AmB was purchased from MedChemExpress (New Jersey, United States). Rabbit monoclonal anti-CD9 and anti-CD63 antibodies were obtained from CST (MA, United States). Recombinant anti-TSG101 and anti-calnexin antibodies were sourced from Abcam (Cambridge, UK). Goat anti-rabbit IgG was obtained from Zhongshan Golden Bridge Biotechnology (Guangdong, China).

The standard strains of Candida parapsilosis ATCC 22019, wild-type (WT) H99 strains of Cryptococcus neoformans, and clinical isolates of Cryptococcus neoformans were obtained from the Shanghai Key Laboratory of Molecular Medical Mycology. All strains were cultured in yeast peptone dextrose (YPD) liquid medium (1% yeast extract, 2% peptone, and 2% glucose) in a shaking incubator at 30°C with 200 rpm.

Induced pluripotent stem cells (iPSCs) were obtained from the Changzhou Xitaihu Institute for Frontier Technology of Cell Therapy and cultured in StemFlex^™ Basal Medium supplemented with 10× StemFlex^™ additives and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific, USA). For exosome collection, DMEM/F12 supplemented with GDEV^™ Medium Supplement 10× (Guodian Pharmaceutical, Beijing, China) was used. Human embryonic kidney cells (HEK 293 T) and RAW264.7 macrophages were obtained from the Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, USA). All cell types were maintained at 37°C in a 5% CO2 atmosphere.

C57BL/6 J mice were obtained from Shanghai Yishang Biotechnology. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) (IACUC-2023-Mi-211) and conducted in accordance with the “Regulations on the Administration of Laboratory Animals” approved by the State Council of the People’s Republic of China.

Initially, the collected iPSC supernatant was centrifuged at 3000 g for 10 min to remove cell debris. Next, the supernatant was filtered using a 0.22-μm filter to sterilize and collect the filtrate. The filtrate was subsequently added to a 100 kDa ultrafiltration tube (Millipore, MA, United States) and centrifuged at 3000 g at 4°C for concentration and purification until all filtrate was processed. Following this, ultracentrifugation was conducted at 100,000 g at 4°C for 90 min, resulting in the precipitation of exosomes at the bottom of the centrifuge tube. Finally, the supernatant was carefully removed to avoid disturbing the pellet, and the pellet was gently resuspended in PBS to obtain the exosome suspension.

We used the Western blot method to identify exosomes by sequentially loading a pre-stained protein ladder (Thermo Fisher Scientific, Waltham, MA, United States), iPSC exosomal protein samples, and control iPSC protein samples (prepared via RIPA lysis buffer, Beyotime, Shanghai, China) into a 12% Tris-Gly SDS-PAGE gel (WSHT, Shanghai, China), with 10 μg of protein per well. The gel was run at 80 V for the stacking phase and 110 V for the separating phase. After electrophoresis, the proteins were transferred to a PVDF membrane in an ice bath at a constant current of 250 mA. Once the transfer was complete, the membrane was blocked in a solution containing 5% non-fat dry milk (Solarbio, Beijing, China) for 1 h. It was then incubated overnight at 4°C with the primary antibodies: CD9 (D8O1A) rabbit mAb (Cell Signaling Technology, Danvers, MA, United States), CD63 (E1W3T) rabbit mAb (Cell Signaling Technology, Danvers, MA, USA), recombinant anti-TSG101 antibody (Abcam, Cambridge, UK), and recombinant anti-calnexin antibody (Abcam, Cambridge, UK), all diluted 1:1000 on a shaking platform. After three washes using TBST (Servicebio, Wuhan, China), the membrane was incubated for 1 h at 4°C with the goat anti-rabbit IgG (ZSGB, Beijing, China) diluted 1:5000 on a shaking platform. Following three additional washes with TBST, the membrane was exposed and developed using a chemiluminescence imaging analysis system (Tanon 4600, Shanghai, China).

