All experimental procedures involving animals were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals and approved by the State Council of the People’s Republic of China. The protocol used was approved by the Institutional Animal Care and Use Committee of Tongji University (Permit Number: TJAA08021101).

HSA powder was dispersed in SDS (0.6%) and DTT (2.4 mM), resulting in a solution with a concentration of 10 mg/mL. According to our previously published method, the mixture was then heated to 90°C and magnetically stirred at 180 rpm for 2 h to obtain reduced HSA (rHSA) (Liu et al., 2020) (Figure 1A). Then, rHSA in MES buffer was magnetically stirred at 350 rpm and 25°C with AN (m/m 50:1) for 2 h to form the AN nanoparticles (AN NP) (Figure 1B). To prepare the HSA nanoparticles (HSA NP), rHSA in MES buffer was magnetically stirred at 350 rpm and 37°C for 4.5 h to form HSA NP. To characterize the nanoparticles, AN NP and HSA NP were dialyzed using commercially available 3KD dialysis bags.

RAW264.7 macrophages were used to evaluate the toxicity of HSA NP. Macrophages (1.25 × 10⁵ cells/mL) in DMEM containing 10% FBS were seeded in 96-well tissue culture plates and different concentrations of HSA NP (experimental) was added. Cytotoxicity was tested using LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China) and LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher, MA, United States) according to manufacturer‘s recommendations. lactate dehydrogenase (LDH) release was assessed by measuring absorbance at 490 and 600 nm and cellular viability (i.e., live/dead) was observed using the Automated Live Cell Imager (Lionheart FX, Biotek, Vermont, United States) using the GFP and PI channels.

Anidulafungin NP particle size was determined by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern, Shanghai, China). The morphology was observed by transmission electron microscopy (TEM, Tecnai-12Bio-Twin, FEI, Netherlands). The effective encapsulation of AN into nanoparticles was confirmed by UV spectral analysis (Varian, Hong Kong, China). To evaluate stability, AN NP was incubated at either 4°C or room temperature for 30 days; alternatively, AN NP was also incubated at 37°C for 48 h. All measurements were conducted in triplicate. The solubility of 125 μg/mL AN with (experimental) or without (control) different ratios of rHSA in PBS was visually observed at 0 and 24 h. Two independent experiments were conducted.

Candida albicans SC5314 was kindly provided by Sanglard D (Centre Hospitalier Universitaire Vaudois). C. albicans ATCC 90029 and clinical strains (379, 385, 395, 537, 538, 541, 0511311, 0710253, 0710419, 0710425, 0710448, 0710452, and 0710492), C. tropicalis standard strains ATCC 20026, ATCC 750, and clinical strains (98F, 267, 785, 909, and 1041), C. glabrata standard strains ATCC 28226, ATCC 1182, and clinical strains (337, 338), C. parapsilosis standard strains ATCC 22019 and clinical strains (642, 644), C. krusei standard strains ATCC 6258, C. guilliermondii standard strains ATCC 6260, C. Auris standard strains 1212 were from the fungi collection of our laboratory. All of other the other standard reference fungal strains (n = 8) were obtained from American Tissue Culture Collection (ATCC). All clinical fungal isolates (n = 23) were isolated from Shanghai Tenth People’s Hospital, Shanghai, China. All fungal strains were routinely cultured on sabouraud dextrose agar (SDA) plates (1% peptone, 4% dextrose, and 1.8% agar) for isolation of individual clones and cultured in yeast peptone dextrose (YPD) liquid medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30°C in a shaking incubator.

RAW264.7 macrophages were obtained from the Cell Resources center of the Chinese Academy of Sciences and were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO₂.

To determine in vitro antifungal activity, a broth microdilution susceptibility assay was conducted in RPMI 1640 medium in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Briefly, strains in RPMI 1640 medium (approximate final concentration = 1 × 10³ CFU/mL) were prepared in 96-well plates; after preparation, antifungal agents (100 μg/mL) were selectively added. The final concentrations ranged from 0.002 to 1 μg/mL for AN and AN NP. HSA NP with the same mass and addition volume as AN NP was used as control to explore their antifungal efficacy against C. albicans SC5314. The plates were then incubated at 35°C for 24 h. MIC results were determined after incubation and were made according to inhibition of visible growth when compared to that of control. The MIC range of fluconazole to C. parapsilosis ATCC 22019 from 0.5 to 1 μg/mL, indicating the test was acceptable. Two independent experiments were conducted and the average used for later analysis.

