one of the most common causes of blindness worldwide4,5. More than 50% of lung-transplant patients might die if infected by invasive aspergillosis. By the end of 2022, the World Health Organization (WHO) released fungal priority pathogens list (FPPL), highlighting 19 fungi that pose the greatest threat to public health, including Candida albicans (C. albicans) and Aspergillus flavus (A. flavus)6. Faced with increasingly severe fungal infections, the available antifungal drugs are very limited7. There are only four classes of antifungal antibiotics available in clinic: polyenes, azoles, flucytosine, and echinocandins. More seriously, fungi are becoming more and more resistant to antifungal antibiotics8. It is urgently demanded to develop new strategies for fungal infection management9,10,11.

Compared to mammalian cells, fungal cells have an additional dense cell wall outside their cell membrane. The fungal cell wall is composed of glucans, chitin, mannans, and glycoproteins, forming a cross-linked multi-layered structure12. The presence of fungal cell wall is a major obstacle of drug penetration into fungal cells13. On the other hand, fungi can accumulate into biofilms to greatly weaken the therapeutic efficacy of antifungal drugs14. Fungal biofilms exhibit multifaceted resistance mechanisms and can be as high as 1000-fold more resistant than planktonic fungi15. The formation of fungal biofilms is a critical factor to the failure of antifungal therapies.

Transition metals such as silver, copper, and zinc were used as antimicrobials thousands of years ago16,17,18. Metal-based antimicrobial nanoagents have been extensively studied and demonstrated excellent antimicrobial properties19,20,21,22. Transition metal ions are promising antifungal agents due to the excellent fungicidal ability and minimal drug resistance, which exhibit a multi-targeted antimicrobial mechanism, including protein dysfunction, cell membrane disruption, and nucleic acid damage23. Unfortunately, the in vivo antifungal application of transition metal ions was never reported in clinic. Dissociative transition metal ions can be easily adsorbed by proteins in physiological environment, leading to the deactivation of metal ions. The design of metal ion delivery systems might be an effective way to tackle this problem. Polyphenols can coordinate with metal ions to form metal-phenolic networks, demonstrating great potential in metal ion delivery24,25,26. Moreover, metal-phenolic coordination self-assembly is dynamically reversible, possessing the potential for controlled release of metal ions in specific microenvironments18,27.

Cooper (Cu) is an essential trace element that plays important roles in human physiology. Meanwhile, Cu is a very important metal-based antifungal agent. Recently, cuproptosis was reported as a new cell death mechanism in 202228. Cuproptosis occurs via Cu2+ binding to lipoylated components of the tricarboxylic acid (TCA) cycle, leading to the accumulation of lipoylated proteins and the loss of Fe-S cluster proteins, triggering a protein toxicity stress response and subsequent cell death. Cuproptosis has become an important therapeutic target in treating many diseases29,30. Very recently, cuproptosis-like bacterial death was reported to combat bacterial infections31,32. However, cuproptosis was not reported in treating fungal infections as far as we know. Cuproptosis is highly related to mitochondrial metabolism. Fungi and mammalian cells are both eukaryotes with mitochondria33. Similar to mammalian cells, the TCA cycle in fungi occurs in mitochondria and is the key metabolic pathway, which provides the possibility of the occurrence of cuproptosis in fungi.

In this contribution, we put forward a strategy to prepare metal-phenolic nanoflowers via lywallzyme (Lyw)-induced self-assembly for antifungal applications. The Cu2+-based nanoflowers are expected to exhibit synergistic fungicidal activity via cuproptosis and cell wall digestion (Fig. 1). Protocatechuic acid (PA)-Cu2+ (PC) nanopetals were prepared via one-pot coordination self-assembly of PA and Cu2+ without additional stabilizers. PC nanopetals could self-assemble into nanoflowers in the presence of Lyw. The Lyw loaded PC (PCW) nanoflowers could effectively adhere to fungal cells. Leveraging the dynamic metal-phenolic coordination interaction, PCW nanoflowers could be disassembled in mildly acidic environments, releasing Lyw and Cu2+. The released Lyw could digest fungal cell walls, which facilitated the invasion of Cu2+ into fungal cells, exhibiting synergistic fungicidal activity. Due to the formation of nanoflowers, PCW maintained excellent antifungal performance even in protein-rich environments. The fungicidal mechanism of PCW nanoflowers was investigated by transcriptome sequencing and quantitative assay kit, which revealed that PCW could induce fungal cuproptosis owing to intracellular overload of Cu2+. The in vivo antifungal performance of PCW was explored by a C. albicans-induced skin infection model and a fungal keratitis model in mice. The Cu2+-based nanoflower provides an antifungal strategy by the combination of cuproptosis and cell wall digestion, which holds great promise in treating both superficial and invasive fungal infections.

Fig. 1: Schematic illustration of the synthesis and fungicidal mechanism of PCW nanoflowers.
figure 1

The PC nanopetals are firstly synthesized via the ont-pot coordination self-aasembly of PA and Cu2+. PCW nanoflowers are then obtained by Lwy induced self-assembly of PC nanopetals. PCW nanoflowers can be decomposed in acidic microenvironment, leading to the release of Cu2+ and Lyw. After incubated with fungal cells, PCW nanoflowers can adhere to the fungal surface. Lyw is then released to digest the dense fungal cell walls. A large amount of Cu2+ is internalized into the fungal interior to induce fungal cuproptosis. PCW nanoflowers are also expected to destroy mature fungal biofilms. The in vivo fungicidal effect of PCW nanoflowers is confirmed by a murine skin fungal infection model and a murine fungal keratitis model.

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Results

Synthesis and characterization of PCW nanoflowers

Metal-phenolic nanoparticles hold great promise in biomedical applications34,35,36. In order to control the overgrowth of metal-phenolic nanoparticles, it is necessary to add additional stabilizers37. In this research, Cu2+-PA nanoparticles were prepared by a simple one-pot method without additional stabilizers. Specifically, ammonia water was firstly added to a copper sulfate solution to obtain a copper-ammonia solution. PA in ethanol was added dropwise (Fig. 2a). Cu2+ and PA gradually coordinated and aggregated to obtain PA-Cu2+ (PC) nanoparticles, which could be well-dispersed in water (Supplementary Fig. 1). The transmission electron microscope (TEM) image in Fig. 2b showed that PC was petaloid with the size of about 50 nm. After positively charged Lyw was added to PC suspension, Lyw would adhere to negatively charged PC surface due to electrostatic interaction, which induced the assembly of petaloid PC into flower-shaped nanoparticles (Fig. 2b, c). Therefore, positively charged Lyw could be loaded onto negatively charged PC nanopetals via electrostatic interaction. The preparation of Lyw-loaded PC (PCW) nanoflowers was optimized by adjusting the feed ratio of Cu2+, PA, and Lyw. Altering the molar ratio of Cu2+ to PA resulted in different sizes of PCW nanoflowers. As the Cu2+ to PA ratio increased from 1:10 to 1:1, the size of PCW nanoflowers reduced from microscale to nanoscale. However, when the ratio of Cu2+ to PA was 1:1, the PCW nanoflowers began to aggregate. Thus, the mole ratio of Cu2+ to PA was fixed as 1:3 to prepare PCW in the following experiments (Supplementary Fig. 2). As the mass ratio of Lyw to PC increased from 1:8 to 1:20, the morphology of PCW was almost unchanged. When Lyw to PC ratio increased to 1:5, PCW was aggregated and precipitated. Therefore, the mass ratio of Lyw to PC was fixed as 1:8 in the following experiments (Supplementary Fig. 3). In this case, PCW nanoflowers were negatively charged with the intensity-average hydrodynamic diameter of 309.6 nm, which was much larger than that of PC (52.9 nm) (Fig. 2d).