For transmission electron microscopy (TEM) analysis, exosome samples were initially fixed with a 2.5% glutaraldehyde solution at 4°C for at least 12 h to preserve their structural integrity. Following fixation, a 5–10 μL aliquot of the exosome suspension was applied to a copper grid and allowed to air-dry at room temperature for 5 min. Any excess liquid was then wicked away using filter paper. The grids, now containing the exosome samples, were stained with saturated uranyl acetate for 1 min to enhance contrast and visualize the exosomal membrane. After staining, the grids were air-dried to remove any residual staining solution. The prepared grids were examined using a Tecnai G2 Spirit TEM (FEI, Hillsboro, Oregon, United States) operated at an acceleration voltage of 80 kV to obtain high-resolution images of the exosomes.

We used the nanoflow device (U30, Fuliu, Xiamen, China) to detect exosomes derived from iPSCs, strictly following the instructions provided in the manual. Initially, specialized quality control standards, including particle concentration and particle size standards, were used to validate the device’s performance. During the quality control process, the device was preheated, and the standards were sequentially loaded while data were recorded to ensure that the measurement results fell within acceptable ranges. Following successful quality control, the extracted iPSC exosome samples were diluted to an appropriate concentration and loaded into the device for detection. Suitable detection parameters were set, and data were recorded in real time. Subsequently, the corresponding software was used to analyze the particle size distribution and concentration of the exosomes.

Following dialysis, the samples were extracted from the dialysis bags and diluted with DMSO. AmB concentration standards were prepared using the same solvent as the samples. The standards and the samples to be analyzed were sequentially added to a microplate. The detection of AmB content was performed using a microplate reader at a wavelength of 405 nm. The amount of AmB loaded into the exosomes was calculated based on the concentration curve derived from the standards.

Drug loading efficiency % = mass of A m B loaded into exosomes / initial mass of A m B added × 100

Drug loading μg A m B / 10 ̂ 9 particles = mass of A m B / number of exosome particles × 10 ̂ 9.

We compared four drug-loading methods: co-incubation, ultrasound, extrusion, and electroporation. Each method used a sample volume of 1 mL, containing iPSC-Exos at a concentration of 50 μg/mL and AmB at a concentration of 10 μg/mL. Finally, the absorbance (optical density, OD) of each well was measured at 450 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, United States). Detailed descriptions of each method are provided in Table 1.

We used the CCK-8 assay to evaluate the proliferation of HEK 293 T and RAW264.7 cells. The CCK-8 kit was purchased from Beyotime (Shanghai, China) and used according to the manufacturer’s instructions. The cells were seeded in 96-well plates during the exponential growth phase at a density of 2 × 10^4 cells per well and treated with various concentrations of amphotericin B and iPSC-Exo/AmB based on the experimental groups. The cells were cultured for 6 h in a 37°C incubator with 5% CO2. At the end of the culture period, cell morphology was observed under a microscope. Subsequently, 10 μL of 5 mg/mL CCK-8 reagent was added to each well and incubated for 1 h in the dark at 37°C with 5% CO2.

In addition, we utilized two cell viability and cytotoxicity detection kits from Beyotime (Shanghai, China) and BestBio (Shanghai, China) to assess cell survival and cytotoxicity. The cells were treated according to the experimental groups and then incubated with calcein AM (1:1000), propidium iodide (PI) (1:1000), and Hoechst 33342 (1:100) in serum-free medium at 37°C for 20–30 min. After washing three times with serum-free medium to remove unbound dye, a complete growth medium was added, and fluorescence images were captured to evaluate cell survival. For apoptosis detection, the cells were washed with PBS, briefly digested with trypsin (without EDTA), and centrifuged at 1000 rpm for 5 min. They were then resuspended in pre-cooled 1× PBS (4°C) and washed again. Following this, the cells were suspended in 300 μL of 1× binding buffer, incubated with 5 μL of Annexin V-FITC in the dark for 15 min, and stained with 10 μL of PI for an additional 10 min. Finally, the samples were analyzed using a flow cytometer.

C57BL/6 J mice were administered AmB, iPSC-Exo/AmB, or iPSC-Exo at a dose of 0.25 mg/kg. Blood samples were collected 48 h post-treatment to assess biochemical parameters. Serum samples were obtained from the mice, and biochemical markers such as blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), and urinary protein (UP) levels were measured to evaluate nephrotoxicity. Hepatotoxicity was assessed by measuring the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), direct bilirubin (DBIL), and total bilirubin (TBIL) in the serum. All analyses were performed in triplicate using commercial assay kits purchased from Jiancheng Bioengineering Institute (Nanjing, China), following the manufacturer’s instructions.