Strains were grown in YEPD medium with a starting inoculum of 5 × 10⁵ CFU/mL. AN and AN NP (0.25 μg/mL for Candida albicans SC5314, 0.5 μg/mL for Candida albicans 538) and PBS (control) were tested for Candida albicans. After incubation with agitation at 30°C and at predetermined time points, 100 μL aliquots were obtained from each solution and serially diluted in PBS. A 100 μL aliquot from each dilution was then spread on a Sabouraud dextrose agar (SDA) plate. Colony counts were determined after incubation at 35°C for 48 h. Each group coated three plates at each predetermined time points. Two independent experiments were conducted and the average used for later analysis.

To examine the effect of AN and AN NP on the growth of different Candida species, we performed a paper disk-diffusion assay in agar. Briefly, a cell suspension of either Candida albicans SC5314 or 538 in PBS (5 mL, 1 × 10⁵ CFU/mL) was poured evenly over a pre-prepared SDA plate. The plate was allowed to stand for 30 min, after which the supernatant was removed. The plate was then allowed to dry for an additional 30 min. After the top layer had solidified, sterile paper disck (8 mm) were placed on the agar surface and test agents were added. Five types of inhibition zones were noted: (a) 1 μg for AN; (b) AN NP containing 1 μg for AN; (c) 2 μg for AN; (d) AN NP containing 2 μg for AN; (e) PBS. After incubating at 35°C for 24 h, the size and pattern of the growth inhibition zone around the disks on the agar were evaluated. Two independent experiments were conducted and the average used for later analysis.

Candida yeast was selected to use during the in vivo assays because it is one of the most common fungal agents that causes life-threatening invasive fungal infections in humans. Of the strains implicated, C. albicans is the most prevalent (Brown et al., 2012a). An established murine model of invasive candidiasis with some modifications was used to evaluate the in vivo efficacy of AN and AN NP. Immunocompetent 7–8-week-old female C57BL/6 mice weighing 18–20 g were used in this study. The mice were randomly divided into groups according to their body weight, and then the random numbers were generated by Excel for simple random grouping. To establish invasive candidiasis, mice were injected with 1 × 10⁶ CFU of C. albicans SC5314 intravenously via the lateral tail vein.

In the survival study, antifungal treatments began 2 h after fungal inoculation as a single dose. Different treatment groups (n = 8 mice per group) were as follows: Vehicle control (PBS), 0.3 mg/kg of AN or AN NP (containing 0.3 mg/kg AN). AN and AN NP were diluted in PBS and intravenously administered via the lateral tail vein in a volume of 0.3 mL. Post-infection, mice were monitored and recorded regarding their survival twice daily for a total of 6 days. Two independent experiments were conducted.

To assess fungal burden, antifungal drug was administered once began 2 h after fungal inoculation and continued for 2 days. Two days after administration, mice were humanely euthanized and kidneys were collected for quantitative determination of tissue fungal burden (n = 5 mice per group) and histological staining (n = 3 mice per group). After kidney weights were determined, tissues were homogenized in 1 mL PBS. Homogenates were serially diluted in 10-fold steps, and aliquots (100 μL) of the resulting homogenates were plated on SDA plates. The plates were incubated at 35°C for 48 h, after which the number of CFU were counted. All fungal burdens are indicated as CFU/g. All SDA plates were performed in duplicate and the average used for later analysis. For histopathology, the kidneys were fixed in 10% neutral formalin for HE and PAS staining.

Time-kill kinetics assay data and fungal burden data were analyzed using one-way analysis of variance (ANOVA), and survival curves were compared using the Log-rank (Mantel–Cox) test. A P- value of ≤0.05 was considered statistically significant. Software Graphpad prism 7 was used to evaluate data.

In this work, we reduced HSA using SDS and DTT to liberate free thiols and allow for the formation of an intermolecular disulfide network. As a result, a nanoassembly was formed, resulting in the obtained HSA NP. Given this, we first sough to determine the safety of our HSA NP. The cytotoxicity of HSA NP was evaluated using a LDH release assay along with cellular viability staining (i.e., determining live/dead cells) in RAW264.7 macrophages. The quantity of LDH released by different concentrations of applied HSA NP was the same as that observed in the negative control. The positive control was approximately of three times that of either HSA NP or NC (Figure 2A). The cellular viability of cells incubated with either HSA NP or NC is shown in Figure 2B. As indicated, live cells are indicated in green and dead cells in red. Importantly, the ratio of living to dead cells across these two groups was consistent and not statistically different from each other.

Figure 2C shows the particle size and representative TEM image of AN NP. AN NP particle size was determined and expressed as mean particle size (in nanometers) ± standard deviation (SD). The particle size was 29 ± 1.5 nm with PDI of 0.173 ± 0.039. TEM imaging revealed that AN NP exhibited uniform and spherical morphology with a diameter of approximately 30–50 nm.