Fig. 2: Synthesis and characterization of PCW Nanoflowers.
figure 2

a Preparation of PCW nanoflowers by self-assembly of PA, Cu2+, and Lyw. Created in BioRender. Ji, J. (2024) BioRender.com/g49x121b TEM images of PC nanopetals and PCW nanoflowers. Inset: The digital photos of PC and PCW in aqueous solution. c Zeta potential values of PC, PCW, PA, and Lyw. d Intensity-average hydrodynamic diameters of PC and PCW. e TEM, HR-TEM, and SAED images of PCW. f TEM mapping analysis of PCW with different colored dots representing C, N, Cu, and S. g Adsorption-desorption isotherm of PCW. h XPS survey spectrum of Cu2p in PCW. i FT-IR spectra of PC, PCW, PA, and Lyw. j UV-vis absorption spectra of PC, PCW, PA, and Lyw. k TEM images of PCW at pH 6.0 and pH 5.0, respectively. l Release curves of Cu2+ from PC and PCW over time in aqueous solution. m Release curves of Cu2+ from PC and PCW over time in MOPS buffer with different pH. b, e, f, k showed one of three independent experiments with similar results. Data for (c) were presented as mean values ± standard deviations (SD) (n = 3 independent experiments). error bars = SD. Source data were provided in Source Data file.

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PC and PCW didn’t show apparent crossed lattice fringes in high-resolution TEM (HR-TEM) images. The selected area electron diffraction (SAED) patterns of PC and PCW did not display diffraction spots (Figs. 2e and S4). However, the X-ray Diffraction (XRD) curves of PC and PCW displayed two peaks at 10° and 13° (Supplementary Fig. 5), indicating a weakly crystallized structure in PC and PCW. The crystallized structure might be helpful in controlling the growth of PC. Energy dispersive spectroscopy (EDS) results in Supplementary Fig. 6 indicated homogeneous distribution of C, O, and Cu in PC. The sulfur (S) element was detected in the EDS image of PCW but was not observed in the EDS image of PC, which confirmed successful loading of Lyw in PCW (Figs. 2f and S7). The Cu contents in PC and PCW were ~32% and 22%, respectively, determined by inductively coupled plasma mass spectrometry (ICP-MS). The loading content of Lyw in PCW was determined to be ~20% (Supplementary Fig. 8). The physicochemical properties of PCW were systematically investigated. The adsorption-desorption isotherm of PCW showed the IUPAC Type IV isotherm38, with the specific surface area (as BET) of 25.1 m2/g (Fig. 2g). Furthermore, the oxidation state of Cu in PCW was investigated by X-ray photoelectron spectroscopy (XPS). The Cu2p3/2 and Cu2p1/2 peaks were found at 935 eV (with two satellite peaks at 940 eV and 944 eV) and 955 eV (with a satellite peak at 963 eV), respectively, which indicated that the electronic structure of Cu in PCW was 3d939. Therefore, Cu in PCW showed Cu(II) oxidation state and formed Cu-O single bonds with PA (Fig. 2h).

To further investigate the coordination interaction between Cu2+ and PA, Fourier-transform infrared spectroscopy (FT-IR) was used to characterize the complexation states of PCW (Fig. 2i). Sharp stretching vibration peaks for the free O-H and the acidic O-H in PA were located at 3571 cm−1 and 3261 cm−1, respectively40. Since PA formed a complex in PCW, these peaks in PCW shifted to broader intermolecular hydrogen bonding O-H stretching vibrations at 3455 cm−1 and 3136 cm−1. The stretching vibration of the carboxyl C=O in PA shifted from 1700–1600 cm−1 to lower wavenumbers (1651 cm−1 and 1529 cm−1), indicating that the carboxyl groups underwent deprotonation due to the coordination interaction between Cu2+ and the carboxyl group of PA41. The UV-visible (UV-vis) spectra were further used to study the coordination interaction between Cu2+ and PA in PCW. PA exhibited characteristic absorption peaks at 249 nm and 287 nm, corresponding to the B and R bands of aromatic acids, respectively (Fig. 2j). The coordination interaction between PA and Cu2+ in PCW led to a redshift of the corresponding characteristic absorption peaks to 272 and 310 nm. Meanwhile, two new absorption peaks at 210 and 235 nm was observed in the UV-vis absorption spectrum of PCW, which were likely corresponding to the absorption bands of Cu-O coordination.

Considering the inherent pH-responsive characteristic of metal-phenolic networks42, the stability of PCW was studied at different pH. The TEM and scanning electron microscope (SEM) images revealed the disassembly of PC and PCW at acidic pH (Figs. 2k and S9). The structure of PCW nanoflowers began to decompose at pH 6.0, with edges becoming blurred. PCW almost lost its nanostructure at pH 5.0, which should be attributed to the disassembly of metal-phenolic networks at acidic pH. The decomposition of PCW at acidic pH resulted in the release of Cu2+. Therefore, the release of Cu2+ from PC and PCW in ultrapure water and 3-morpholinopropane sulfonic acid (MOPS) buffer solution at different pH was studied. PCW was very stable in water with less than 8.5% release of Cu2+ within 700 min (Fig. 2l). When PCW was incubated in MOPS buffer at pH 7.4, despite the influence of high ion concentration on the coordination interactions, Cu2+ release remained relatively low (14% after 700 min incubation). However, more than 70% of Cu2+ was released from PC and PCW within 450 min at pH 6.0, exhibiting efficient pH-triggered Cu2+ release (Fig. 2m).

In vitro fungicidal activity of PCW

The in vitro antifungal property of PCW was evaluated by a standard plate count method using C. albicans as a model fungal strain. As shown in Fig. 3a, PC and PCW were more effective to eradicate C. albicans than free Cu2+. Even at a very low Cu2+ concentration of 1 µg/mL, PCW achieved a 99% reduction of C. albicans population. PA and Lyw didn’t show obvious fungicidal activity when the concentration was as high as 200 μg/mL (Supplementary Figs. 10, 11). The minimum fungicidal concentration (MFC, kill 99.9% fungi) of Cu2+, PC, and PCW were 15, 5, and 2 µg/mL, respectively. Furthermore, based on the pH-responsive drug release behavior of PCW, the fungicidal activity of PCW was investigated in MOPS buffer with different pH. As expected, PCW exhibited much higher fungicidal rate than free Cu2+ and PC at both pH 7.4 and 6.0 (Fig. 3b). Although high salt concentration in MOPS can interfere the coordination self-assembly of Cu2+ and PA, PCW maintained a very high fungicidal rate at pH 7.4, with 2 logs CFU reduction at 5 μg/mL Cu2+ equivalent. Owing to the disassembly of PCW and subsequent rapid release of Cu2+ at pH 6.0, PCW showed stronger fungicidal capability at pH 6.0 than that at pH 7.4. PCW led to 3 logs CFU reduction of C. albicans population at pH 6.0 when Cu2+ equivalent was as low as 5 μg/mL, whereas free Cu2+ could only reduce 22% C. albicans population. The time-dependent fungicidal evaluation in Supplementary Fig. 12 also confirmed the excellent fungicidal activity of PCW.

Fig. 3: The in vitro fungicidal evaluation of PCW.
figure 3

a The in vitro fungicidal evaluation in aqueous solution. C. albicans was incubated with different concentrations of Cu2+, PC, and PCW (1, 2, 5, 10, 15 μg/mL, Cu2+ equivalent). b The in vitro fungicidal evaluation in MOPS buffer with different pH. C. albicans was incubated with different concentrations of Cu2+, PC, and PCW in MOPS buffer at different pH (5, 10, 20 μg/mL, Cu2+ equivalent). c The in vitro fungicidal evaluation in FBS. C. albicans was incubated with Cu2+, PC, and PCW in various volume ratios (20%, 40%, 60%, 80%, 100%) of FBS/H2O (20 μg/mL, Cu2+ equivalent). d The in vitro fungicidal evaluation in SDB. C. albicans was incubated with different concentrations of Cu2+, PC, and PCW in 20% SDB (5, 10, 20 μg/mL, Cu2+ equivalent). e Additive effect of Cu2+ and Lyw against C. albicans determined by checkerboard microdilution assays. f Synergistic effect of PC and Lyw against C. albicans determined by checkerboard microdilution assays. g Plates images of C. albicans and A. flavus after incubation with MOPS (control), Cu2+, Cu2++Lyw, PC, PCW, and AmB. h Live (green)/dead (red) staining images of C. albicans after incubation with MOPS (control), Cu2+, Cu2++Lyw, PC, PCW, and AmB. The merged images were presented as mixed channels of NO1 (green) and PI (red). g, h showed one of three independent experiments with similar results. Data for (a–d) were presented as mean values ± SD (n = 3 independent experiments). error bars = SD. Significance of (a–d) were calculated by one-way ANOVA with Tukey’s post hoc test. Source data were provided in Source Data file.