The minimum inhibitory concentration (MIC) assay was conducted to evaluate the sensitivity of antifungal drugs, as described by Liu et al. (2020). The MIC values for iPSC-Exo/AmB and AmB were determined using the broth microdilution method, following the Clinical and Laboratory Standards Institute (CLSI) guideline M27-A3. A series of diluted iPSC-Exo/AmB and AmB solutions were mixed with RPMI 1640 liquid culture medium at 30°C, resulting in a final yeast suspension of 1 × 10^3 cells/mL in a 96-well microtiter plate. For Candida species, the MIC was measured after 24 h of incubation, while for other fungi, it was determined after 72 h. Each antifungal susceptibility test included the quality control strain C. parapsilosis ATCC 22019 to ensure assay accuracy. The MIC range of fluconazole against C. parapsilosis ATCC 22019 was found to be between 0.5 μg/mL and 4 μg/mL, confirming that the test results were within an acceptable range.

To evaluate the therapeutic effects of drugs on CM, this study utilized female C57BL/6 J mice aged 6–8 weeks. A systemic infection model was established by intravenously injecting 200 μL of a suspension containing the viable C. neoformans H99 strain (1 × 10^6 CFU) via the lateral tail vein. Two hours post-infection, the mice were treated with AmB, iPSC-Exo/AmB, or iPSC-Exos at a dose of 0.25 mg/kg (iPSC-Exos were used at the same particles), while untreated mice served as controls. At 48 h after infection, the mice were euthanized, and brain tissues were collected for fungal burden measurement and periodic acid–Schiff (PAS) staining. The serum was harvested, and commercial assay kits procured from Jiangsu Sumeike Biological Technology (Yancheng, China) were used to measure the concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) according to the manufacturer’s instructions. All experiments were performed in triplicate to ensure the accuracy and reproducibility of the results.

To investigate the biodistribution of iPSC-Exo/AmB and AmB in C57 BL/6 J mice, we administered a 1 mg/kg injection and sacrificed the mice at 1, 3, 6, 9, and 12 h post-injection (n = 3). The kidneys, liver, and brain tissues were harvested and processed with acetonitrile for ultrasonic homogenization at 70 Hz for 2 min. The samples were then centrifuged at 13,000 r/min for 15 min at 4°C, and the supernatant was filtered through a 0.22-μm membrane.

Sample analysis was conducted using a solid-phase chromatography column (Waters HSS T3, 2.1 × 100 mm, 1.8 μm, Waters, United States) coupled with ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS, AB Sciex 4,000 QTRAP, United States). The chromatographic conditions included a column temperature of 30°C and a flow rate of 0.3 mL/min, using a gradient elution process. The mobile phase began with 95% ultrapure water and 5% acetonitrile, adjusted to 40% ultrapure water and 60% acetonitrile at 1.00 min, and finally transitioned to 5% ultrapure water and 95% acetonitrile at 3.00 min.

The data were statistically analyzed using SPSS 23.0 software. Data normality was assessed using the Kolmogorov–Smirnov test. For normally distributed data, numerical variables are presented as mean ± standard deviation (x ± s). One-way analysis of variance (ANOVA) was used for group comparisons, and pairwise comparisons between groups were conducted using the least significant difference (LSD) t-test. A p-value of less than 0.05 was considered statistically significant.

Exosomes were successfully extracted from iPSCs using ultrafiltration and ultracentrifugation techniques. TEM revealed that these exosomes possess a distinct membrane structure and exhibit typical disk-like morphology with well-defined edges (Figure 1A). Further analysis using nanoflow cytometry (nFCM) indicated that the extracted exosomes have a median diameter of 62.75 nm, an average diameter of 64.49 nm, and a standard deviation of 9.34 nm (Figure 1B), with an average particle concentration of 3.03E+11 particles/mL (Figure 1C). Western blotting confirmed the presence of the exosomal marker proteins CD9, CD63, and TSG101, while the endoplasmic reticulum protein calnexin was not detected (Figure 1D). These results demonstrate that the iPSC-Exos conform to the standard characteristics of exosomes, providing a solid foundation for subsequent experimental research.

