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. 2026 Jul 10;19(7):e70411. doi: 10.1111/1751-7915.70411

From Mycosynthesis to Immunomodulation: Exploring the Safety, Adjuvant Potential and Microbiota Impact of Biogenic Silver Nanoparticles

Ana Clara Muniz‐Lagos 1,2,3,4, Diego M Roldán 1, Paula Arbildi 2,3,4, Gustavo Mourglia‐Ettlin 2,3,4,✉, Silvana Alborés 1,✉
PMCID: PMC13354849  PMID: 42432985

ABSTRACT

Subunit vaccines are considered the safest immunization platforms but typically require adjuvants to elicit robust immune responses. Although nanoparticles have emerged as promising adjuvants, biogenic silver nanoparticles (AgNPs) produced by mycosynthesis remain largely unexplored. Herein, nine different AgNPs were mycosynthesized using local strains of Phanerochaete chrysosporium, Penicillium expansum and Punctularia atropurpurascens grown in three different culture media (PDB, MEB, MGYP). Mycosynthesized AgNPs were all shown to be spherical, negatively charged and containing fungal proteins within the biomolecular capping. Additionally, although in vitro biocompatibility assessments showed AgNPs‐specific differences in haemolytic and cytotoxic activities, no signs of acute toxicity were observed in mice. The adjuvant potential of AgNPs was further evaluated in ovalbumin‐immunized mice, showing that four AgNPs displayed significant immunoenhancement activity with an outstanding IgG2a‐polarizaing effect. Adjuvant and IgG subclass polarizing activities were both shown to be dose dependent. Finally, distal effects induced by selected AgNPs were assessed through faecal microbiota analyses. Remarkably, while Firmicutes and Bacteroidota remained the dominant phyla, AgNPs‐treated groups showed compositional shifts consistent with mild immunomodulation. Summing up, our results on biocompatibility, adjuvant capacity, strong and dose‐dependent IgG2a‐polarizing activity, as well as limited impact on gut microbiota, support mycosynthesized AgNPs as versatile next‐generation platforms for developing novel vaccine adjuvants.

Mycosynthesized silver nanoparticles from fungal strains act as safe, biocompatible vaccine adjuvants. They enhance immune responses in mice, promoting strong, dose‐dependent IgG2a polarization with minimal toxicity and limited gut microbiota disruption, highlighting their potential as versatile next‐generation platforms for subunit vaccine development.

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1. Introduction

Successful immunization with protein antigens usually requires the use of adjuvants to trigger and enhance immune responses, being thus crucial for the effective performance of most subunit vaccines. Although a wide variety of compounds with adjuvant properties—through diverse mechanisms of action—have been described, Alum remained the only clinically approved adjuvant for almost a century. Currently, few more vaccine adjuvants have been approved by the FDA for human use, including AS04, MF59, AS01 and CpG 1018; and notably, most of them are typically formulated as nanoparticles (Marques Neto et al. 2017; Nooraei et al. 2023).

Nanoscaled adjuvants (1–100 nm) have been proposed to potentiate vaccine‐induced immunity, since nanoparticles (NPs) usually enhance antigen‐specific immune responses through modulation of antigen uptake, processing and presentation, as well as through polarization of adaptive immunity. Additionally, NPs serve as antigen delivery systems, reducing antigen dose and limiting potentially associated (cyto)toxicities. Moreover, and through antigen conjugation, NPs have been shown to improve recognition and interaction with antigen‐presenting cells, thereby promoting both humoral and cell‐mediated immune responses; ultimately, increasing the overall vaccine efficacy (Sekimukai et al. 2020; Ahmadbeigi et al. 2024; Buchhorn de Freitas et al. 2024). Among nanoscaled adjuvants, metal‐based NPs have attracted considerable attention due to their proved capacity to enhance the immunogenicity of subunit (protein‐based) vaccines (Li, Chen, et al. 2025); and particularly, silver NPs (AgNPs) were shown to improve vaccine efficacy and effectiveness, as well as antigen delivery and biodistribution (Sanchez‐Guzman et al. 2019).