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It is well-known that Cu2+ shows strong interaction with proteins, which is an important reason to make Cu2+ invalid in antimicrobial applications43. The fungicidal effect of PC and PCW was studied in protein-rich fetal bovine serum (FBS) solution. As shown in Fig. 3c, free Cu2+ gradually lost its fungicidal ability with the increase of FBS concentration. However, the fungicidal effect of PCW was almost unaffected by FBS. PCW at 20 μg/mL Cu2+ equivalent could lead to 2 logs CFU reduction in 100% FBS solution. More importantly, PCW showed excellent fungicidal capability in Sabouraud Dextrose Broth (SDB) medium containing various proteins, amino acids, and inorganic salts, whereas dissociative Cu2+ completely lost its fungicidal capability (Fig. 3d). The excellent fungicidal capability of PCW nanoflowers in protein-rich environments might be attributed to the direct release of Cu2+ onto fungal cell surface, which avoided the deactivation by proteins.

The synergistic fungicidal mechanism of Cu2+ and Lyw in PCW

Based on the superior fungicidal efficacy of PCW, the fungicidal mechanism of PCW was investigated. A checkerboard microdilution assay was utilized to investigate the synergy between Cu2+ and Lyw in PCW. Initially, the minimum inhibitory concentrations (MIC, inhibit 90% fungi) of Cu2+ equivalent for PCW, PC, and dissociative Cu2+ were 25 µg/mL, 50 µg/mL, and 400 µg/mL, respectively (Supplementary Fig. 13). Subsequently, the fractional inhibitory concentration (FIC) indexes of FIC (Cu2+/Lyw) and FIC (PC/Lyw) were calculated to be 1 and 0.375, respectively (Fig. 3e, f), which indicated a synergistic interaction between Cu2+ and Lyw in PCW, in contrast to the merely additive interaction between dissociative Cu2+ and Lyw. The excellent fungicidal effect of PCW was intuitively observed from the digital photos of plate counting assays. As shown in Fig. 3g, C. albicans and A. flavus exhibited the least colony growth after treated with PCW. The fungicidal ability of PCW was comparable to that of the commonly used antifungal antibiotic Amphotericin B (AmB). The live/dead fluorescent staining assay also validated the strongest fungal eradication ability of PCW among all the groups (Figs. 3h and S14).

In order to explore the role of PA in the fungicidal process of PCW nanoflowers, the interaction between PCW and C. albicans was studied. As shown in Fig. 4a, PC and PCW could effectively adhere to C. albicans, confirmed by the colocalization of Rhodamine B (RhoB) labeled PC, FITC labeled Lyw, and DAPI stained C. albicans. Meanwhile, Red fluorescence was not observed in the untreated C. albicans stained with DAPI (Supplementary Fig. 15). Since both PCW and C. albicans were negatively charged (Fig. 2c), the adhesion of PCW with C. albicans was not induced by electrostatic interaction. The effective adhesion of PCW with C. albicans might be attributed to the strong interaction between PA of PCW and C. albicans because polyphenols were reported to adhere to fungi44,45. TEM images also revealed that both PC nanopetals and PCW nanoflowers could adhere to fungal surface, which played an important role for the excellent fungicidal ability of PCW since it could largely increase local drug concentration around fungi. (Fig. 4b).

Fig. 4: The in vitro interaction mechanism of PCW with fungi.
figure 4

a Fluorescent images of C. albicans after incubation with PCW (RhoB labeled PC, FITC labeled Lyw). C. albicans was stained by DAPI. b TEM images of C. albicans after incubation with Cu2+, PC, and PCW. c Fluorescent images of C. albicans stained with trypan blue after incubation with Cu2+, PC, and PCW. d Total carbohydrate concentration of C. albicans after incubation with Cu2+, PC, and PCW measured by the phenol-sulfuric acid method (n = 6 independent experiments). e TEM images of C. albicans sections after incubation with Cu2+, PC, and PCW. f Cu element mapping images from TEM and SEM of C. albicans after incubation with Cu2+, PC, and PCW. g Semi-quantitative results of Cu element from SEM mapping of C. albicans after incubation with Cu2+, PC, and PCW (n = 5 independent experiments). h SEM images of C. albicans after incubation with Cu2+, PC, and PCW. i Schematic illustration of the interaction mechanism between PCW and C. albicans. MOPS treatment was used as control. a, b, c, e, f, h showed one of three independent experiments with similar results. Data for (d, g) were presented as mean values ± SD. error bars = SD. Significance of (d, g) were calculated by one-way ANOVA with Tukey’s post hoc test. Source data were provided in Source Data file.

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Lyw is a lysing enzyme that can induce fungal cell wall digestion. After incubating C. albicans with PCW for 6 h and staining with trypan blue, the green fluorescence of trypan blue was diminished, which implied that Lyw released from PCW could digest fungal cell walls (Fig. 4c). Due to the digestion of cell walls by Lyw, PCW nanoflowers significantly reduced the total carbohydrate content of C. albicans (Figs. 4d and S16). The digestion of fungal cell walls by PCW was also observed by TEM images of fungal sections. As shown in Fig. 4e, PCW could adhere around C. albicans and lead to a blurred outer cell wall structure, indicating the digestion of fungal cell walls.

Furthermore, it was very glad to see that more copper signals were observed in PCW treated C. albicans than other groups (Figs. 4f, g and S17–19), which might be ascribed to effective adhesion of PCW on C. albicans and enhanced invasion of Cu2+ into C. albicans after digestion of fungal cell walls by Lyw. The overload of Cu2+ might be an important reason for the excellent fungicidal performance of PCW. C. albicans incubated with PCW exhibited dramatic morphological changes, resulting in fragmented fungal cells (Fig. 4h). In summary, PCW nanoflowers were initially adhered to the fungal surface. Lyw could then be released to digest the dense fungal cell walls, facilitating the influx of a large amount of Cu2+ into the fungal interior to exert the fungicidal effect (Fig. 4i).

In vitro antibiofilm and biosafety evaluation of PCW

Fungal biofilms exhibit formidable resistance to the gamut of antifungal drugs, which can become 1000 times more resistant than planktonic fungi15. The potential of PCW nanoflowers in eradicating mature C. albicans biofilms was investigated. The antibiofilm effect of different treatments was firstly evaluated by measuring biofilm biomass using crystal violet staining. As shown in Fig. 5a, b, C. albicans biofilms remained dense and intact after incubated with dissociative Cu2+, indicating very weak antibiofilm ability of dissociative Cu2+. In contrast, there was a significant reduction in biofilm biomass after C. albicans biofilms were incubated with PC nanopetals. Mature C. albicans biofilms treated with PCW nanoflowers were almost non-visible, exhibiting much better antibiofilm ability. The excellent antibiofilm effect of PCW was also confirmed by live/dead staining of biofilms and biofilm thickness measurement after different treatments (Fig. 5c, d). Meanwhile, PCW nanoflowers exhibited negligible cytotoxicity and hemolysis when the equivalent Cu2+ concentration was as high as 50 µg/mL (Fig. 5e, f). However, PCW nanoflowers at 2 µg/mL were able to eradicate more than 99.9% C. albicans, indicating excellent biosafety of PCW in antifungal applications.