In this study, we investigated the hydrophobicity of AmB and evaluated its loading efficiency and purification process in nanodrug carriers. We referenced the preparation and purification methods of AmB described in relevant patents (CN108714151 B and CN111529506 A). A 48-h dialysis experiment was conducted at 25°C using a 14 kDa dialysis bag with three concentrations of AmB: 20, 10, and 5 μg/mL. The results showed that the residual rate of AmB at a concentration of 20 μg/mL was 19.5%, while the residual amounts at 10 μg/mL and 5 μg/mL were below the detection limit (as shown in Table 2). Based on these findings, we determined that 10 μg/mL is the optimal feeding concentration for AmB. This concentration not only enhances loading efficiency but also facilitates subsequent purification steps, thereby improving the overall efficiency and purity of the preparation process.

To enhance the drug loading capacity and efficiency of iPSC-Exos for AmB, we evaluated four drug-loading methods: co-incubation, ultrasound, extrusion, and electroporation. Figure 2 displays the particle size distribution post-treatment as measured by nFCM, facilitating a comparative analysis of these drug delivery strategies. Our analysis revealed that all methods increased particle size, with co-incubation showing a significant median diameter increase of 15 nm (p < 0.0001). Co-incubation not only elevated the drug loading efficiency to 43.6% but also achieved a substantial loading capacity of 4.6 μg AmB per 10^9 particles, as reported in Table 3. This enlargement exceeds the renal excretion threshold, suggesting that the iPSC-Exo/AmB complex formed via co-incubation could significantly reduce AmB accumulation in the kidneys, thereby potentially mitigating its nephrotoxic effects.

Based on the preceding research, we obtained iPSC-Exos at a concentration of 3.03E+11 particles/mL. Co-incubation method revealed that each 10^9 particles were found to carry 4.6 μg of AmB. We then dissolved the iPSC-Exo/AmB in PBS and diluted it to prepare a stock solution with a concentration of 50 μg/mL of AmB. AmB is known to interact with cholesterol-containing cell membranes, leading to damage in host cells. As shown in Figure 3A, our hemolysis activity test results indicated that iPSC-Exo/AmB significantly reduced hemolytic activity compared to AmB at all tested concentrations (p < 0.0001), demonstrating lower destructiveness to rabbit red blood cells.

The CCK-8 assay results, as presented in Figures 3B,C, revealed the dose-dependent toxicity of AmB on HEK293T and RAW264.7 cells. At a concentration of 25 μg/mL, the viability of HEK293T cells decreased to 60.25%, while RAW264.7 cells exhibited a viability of 56.11%. Increasing the AmB concentration to 50 μg/mL further reduced viability to 42.66 and 44.29%, respectively, highlighting the significant inhibitory effect of high AmB concentrations. In contrast, cells treated with iPSC-Exo/AmB displayed significantly higher viability at the same concentrations (p < 0.05). At 25 μg/mL, the viability of HEK293T and RAW264.7 cells was maintained at 73.32 and 81.44%, respectively. At 50 μg/mL, these rates were 73.32% for HEK293T cells and 67.56% for RAW264.7 cells. These findings indicate that iPSC-Exo/AmB effectively mitigates the cytotoxic effects of AmB.

In addition, at the working concentration of 25 μg/mL, live–dead cell staining (Figure 3D) and flow cytometry (Figures 3E,F) results demonstrated that iPSC-Exo/AmB significantly reduced the rate of cell apoptosis. Collectively, these results suggest that iPSC-Exo/AmB offers significant advantages over traditional AmB in reducing toxicity to HEK293T and RAW264.7 cells, thereby validating its safety and potential as an antifungal agent.