The aggregation state of AN in rHSA (AN NP) was evaluated by measuring UV-visible absorbance (Figure 2D). AN and HSA NP was used as contrast. The absorption spectra of AN and HSA NP showed separate, highly pronounced peaks at 310 and 277 nm, respectively. The absorption spectrum of AN NP showed two highly pronounced peaks at 280 and 306 nm.

The behavior of AN with or without different ratios of rHSA was measured in PBS at room temperature (Figure 2E). When the ratio of rHSA and AN was 0, 1:5, 1:1, or 2:1, the mixed liquid went from turbid to clear at 0 h and a white precipitate gradually formed in less than 24 h. PBS was used as the negative control. In addition, long-term stability analysis indicated that the size distribution of AN-NP did not change markedly within 30 days—either at 4°C or room temperature (Figure 3A) or at 37°C within 48 h (Figure 3B). Considering that poor dispersibility has limited the in vitro and in vivo antifungal efficacies of AN, we hypothesized that the improved solubility and dispersibility of AN-NP would predict its superior antifungal activity compared to AN.

Anidulafungin had low solubility and poor dispersibility in aqueous solution (Patel and Charneski, 2006). Our results indicated that nanoassembly with HSA markedly improved the dispersibility of AN, making it possible to enhance its antifungal activity (Figure 2E). Given this, we first compared the MIC of AN and AN-NP against the common pathogenic Candida spp., including 9 standard reference strains and 23 clinical isolates. This was done using a broth microdilution susceptibility assay according to CLSI guidelines M27-A3. First of all, we determined the value of HSA NP was the same as that in the non-dosing group. Next our results showed that AN and AN NP were active against all 32 strains tested, with MIC values ranging from 0.125 to 2 mg/L and 0.0313 to 0.5 mg/L, respectively (Table 1). Obviously, the efficacy of AN NP against Candida strains was found to be superior to AN, with the exception of the C. tropicalis standard strain ATCC 20026. AN NP exhibited a stronger effect against Candida species (8 standard reference strains and 23 clinical isolates), with MIC values 4- to 32-fold lower than those of AN.

Furthermore, we investigated the in vitro drug susceptibility of AN and AN NP using a time-kill kinetics assay along with a paper disk agar diffusion test. The time-kill kinetics assay was conducted against C. albicans SC5314 and 538. As shown in Figure 4A and when compared with control, AN and AN NP significantly inhibited the growth of C. albicans SC5314 and 538. AN NP showed a stronger inhibitory effect relative to AN and exhibited fungistatic effect against both strains.

The paper disk ager diffusion test revealed that AN and AN NP significantly inhibited the growth of C. albicans SC5314 and 538 when compared with PBS (Figure 4B). Compared with AN (1 μg or 2 μg), AN NP containing either 1 μg or 2 μg of AN revealed a dose-independent increase in zone diameter. Meanwhile, AN and AN NP exhibited fungistatic effects against both strains.

In the 6-day survival experiment, AN NP exhibited potent efficacy in our in vivo, invasive candidiasis model. We assessed the in vivo effects of AN NP at a dose of 0.3 mg/kg, with AN and PBS as controls (Figure 5A). The result showed that 0.3 mg/kg of AN NP protected the mice through the observation period and somewhat improved their overall survival rate. All mice in the control groups were dead at 3 days post-inoculation, with a median survival time of 2.5 days. Compared with the survival time for the control groups, AN NP significantly prolonged the survival time in mice (P < 0.005). The median survival time of mice in the AN NP group was 4 days.

To further examine the protective role of AN NP in host defense against C. albicans infection, we surveyed the fungal burden and histopathology of the kidneys, which are the organs most vulnerable to C. albicans infection. For this assessment, AN and PBS were used as controls. Following 2 days of antifungal treatment, we examined changes in the fungal burden in the kidneys. As indicated by the results shown in Figure 5B, a significant reduction in fungal burden was observed after AN NP treatment. The CFU counts for the AN NP-treated group (0.3 mg/kg) were significantly lower than those of the AN group. More specifically, the mean value for the AN NP group was 0.6 × 10⁷ CFU/g while the mean value for the AN group was 2.5 × 10⁷ CFU/g (P < 0.0001). Histopathological studies of renal tissue of all mice treated with AN NP at 0.25 mg/kg showed improved infection status compared to the control groups (Figure 5C). Signs of improved infection status in the renal tissue of mice treated with AN NP included decreased areas with inflammatory cells and reduced numbers of fungal hyphae (surrounding tubules and glomeruli).