Fig. 5: The in vitro antibiofilm and biosafety evaluation of PCW.
figure 5

a Crystal violet staining images of C. albicans biofilms after treated with Cu2+, Cu2++Lyw, PC, PCW, and AmB. b Quantitative analysis of crystal violet absorbance (n = 8 independent experiments). c Live (green)/dead (red) staining images of C. albicans biofilms after incubation with Cu2+, Cu2++Lyw, PC, PCW, and AmB. The images were presented as mixed channels of NO1 (green) and PI (red). d Quantitative analysis of biofilm thickness (n = 3 independent experiments). e Viability of NIH-3T3 cells after incubation with different concentrations of Cu2+, PC, and PCW (1.56, 3.13, 6.25, 12.5, 25, 50 μg/mL, Cu2+ equivalent) (n = 5 independent experiments). f The hemolysis rate of red blood cells after incubation with different concentrations of Cu2+, PC, and PCW (1.56, 3.13, 6.25, 12.5, 25, 50 μg/mL, Cu2+ equivalent) (n = 3 independent experiments). MOPS treatment was control. a, c Showed one of three independent experiments with similar results. Data for (b, d–f) were presented as mean values ± SD. error bars = SD. Significance of (b, d) were calculated by one-way ANOVA with Tukey’s post hoc test. Source data were provided in Source Data file.

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PCW induced cuproptosis of fungi

Cu is an essential element for the growth and reproduction of fungi. Fungi possess a sophisticated regulatory system to maintain intracellular Cu2+ homeostasis46. Cu2+ overload can induce fungal death. To further investigate the microbiological mechanisms of PCW nanoflowers on fungal cells, transcriptomic sequencing analysis was conducted on C. albicans incubated with dissociative Cu2+, PC nanopetals, and PCW nanoflowers. Compared to the control group, PC and PCW groups exhibited 2526 and 2657 differentially expressed genes (DEGs), respectively, while the dissociative Cu2+ group only showed 1019 DEGs (Figs. 6b and S20, S21). PCW treatment resulted in 1032 downregulated genes and 893 upregulated genes in C. albicans compared to dissociative Cu2+ (Fig. 6c). Furthermore, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to investigate the change of functional pathways in C. albicans after treated with dissociative Cu2+, PC nanopetals, and PCW nanoflowers (Figs. 6d, e and S22, S23). The top 20 most significant pathways from the KEGG enrichment analysis were displayed in a bubble chart format. Compared to the control group, PCW nanoflowers significantly affected C. albicans pathways related to protein processing, peroxidases, TCA cycle, pyruvate metabolism, and the metabolism of starch and sucrose (Fig. 6d). Importantly, the enrichment score for the TCA cycle pathway of PCW and Cu2+ groups showed padj <0.1, suggesting that the excellent fungicidal performance of PCW was highly related to TCA cycle (Fig. 6e). It was reported that cuproptosis is dependent on mitochondrial respiration, where excess Cu promotes the acylation of pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-KDH), thereby affecting the TCA cycle. Therefore, PCW might induce fungal death by cuproptosis. The GO analysis also illustrated significant impacts of PCW nanoflowers on fungal transmembrane transport and mitochondrial-related pathways (Figs. 6f and S24, S25). Additionally, significant difference in the ion transport process was verified between PCW and Cu2+ groups, likely because the enhanced adhesion of PCW nanoflowers facilitated the internalization of Cu2+, leading to an imbalance in the internal ion transport of the fungus (Supplementary Fig. 24). The subsequent classification and heatmap for DEGs based on gene function after treated with PCW was shown in Fig. 6g. Specifically, genes related to Cu2+ uptake, such as CTR1, were significantly downregulated in PCW treated group, whereas genes associated with Cu2+ transport and copper resistance (ATX1, CCC2, and CRF1) were notably upregulated, indicating an overload of intracellular copper in C. albicans47,48,49. Moreover, genes associated with PDH expression, such as PDX1, PDA1, and PDA2, were significantly downregulated after PCW treatment, which further validated that PCW could greatly affect TCA cycle28.

Fig. 6: Transcriptome sequencing analysis results.
figure 6

a Schematic illustration of fungal cuproptosis after treated with PCW. CTR1, ATX1, CCC2 the gene names of C. albicans, LPO lipid peroxidation, PDH Pyruvate Dehydrogenase, TCA Tricarboxylic Acid cycle. b Volcano plot of DEGs in C. albicans comparing with PCW and control. c Volcano plot of DEGs in C. albicans comparing with PCW and Cu2+. d KEGG enrichment analysis of DEGs in C. albicans (PCW vs control). e KEGG enrichment analysis of downregulated DEGs in C. albicans (PCW vs Cu2+). f GO annotation analysis of DEGs in C. albicans (PCW vs control). g Heatmap of key DEGs changes. The heatmap shows the upregulation or downregulation of DEGs related to Cu2+ uptake, transport, tolerance, PDH complex, TCA cycle enzymes, Fe-S cluster proteins, cell wall synthesis, DNA damage, fungi and biofilm development. h PDH activity of C. albicans after incubation with Cu2+, PC, and PCW (10 μg/mL, Cu2+ equivalent) measured by the PDH activity assay kit. i MDA concentration in C. albicans after incubation with Cu2+, PC, and PCW (10 μg/mL, Cu2+ equivalent) measured by the MDA assay kit. Data for (h, i) were presented as mean values ± SD (n = 3 independent experiments). error bars = SD. Significance of (h, i) were calculated by one-way ANOVA with Tukey’s post hoc test. Source data were provided in Source Data file.

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According to the above transcriptomic analysis, the fungicidal effect of PCW was highly related to TCA cycle, which implied the occurrence of fungal cuproptosis. In order to validate PCW-induced fungal cuproptosis, the PDH activity in C. albicans was measured by a PDH activity assay kit after different treatments, which is an important enzyme that can be down-regulated in cuproptosis. As shown in Fig. 6h, the PDH activity was significantly reduced after C. albicans was treated with PCW nanoflowers. Furthermore, the expression of genes related to Fe-S cluster proteins (ISA1, ISA2) was notably reduced, which was an important hallmark of cuproptosis (Fig. 6g)28. Lipid peroxides (LPO) accumulation was another key signal of cuproptosis. The level of malondialdehyde (MDA), which was the decomposition product of LPO, was remarkably increased after PCW treatment, implying significant LPO accumulation (Fig. 6i)31. Collectively, these results implied that PCW nanoflowers exerted the fungicidal effect via cuproptosis.

The downregulation of genes related to fungal cell wall formation suggested that PCW nanoflowers could digest fungal cell walls due to the presence of Lyw in PCW (Figs. 6g and S23, S25). Due to the synergistic effect of cuproptosis and cell wall digestion, PCW disrupted multiple metabolic functions of C. albicans, including mitotic inhibitors, DNA damage, fungal and biofilm development. The comprehensive analysis of transcriptomic sequencing results summarized the action mechanism of PCW nanoflowers on fungal cells as depicted in Fig. 6a. PCW nanoflowers adhered to the fungal surface, increasing the local concentration of Cu2+, resulting in an overload of Cu2+ within the fungus. It was followed by a decrease of PDH activity, a restricted TCA cycle, reduced Fe-S cluster proteins, and significant LPO accumulation, indicating that fungal cells underwent cuproptosis after treated with PCW nanoflowers.

In vivo therapeutic effect of PCW against wound fungal infections

Based on the superior fungicidal ability of PCW nanoflowers in vitro, a murine wound infection model was established by inoculating C. albicans on the superficial wound to explore the in vivo antifungal potential of PCW nanoflowers. The C. albicans infected ICR mice were randomly divided into 6 groups (9 mice per group) and treated with different formulations through local administration, including saline, dissociative Cu2+, mixture of dissociative Cu2+ and Lyw, PC, PCW, and AmB, respectively (Fig. 7a). The wounds were photographed and measured daily to compare the therapeutic efficacy among different groups (Fig. 7b). Initially, all wounds except the uninfected group (denoted as Un-inf) showed clear exudation and suppuration, confirming C. albicans proliferation at the wound sites. Scab was formed on the 3rd day post-treatment, with the PCW group displaying the smallest scab area. The residual fungi in wound tissues were measured by a standard plate count method on the 3rd day post-treatment. As indicated in Fig. 7c, dissociative Cu2+ didn’t exhibit obvious fungicidal ability in vivo, probably owing to the interference by surrounding proteins and mammalian cells. Because PC could adhere to fungal cells to realize effective accumulation of Cu2+ on fungal surface, PC exhibited much better fungicidal ability than dissociative Cu2+, with the decrease of more than 90% fungal population. PCW showed better in vivo fungicidal effect than PC due to the synergy between Cu2+ and Lyw, with more than 99.9% reduction of fungal population. Due to the superior in vivo fungicidal ability of PCW, PCW treatment could achieve fast wound healing. On the 7th day post-treatment, wounds were almost invisible in PCW treated group, and mouse hair growth was lush, similar with uninfected group (Fig. 7d, e). However, significant scabs could still be observed in other groups, including AmB treated group.