AmB is often limited in clinical applications due to its significant nephrotoxicity. In this study, we developed iPSC-Exo/AmB, with a particle size of approximately 80 nm, which exceeds the renal excretion threshold but remains within the hepatic excretion range (Liu et al., 2020). To elucidate the in vivo distribution of the drug, we used the UPLC-MS/MS method to compare the concentrations of AmB in the liver and kidneys of mice after intravenous administration of AmB and iPSC-Exo/AmB (see Figures 4A,B). The results indicated that both formulations achieved their maximum concentrations at 3 h. Notably, compared to AmB alone, the administration of iPSC-Exo/AmB resulted in significantly higher drug concentrations in the liver and significantly lower concentrations in the kidneys, with a relatively slow and weak accumulation process observed in the latter.

To evaluate the safety and efficacy of iPSC-Exo/AmB, we used C57BL/6 J mice and administered AmB, iPSC-Exo/AmB, or iPSC-Exo at a dose of 0.25 mg/kg (with iPSC-Exos used at the same particle concentrations). After 48 h, we collected blood samples to measure various biochemical indicators. The results showed that (Figures 4D–K), compared to the PBS control group, mice treated with AmB had significant increases in BUN, CRE, UA, UP, AST, ALT, DBIL, and TBIL, indicating adverse effects on liver and kidney functions. In contrast, the iPSC-Exo group showed no significant changes, suggesting that iPSC-Exo does not burden these organs. Moreover, mice treated with iPSC-Exo/AmB exhibited significant reductions in BUN, CRE, UA, UP, AST, ALT, DBIL, and TBIL compared to the AmB-only group, although iPSC-Exo/AmB is more widely distributed in the liver (Figure 4B). These findings indicate that the iPSC-Exo/AmB complex effectively mitigates the adverse effects of AmB on liver and kidney functions.

In this study, we assessed the minimum inhibitory concentrations (MICs) of traditional AmB and iPSC-Exo/AmB against C. neoformans, including the standard strain H99 and clinical isolate, through in vitro experiments. The microbroth dilution method was used, adhering strictly to the CLSI M27-A3 guidelines. Fluconazole’s MIC against C. parapsilosis ATCC 22019, which is 0.5 μg/mL, served as a quality control standard. The results (see Table 4) indicated that iPSC-Exo/AmB demonstrated at least eight times greater antimicrobial efficacy than traditional AmB in inhibiting C. neoformans in vitro, significantly enhancing its antimicrobial activity.

The CNS is the primary target of C. neoformans infection. UPLC-MS/MS analysis revealed that in C57BL/6 J mice injected intravenously with AmB and iPSC-Exo/AmB, the brain distribution of iPSC-Exo/AmB was significantly higher than that of AmB at 3 h. Moreover, at 6 h, this difference became even more pronounced, with the AmB concentration in the iPSC-Exo/AmB group increasing by approximately sixfold (Figure 4C). We established a systemic infection model in C57BL/6 J mice by intravenously administering C. neoformans H99 at a dose of 1 × 10^6 CFU per mouse. The mice were treated with either AmB, iPSC-Exo/AmB, or iPSC-Exo alone at a dose of 0.25 mg/kg. The mice were euthanized 48 h post-infection, and their brains were harvested for analysis. Our results indicated that the administration of iPSC-Exo/AmB significantly reduced fungal load in the brain compared to traditional AmB treatment (p < 0.0001). Furthermore, the administration of iPSC-Exo alone also resulted in a significant decrease in brain fungal burden compared to the model group (p < 0.01; see Figures 5A,B). Histological examination confirmed these findings; periodic acid–Schiff (PAS) staining of brain tissue specifically from the areas surrounding the cerebral cortex of mice treated with iPSC-Exo/AmB revealed a markedly reduced number of C. neoformans colonies compared to the AmB treatment group (see Figure 5C).

Although the assessment of the brain organ coefficient and changes in body weight did not reveal significant differences among the treatment groups (data not shown), the levels of inflammatory factors, such as TNF-α and IL-6, in the serum of mice treated with iPSC-Exo/AmB were markedly lower than those in the AmB treatment group. This suggests that iPSC-Exo/AmB may have superior anti-inflammatory effects, as illustrated in Figures 5D,E. These findings indicate that iPSC-Exo/AmB represents a novel therapeutic strategy with significant potential for treating CNS infections caused by C. neoformans.