Fig. 7: The in vivo antifungal ability of PCW on a C. albicans-induced wound infection model in mice.
figure 7

a The scheme illustrated the construction of the murine skin fungal infection model and the treatment protocol with different formulations. b Photos of mice wounds after different treatments on the 1st, 2nd, 3rd, 5th, and 7th day. c C. albicans colony counting of wound tissue after different treatments on the 3rd day (n = 3 independent experiments). d Visual graph of wound area changes after different treatments. e Statistical results of wound areas after different treatments (n = 6 independent experiments). f SEM images of wound tissues after different treatments on the 7th day. g H&E staining and IL-6 staining images of wound tissues after different treatments. HE a common histological staining technique; IL-6 inflammatory cytokines. f, g showed one of three independent experiments with similar results. Data for (c, e) were presented as mean values ± SD. error bars = SD. Significance of (c, e) were calculated by one-way ANOVA with Tukey’s post hoc test. Source data were provided in Source Data file.

Full size image

SEM images provided a vivid view of the residual fungi in the tissues after different treatments. Large amounts of fungal remnants were present in the saline and dissociative Cu2+ treated wound tissues, indicating ongoing severe fungal infections. Partial fungal remnants were observed in PC and AmB treated groups, while fungi were almost not observed in the PCW treated group (Fig. 7f). Further wound healing assessment was performed through pathological section analysis. The epidermal layer in the Cu2+, Cu2++Lyw, and PC groups was either incomplete or had granulation tissue, indicating incomplete wound healing (Fig. 7g). However, wounds treated with PCW nanoflowers were recovered, featuring distinct layers of epidermis, dermis, and subcutaneous muscle, even with the formation of hair follicles. Meanwhile, the cells in the dermis layer were loosely arranged without the aggregation of inflammatory cells in the PCW treated group (Figs. 7g and S26). The inflammatory factor levels in the subcutaneous wound tissue, including IL-6 and TNF-α, were much lower in PCW treated group than other groups (Figs. 7g and S27). These results indicated that PCW nanoflowers exhibited exceptional fungicidal effects and promoted wound healing in a murine superficial fungal infection model.

In vivo therapeutic effect of PCW against fungal keratitis

After proving the excellent fungicidal effect of PCW in treating superficial fungal infections, the feasibility of PCW nanoflowers in treating more challenging invasive fungal infections (IFI) was explored. Fungal keratitis is a challenging ophthalmic disease caused by the extensive proliferation of fungi on the cornea, leading to eye pain, reduced vision, corneal perforation, and even blindness4. Conventional eye drops can be easily washed away by tears, resulting in very frequent drug administration44. The adhesion of PCW nanoflowers to fungal surface might increase drug retention in infected cornea, which is critical for enhanced therapeutic efficacy. RhoB labeled PCW (RhoB-PCW) was instilled as eye drops to evaluate the retention ability of PCW in C. albicans infected cornea. As illustrated in Fig. 8a, b, free RhoB was rapidly cleared from eyes within 1 h. PCW demonstrated prolonged retention at the C. albicans-infected cornea. The fluorescent signal of RhoB-PCW was detectable even after 24 h, which indicated that PCW nanoflowers could be effectively retained at the C. albicans infected cornea due to their strong adhesion to fungal surface (Supplementary Fig. 29).

Fig. 8: The in vivo antifungal ability of PCW on a C. albicans induced keratitis model.
figure 8

a Real-time ocular fluorescence imaging of RhoB and PCW@ RhoB at different time points. b Fluorescent intensity decay curves of RhoB and PCW@ RhoB in the eye area over time. c Schematic diagram of murine keratitis infection with C. albicans and treatment protocol with different formulations. d Slit lamp photos of eyes after different treatments on the 1st, 2nd, 3rd, 6th, 7th, 9th, and 12th day. e Ocular clinical scores after different treatments. f C. albicans colony counting of corneal tissue after different treatments on the 6th day. g SEM images of corneal tissues after different treatments on the 12th day. f, g showed one of three independent experiments with similar results. Data for (b, e, f) were presented as mean values ± SD (n = 3 independent experiments). error bars = SD. Significance of (b) was calculated by two-sided Student’s t-test. Significance of (f) was calculated by one-way ANOVA with Tukey’s post hoc test. Source data were provided in Source Data file.

Full size image

A murine C. albicans keratitis model was fabricated to evaluate the potential of PCW nanoflowers in treating invasive fungal infections (Fig. 8c). After the fungal keratitis model was established, the mice were randomly divided into six groups (12 mice per group) and received with different treatments by instillation as eye drops, including saline, dissociative Cu2+, mixture of dissociative Cu2+ and Lyw, PC, PCW, and AmB, respectively. The ocular conditions after different treatments were recorded using a slit lamp (Fig. 8d). Severe corneal lesions were observed on the 1st day, including swelling, congestion, opacity, and exudation, with clinical scores nearly 12 points for all groups (Fig. 8e). The PCW treated group showed significant improvement in corneal infection on the 3rd day post-treatment, with relatively clear eyeballs and reduced C. albicans biofilms. The clinical score of the PCW treated group dropped to 4 points. However, serious infection could still be observed in other groups. The ocular residual fungi after different treatments were measured by a standard plate count method on the 3rd day post-treatment (Fig. 8f). PCW nanoflowers eradicated more than 99.9% of C. albicans from the cornea, whereas the dissociative Cu2+ treated group still showed a substantial amount of residual C. albicans. It should be noted that AmB showed much weaker therapeutic efficacy than PC and PCW, probably owing to fast clearance from eyes and low drug bioavailability. The corneas treated with PCW was nearly recovered on the 6th day. SEM images also revealed complete eradication of C. albicans in the PCW treated group (Fig. 8g).

Fluorescein sodium (FLS) staining was adopted to highlight differences of corneas after different treatments. Longer retention time of FLS on the eyeballs indicates rougher corneal surfaces and larger damage areas. As shown in Fig. 9a, b, the corneas could hardly be stained by FLS in PC and PCW treated groups, indicating relatively smooth corneal surfaces with low corneal damage areas. Optical coherence tomography (OCT) imaging showed that the cornea was swollen and opaque with significant indentations in the dissociative Cu2+ treated group, with the corneal thickness of about 145 nm, much thicker than the normal cornea (∽45 nm) (Fig. 9c, d). Conversely, intact cornea and iris structures were observed in the PCW treated group with the corneal thickness of about 50 nm, indicating much better corneal condition. The excellent therapeutic efficacy of PCW was further confirmed by histological and immunohistochemical analysis (Fig. 9e). After treated with PCW nanoflowers, the cornea was restored to normal thickness with lower cell density, displaying clear epithelial, stromal, and endothelial layers (Fig. 9f). Periodic acid-Schiff (PAS) staining showed that PCW nanoflowers possessed excellent fungicidal performance, with PAS expression similar to the uninfected group (Fig. 9g). Furthermore, severe inflammatory infiltration and high IL-6 expression were observed in saline, dissociative Cu2+, Cu2++Lyw, PC, and AmB groups, whereas PCW nanoflowers remarkably alleviated inflammation (Fig. 9h). Moreover, the body weights of mice treated with PC and PCW were very close to that of healthy mice (Supplementary Fig. 30). The H&E staining results of main organs (heart, liver, spleen, lung, and kidney) after treated with PC and PCW were shown in Supplementary Fig. 31. Compared with the control group, obvious organ lesions were not observed in PC and PCW treated groups, implying excellent biosafety of PC and PCW.

Fig. 9: Ophthalmic pathological characterization of C. albicans keratitis model.
figure 9

a Slit lamp photos of corneas stained with FLS after different treatments on the 12th day. b Statistical results of corneal injury areas from FLS staining images after different treatments. c OCT imaging photos after different treatments on the 12th day. d Statistical results of corneal thickness from OCT images after different treatments. e H&E staining, PAS staining, and IL-6 staining images of ocular sections after different treatments. HE a common histological staining technique, PAS Periodic Acid-Schiff staining, IL-6 inflammatory cytokines. f Cell density in the corneal stroma layer after different treatments calculated from H&E staining images. g Relative expression levels of PAS after different treatments. h Relative expression levels of the inflammatory factor IL-6 after different treatments. e showed one of three independent experiments with similar results. Data for (b, d, f, g, h) were presented as mean values ± SD (n = 3 independent experiments). error bars = SD. Significance of (b, d, f, g, h) were calculated by one-way ANOVA with Tukey’s post hoc test. Source data were provided in Source Data file.

Full size image

Discussion

In summary, PC nanopetals were successfully prepared by the coordination interaction between Cu2+ and PA without the addition of stabilizers, which were further assembled into PCW nanoflowers by Lyw-induced self-assembly. PCW nanoflowers were stable at neutral pH and could be rapidly decomposed in mildly acidic environments, leading to effective release of Cu2+ and Lyw. The in vitro synergistic fungicidal effect of Cu2+ and Lyw in PCW was confirmed by a checkerboard microdilution assay. PCW showed much better fungicidal ability than free Cu2+ and PC in aqueous solution. More importantly, PCW nanoflowers maintained exceptional fungicidal activity even in protein-rich and high-salt conditions, while dissociative Cu2+ completely lost its fungicidal activity. The exceptional fungicidal activity of PCW might be attributed to effective fungal adhesion and rapid drug release, which increased the local concentration of Cu2+ around fungi. PCW nanoflowers also exhibited excellent fungal biofilm disruption capability. The fungicidal process of PCW involved initial adhesion to the fungal surface and gradual decomposition of PCW in acidic pH, releasing Lyw to digest the fungal cell walls. The released Cu2+ could then be internalized into fungal cells, exerting its fungicidal effect. Transcriptomic sequencing analysis showed that PCW nanoflowers could lead to a decrease of PDH activity, a restricted TCA cycle, reduced Fe-S cluster proteins, and significant LPO accumulation, which implied that PCW nanoflowers exerted the fungicidal effect via cuproptosis. PCW nanoflowers showed negligible in vitro cytotoxicity and hemolysis. In the mouse model of C. albicans skin infection, PCW nanoflowers exhibited significant fungal clearance and wound healing effects, showing promise for treating superficial fungal infections. In the murine fungal keratitis model, PCW nanoflowers could be retained in the infected cornea for over 24 h. Corneal fungi could be effectively eliminated by PCW nanoflowers and corneal tissues could be healed, suggesting a potential therapeutic strategy against invasive fungal infections. This research demonstrated the excellent therapeutic efficacy in treating in vivo fungal infections, providing an effective strategy for future treatment of fungal infections. Since local administration is widely accepted in treating microbial infections, such as eyedrop instillation and nebulization, PCW nanoflowers were used to treat in vivo fungal infectons by local administration. Surface modification of PCW nanoflowers might be necessary to achieve systemic administration by intravenous injection.

Methods

Synthesis of PCW nanoflowers

The synthesis of PCW nanoflowers was carried out in two phases: Firstly, PC nanopetals were prepared using a one-pot self-assembly method with CuSO4 and protocatechuic acid (PA). Initially, CuSO4 aquesou solution (10 mg/mL) and PA in ethanol (50 mg/mL) were prepared. The process began by mixing 2 mL of ammonia water with 600 μL of CuSO4 solution, forming a blue copper-ammonia complex. Addition of 300 μL of PA solution changed the color first to yellow, then to red-brown (shaking for 30 s). This solution was then centrifuged, washed, and ultrasonically dispersed in 10 mL of H2O to form the PC solution. Secondly, 10 mL of the PC solution (200 μg/mL) was mixed with 250 μL of lywallzyme solution (1 mg/mL) and stirred at room temperature for 1 h. The resulting PC solution with lywallzyme was then centrifuged, washed, and ultrasonically dispersed in 5 mL H2O to obtain PCW. By varying the molar ratio of PA/Cu2+ (10:1, 8:1, 5:1, 3:1, 1:1) and the mass ratio of PC/lywallzyme (20:1, 10:1, 8:1, 5:1), PCW nanoflowers with different compositions were obtained. The synthesis of PCW was repeated for more than 10 times with very high repeatability.

Detection of drug contents in PC and PCW

1 mg of PC and PCW reddish-brown powder was ultrasonically dispersed in 1 mL of H2O. To degrade the nanostructures completely, 10 µL of concentrated nitric acid was added. The Cu2+ content was determined using ICP-MS. Similarly, 1 mg PCW powder was dispersed in 1 mL H2O and 5 μL HCl added to disassemble the nanostructures. After undergoing dialysis for 48 h to remove PA (molecular weight cutoff: 5000 Da), the absorbance at 283 nm was measured. Comparing with a standard curve of lywallzyme, the loading content of lywallzyme in PCW was calculated.

pH-responsive decomposition of PCW

To investigate the dissociation of PCW and Cu2+ release behavior under different pH conditions, 1 mL aqueous dispersions of PC and PCW (1 mg/mL) were placed inside dialysis bags (molecular weight cutoff: 5000 Da). These samples were then immersed in 50 mL of dialysis solutions—either H2O or MOPS buffer at pH 7.4 and 6.0. At predetermined time intervals (15, 60, 120, 180, 240, 300, 360, 450, 540, and 720 min), 100 µL samples were removed from the dialysis solutions and 100 µL fresh medium was supplemented. The Cu2+ concentration was measured by ICP-MS. Additionally, the morphological changes of PC and PCW in MOPS at pH 6.0 and 5.0 were characterized using TEM and SEM.

In vitro fungicidal evaluation of PCW

Initially, the fungicidal properties of free Cu2+, PC, and PCW in an aqueous environment were assessed. 200 μL of C. albicans suspension (107 CFU/mL, dispersed in sterile H2O) was mixed with 200 μL of solutions containing Cu2+, PC, and PCW (1, 2, 5, 10, 15 μg/mL, Cu2+ equivalent, control: H2O). After mixing the fungi and materials, they were incubated on a shaker at 37 °C for 6 h. The number of surviving fungi was counted using a standard plate count method. Similarly, the antifungal properties of different concentrations (25, 50, 75, 100 μg/mL) of PA and lywallzyme were tested. Each condition was replicated in three parallel samples. Subsequently, to investigate the antifungal capabilities of PCW in complicated environments, 200 μL of C. albicans suspension (107 CFU/mL, dispersed in 20% SDB, MOPS buffer at pH 7.4 and 6.0) was mixed with 200 μL of solutions containing Cu2+, PC, and PCW (5, 10, 20 μg/mL, Cu2+ equivalent, control: solvent). Futhermore, C. albicans (107 CFU/mL) were dispersed in various volume ratios (20%, 40%, 60%, 80%, 100%) of FBS/H2O. 200 μL of the fungal suspension was mixed with 200 μL of solutions containing Cu2+, PC, and PCW (20 μg/mL, Cu2+ equivalent, control: solvent). The incubation and counting processes described above were repeated for these conditions.

Checkerboard microdilution assays

Checkerboard microdilution assays were employed to evaluate the synergistic effects between PC and lywallzyme within PCW, as well as between free Cu2+ and lywallzyme. In these assays, 80 μL of different concentrations of PC (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 μg/mL, Cu2+ equivalent) were placed into the wells of a 96-well plate. This was followed by the addition of 80 μL of different concentrations of lywallzyme (0, 31.25, 62.5, 125, 250, 500, 1000 μg/mL), and finally, 80 μL of C. albicans suspension (105 CFU/mL, dispersed in SDB medium). After thorough mixing and incubating at 37 °C for 12 h, the optical density at 590 nm was measured to determine the inhibition rates. The procedures for the checkerboard microdilution assays involving free Cu2+ and lywallzyme mirrored the above. Each experiment was conducted using three parallel samples. The Fractional Inhibitory Concentration (FIC) values for Cu2+/lywallzyme and PC/lywallzyme were determined by formula (1).

$${{\rm{FIC}}}=\frac{{{\rm{MIC}}}\; {{\rm{of}}}\; {{\rm{drug}}}\; {{\rm{A}}}\; {{\rm{in}}}\; {{\rm{combination}}}} {{{\rm{MIC}}}\; {{\rm{of}}}\; {{\rm{drug}}}\; {{\rm{A}}}\; {{\rm{alone}}}}+\frac{{{\rm{MIC}}}\; {{\rm{of}}}\; {{\rm{drug}}}\; {{\rm{B}}}\; {{\rm{in}}}\; {{\rm{combination}}}} {{{\rm{MIC}}}\; {{\rm{of}}}\; {{\rm{drug}}}\; {{\rm{B}}}\; {{\rm{alone}}}}$$
(1)

Live/Dead fungal fluorescent imaging

1 mL of C. albicans suspension (108 CFU/mL, dispersed in MOPS) was incubated with 1 mL of MOPS, free Cu2+ (20 μg/mL), lywallzyme alone (20 μg/mL), a mixture of Cu2+ and lywallzyme (each at 20 μg/mL), a mixture of Cu2+ and lywallzyme (each at 10 μg/mL), PC (66 μg/mL, equivalent to 20 μg/mL Cu2+), PCW (90 μg/mL, equivalent to 20 μg/mL Cu2+), PA (90 μg/mL), and AmB (90 μg/mL) at 37 °C for 6 h. After incubation, live/dead bacterial stain (NO1 and PI) was added, and the mixture were stained for 20 min. The suspension was then washed three times with sterile MOPS to remove any residual dye. Finally, the C. albicans suspension was centrifuged and concentrated, then redispersed in 50 μL of MOPS, and fluorescent images were captured using a laser scanning confocal microscope.

Adhesion of PCW on fungal surface

Firstly, DAPI staining solution was added to 1 mL of C. albicans suspension (107 CFU/mL, dispersed in MOPS) and left to stain for 20 min. The stained fungi were then washed three times with sterile MOPS to remove residual dye, yielding DAPI stained C. albicans. Additionally, 10 μL of RhoB solution (1 mg/mL) was mixed into 1 mL of PC (1 mg/mL) and allowed to stain for 4 h. The mixture was then washed three times with MOPS to remove excess RhoB. Similarly, 10 μL of FITC solution (1 mg/mL) was added to 1 mL of lywallzyme (1 mg/mL) and left to stain for 1 h before being washed three times. This process resulted in the preparation of PCW labeled with RhoB and FITC simultaneously. Then, 200 μL of DAPI-stained C. albicans was mixed with 200 μL of RhoB-labeled PC (100 μg/mL), FITC- labeled lywallzyme (100 μg/mL), and PCW labeled with both RhoB and FITC (100 μg/mL) at 37 °C for 6 h. These mixtures were then characterized using a confocal microscope to observe the interactions between PCW and C. albicans.

Fungal morphology characterization

1 mL of C. albicans suspension (~107 CFU/mL, dispersed in MOPS) was mixed with 1 mL of MOPS, Cu2+, PC, and PCW (10 μg/mL, Cu2+ equivalent) and incubated at 37 °C for 6 h. After incubation, the suspension was diluted 100 times and characterized using TEM to observe the overall morphology of C. albicans and PCW. For internal and surface morphology characterization of C. albicans, the fungi were further observed using SEM.

Trypan blue staining

Trypan blue was used to specifically stain fungal cell walls and extracellular polysaccharides. 1 mL of C. albicans suspension (108 CFU/mL, dispersed in MOPS) was mixed respectively with 1 mL of MOPS, Cu2+, PC, and PCW (20 μg/mL, Cu2+ equivalent) and incubated at 37 °C for 6 h. After incubation, 10 μL of trypan blue dye (1 mg/mL) was added to the C. albicans suspension and stained for 10 min before fluorescence images were captured using a confocal microscope.

Crystal violet staining

200 µL of C. albicans suspension (106 CFU/mL, dispersed in SDB) was added in a 96-well plate and incubated at 37 °C for 48 h. The supernatant was gently removed, fresh medium was added. The incubation continued for another 48 h to allow biofilm formation at the bottom of the wells. Subsequently, 200 µL of various treatments including MOPS, free Cu2+ (20 μg/mL), a combination of Cu2+ and lywallzyme (each at 20 μg/mL), PC (66 μg/mL, equivalent to 20 μg/mL Cu2+), PCW (90 μg/mL, equivalent to 20 μg/mL Cu2+), and AmB (90 μg/mL) was introduced to the formed C. albicans biofilm. The biofilm was then incubated at 37 °C for 6 h. After the incubation, the supernatant was carefully poured off and the biofilm was gently rinsed with sterile MOPS to remove unbound cells and materials. Each well was stained with 100 µL of 1% crystal violet for 20 min. The stain was then carefully removed, and the wells were rinsed with sterile MOPS to eliminate any excess stain. Once the wells had dried, photographs were taken to document the stained biofilms. For semi-quantitative analysis, 200 µL of 33% acetic acid solution was added to each well to dissolve the bound crystal violet, and the absorbance at 590 nm was measured. Each treatment group was replicated in eight parallel samples.

Live/dead biofilm staining

Morphological images and thickness of the biofilms were obtained using live/dead biofilm staining. After an 8 h incubation of the C. albicans biofilms with various treatments, the supernatant was gently removed and the biofilm was rinsed with sterile MOPS. Then, 100 µL of live/dead bacterial staining dye (NO1 and PI) was added to the biofilms, staining for 20 min. Finally, images of the biofilms were captured using a laser scanning confocal microscope.

In vitro cytotoxicity test

The cytotoxicity of free Cu2+, PC, and PCW was evaluated using the CCK-8 assay kit. NIH-3T3 cells were seeded at a density of 6000 cells per well in a 96-well plate and incubated for 24 h. Different concentrations of Cu2+, PC, and PCW solutions (1.56, 3.13, 6.25, 12.5, 25, 50 µg/mL, Cu2+ equivalent) were then added to fresh DMEM high-glucose medium. The control group received an equal volume of PBS and cells, while the blank group consisted only of the medium without any cells. These mixtures, along with the freshly prepared DMEM high-glucose medium and cells, were incubated for another 48 h. After this incubation period, the medium was replaced with fresh DMEM high-glucose medium containing 10% CCK-8, and the plates were incubated for an additional 2 h at 37 °C. Absorbance at 450 nm was then measured using a microplate reader to assess cell viability. Cell viability of the NIH-3T3 cells was calculated using formula (2). Each group was replicated in five parallel samples.

$${{\rm{Cell\; Viability}}}=\frac{{As}-{Ab}}{{Ac}-{Ab}}\times 100\%$$
(2)

As was the absorbance of the experimental group, Ac was the absorbance of the PBS group (no drug), and Ab was the absorbance of the blank group (no cells, no drug).

In vitro hemolytic activity test

The hemolytic activity of free Cu2+, PC, and PCW was assessed using rabbit red blood cells (RBCs). 1 mL of RBCs was centrifuged at 825 g for 5 min, discarding the supernatant, and the cells were resuspended in 20 mL of PBS buffer to create a 5% v/v suspension. RBCs suspension was mixed with different concentrations of free Cu2+, PC, and PCW (1.56, 3.13, 6.25, 12.5, 25, 50 µg/mL, Cu2+ equivalent). Triton X-100 (5 µg/mL) served as the positive control for complete hemolysis, and PBS as the negative control for no hemolysis. After incubating at 37 °C for 1 h, the solutions were centrifuged at 825 g for 10 min. The absorbance of the supernatant was then measured at 570 nm and the hemolysis rate was calculated using formula (3). Each group was replicated in three parallel samples.

$${{\rm{Hemolysis\; Rate}}}=\frac{{Am}-{An}}{{Ap}-{An}}\times 100\%$$
(3)

Am was the absorbance of the experimental group, An was the absorbance of the PBS group, and Ap was the absorbance of the Triton X-100 group.

Fungal transcriptomic sequencing analysis

1 mL of C. albicans suspension (108 CFU/mL, dispersed in H2O) was mixed with 1 mL of H2O, Cu2+, PC, and PCW (20 μg/mL, Cu2+ equivalent) and incubated at 37 °C for 6 h. The samples were immediately frozen in liquid nitrogen before being sent to Novogene Co., Ltd. (Beijing, China) for prokaryotic transcriptome sequencing, which included RNA extraction, sequencing, and preliminary analysis. The obtained sequencing results were processed using the DAVID bioinformatics resources (https://david.ncifcrf.gov/). The selection criteria for differentially expressed genes (DEGs) were set as |log2FoldChange| > 1 and padj < 0.05. Functional annotation clustering was achieved through the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The determination of Malondialdehyde (MDA) content and Pyruvate Dehydrogenase (PDH) activity were carried out according to the instructions provided in the respective assay kits. Each group was replicated in three parallel samples.

In vivo antifungal evaluation on a C. albicans induced wound infection model

Healthy female ICR mice (aged 6–8 weeks, weighing ~20 g) were used in this experiment. The mice were housed in a lighted environment with a temperature of 25 °C and a humidity level of 50%. On day 0, the mice were anesthetized using isoflurane, and the hair on their backs was shaved off. A full-thickness skin wound ~10 mm in diameter was created using sterilized medical scissors. The un-infection group refers to only wounds but no infection. Subsequently, 10 μL of C. albicans suspension (108 CFU/mL, dispersed in MOPS) was applied and evenly spread over the wound surface; 10 μL of saline was applied as the control group. After the liquid on the wound surface had dried, the wound was covered with an airtight medical PU film. The wounds were photographed and the weight was measured daily. Significant tissue exudate and cloudy fungal fluid were observed on the wound surface after 24 h, the C. albicans-induced wound infection was considered successfully established. On day 1, the infected mice were randomly divided into 6 groups (9 mice per group, 3 mice were used for C. albicans colony counting of wound tissue after different treatments. 3 mice were used for tissue section and 3 mice were used for SEM images) and treated with 10 μL of saline, free Cu2+ (20 µg/mL), a mixture of Cu2+ and lywallzyme (each at 20 µg/mL), PC (65 µg/mL, equivalent to 20 µg/mL Cu2+), PCW (90 µg/mL, equivalent to 20 µg/mL Cu2+), or AmB (90 µg/mL) on the wound surface. After the treatment had dried naturally, the wound was covered with a new piece of PU film. The treatment was repeated at the same time on day 2. On day 3, three mice from each group were euthanized, and their wound tissues were soaked in 10 mL PBS buffer. The tissues were then homogenized using a tissue homogenizer (12,850 g, 1 min). The bacterial counts in the wound homogenates were then determined by a standard plate counting method. Each group was replicated in three parallel samples. On day 7, the remaining mice from each group were euthanized, and their wound tissues were collected for histological analysis and SEM observation. For histology, the tissues were fixed in 2.5% glutaraldehyde solution, embedded in resin, sectioned into ultrathin sections, and stained with H&E/IL-6/TNF-α for observation to evaluate tissue morphology and inflammation levels. For SEM preparation, the tissues were fixed overnight in 2.5% glutaraldehyde solution. The next day, the fixative was discarded, and the tissues were dehydrated in a graded series of ethanol solutions (70%, 90%, 100%) for 15 min, followed by freeze-drying for SEM observation. Each group was replicated in three parallel samples.

In vivo antifungal evaluation on a C. albicans induced keratitis model

Healthy female C57 mice (aged 6–8 weeks, weighing ~20 g) were used in this experiment. The mice were housed in a lighted environment with a temperature of 25 °C and a humidity level of 50%. On day 0, the mice were anesthetized, and their eyelashes were trimmed. The right cornea was then scratched using an insulin syringe needle, and 10 μL of C. albicans suspension (107 CFU/mL, dispersed in MOPS) was applied to the corneal surface. The mice were returned to their cages after the liquid on the corneal surface had dried. After 24 h, the turbid and whitened corneal surfaces were considered to get successfully established C. albicans-induced keratitis model. The infection and recovery status of the eyes were photographed daily using a slit-lamp microscope. The clinical scores (full score was 12) were assigned and the weight of the mice was measured daily. To determine the retention of PCW in the eye, 10 μL of RhoB or PCW loaded with RhoB were dropped to the eye on day 1. The ocular fluorescence was measured at 0, 1, 6, 12, and 24 h by an in vivo imaging system (Lumina LT, PerkinElmer). The eyeballs were then fixed, and the corneas were prepared for SEM observation to examine the adhesion of the nanoflowers on the surface. On day 1, the infected mice were randomly divided into 6 groups (12 mice per group, 3 mice were used for C. albicans colony counting of corneal tissue after different treatments. 3 mice were used for tissue section. 3 mice were used for SEM images and another 3 mice were used for OCT images.) and treated with 10 μL of saline, free Cu2+ (20 µg/mL), a mixture of Cu2+ and lywallzyme (each at 20 µg/mL), PC (65 µg/mL, equivalent to 20 µg/mL Cu2+), PCW (90 µg/mL, equivalent to 20 µg/mL Cu2+), or AmB (90 µg/mL) onto the corneal surface. After the treatment had dried naturally, the mice were returned to their cages. The medication was reapplied at the same time on day 2 and 3. On day 6, three mice from each group were euthanized, and their eyeballs were soaked in 10 mL PBS buffer. The eyeballs were then thoroughly homogenized using a tissue homogenizer (12,850 g, 1 min), and the number of C. albicans in the eyeball homogenates was counted by a standard plate counting method. Each group was replicated in three parallel samples. On day 12, the remaining mice were anesthetized, and the corneal thickness of each group was photographed using Optical Coherence Tomography (OCT). Subsequently, 10 μL of fluorescein sodium solution (5 mg/mL) was applied to the corneal surface and left for 10 min. The corneal fluorescence images were photographed using a slit-lamp equipped with a filter. Finally, the remaining mice from each group were euthanized, and their eyeballs were collected for histological analysis and SEM observation. For histology, the obtained eyeballs were soaked in an appropriate eye fixation solution, embedded in resin, and sliced into ultrathin sections. The sections were then stained with H&E/IL-6/PAS and observed to evaluate tissue morphology or inflammation levels. For SEM preparation, the corneas were fixed overnight in the appropriate eye fixation solution. The next day, the fixative was discarded, and the tissues were dehydrated using a graded series of ethanol solutions (70%, 90%, 100%) for 15 min, followed by freeze-drying for SEM observation. Each group was replicated in three parallel samples.

Inclusion and ethics

All animal experiments were conducted following the guidelines for Animal Care and Use Committee, Zhejiang University and the Principles of Laboratory Animal Care (NIH publication no.86-23, revised 1985). All animal experiments were approved by Animal Care and Use Committee of Zhejiang Academy of Medical Sciences. The assigned approval number was ZJCLA-IACUC-20010667. All the mice were obtained from the animal center of Zhejiang Academy of Medical Sciences.

Statistical analyses

The results were presented as the mean ± standard deviation (SD). Statistical analysis was performed using the GraphPad Prism or Origin software. An independent-sample t-test was used to compare between two groups. For the comparison among (more than) three groups, comparative studies of means were carried out using one-way ANOVA analysis with Tukey’s post hoc test.

Reporting summary

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