The biological synthesis of AgNPs using either plants, fungi, algae, or bacteria offers a promising, eco‐friendly and cost‐effective alternative (Balakumaran et al. 2016; Saeed et al. 2020). Furthermore, biosynthesis ensures colloidal stability of AgNPs and allows for the control over different physicochemical parameters, like size and shape (Ibrahim et al. 2021). Fungal‐mediated AgNPs biosynthesis (mycosynthesis) is particularly advantageous because fungi are effective reducing and stabilizing organisms due to their metal tolerance and bioaccumulation ability. Fungi also produce abundant extracellular proteins and enzymes and can be easily cultured to produce large and well‐dispersed AgNPs (Madakka et al. 2018; Sanguiñedo et al. 2018; Barabadi et al. 2019; Guilger‐Casagrande and de Lima 2019). Mycosynthesized metal NPs are stabilized by a biomolecular capping that protects them against oxidation. This capping is formed by fungal biomolecules, which are influenced by the growth culture medium and the mycosynthesis conditions used, like temperature, pH, biomass and silver concentration (Saxena et al. 2016). Thus, depending on both the fungal species and its growth characteristics, the biomolecular capping of AgNPs may provide diverse biological activities as well as reduced potential cytotoxicity and genotoxicity, thus making mycosynthesized AgNPs an attractive biomedical nanomaterial (Guilger‐Casagrande and de Lima 2019; Durán et al. 2023).

The evaluation of new adjuvants should not only focus on their ability to enhance the desired specific immune response, but also on potential impacts in other physiological systems such as the intestinal microbiota, a recognized central regulator of the host immune and metabolic homeostasis (Lynn et al. 2022; Zimmermann 2023; Bowser et al. 2024). For instance, it has been postulated that the initial response to vaccination—enhanced by adjuvants—can derive in immune responses that induce changes to the microbial metabolites, microbiota composition and its prevalence over time. This observed shifts also condition the immune response and have an effect on the vaccine efficacy (Loddo et al. 2025). In this regard, it has been reported that exposure to metal NPs, even via non‐oral routes, can exert profound distal effects on the gut microbiota. Relative abundances of Firmicutes and Bacteroidota phyla serve as biomarkers of dysbiosis, and a decrease in Firmicutes coupled to an increase in Bacteroidota has been a well‐documented pattern linked to NPs‐induced metabolic stress and pro‐inflammatory immunomodulation (Shin et al. 2015; Li et al. 2023). Such local gut alterations can manifest systemically through the translocation of microbes or their products into the bloodstream (Roy and Trinchieri 2017), confirming that—regardless the location of an immune stimulus—gastrointestinal consequences may occur.

In the present work, biogenic AgNPs were synthesized using local strains of filamentous fungi grown in different culture media. Obtained AgNPs were physiochemically characterized and their biocompatibility profiles were assessed in vitro and in vivo. The adjuvant activity of AgNPs was further evaluated in immunized mice, and their potential distal effects were finally explored through faecal microbiota analysis. Our results support mycosynthesized AgNPs as interestingly next‐generation platforms for the development of novel vaccine adjuvants.

2. Experimental Procedures

2.1. Reagents, Materials and Fungal Strains

Potato Dextrose Agar/Broth, Malt Extract and Yeast Extract were from Oxoid. Bacto Peptone, Nunc 96‐well plates and the Micro BCA kit were from Thermo Fisher. Silver nitrate, albumin from chicken egg white (OVA), bovine serum albumin (BSA), thiazolyl blue tetrazolium bromide (MTT), DMEM high glucose medium, RPMI culture medium, 3,3′, 5,5′‐tetramethylbenzidine (TMB) and Tween‐20 were all from Sigma‐Aldrich. Goat anti‐mouse IgG‐, IgG1‐, IgG2a‐, IgG2b‐ or IgG3‐HRP were from Southern Biotech. Sterile saline and Alhydrogrel (Alum) were from Fármaco Uruguayo and InvivoGen, respectively. Phosphate buffer saline and foetal bovine serum were from Capricorn Scientific, and membrane filters from Millipore.

Fungal strains Phanerochaete chrysosporium 12G, Penicillium expansum 14S and Punctularia atropurpurascens H2126 belonged to the CCMG Collection from Facultad de Química—Universidad de la República (Uruguay).

2.2. Animals and Ethics Statement

Animal experiments were performed in compliance with Comisión Honoraria de Experimentación Animal (CHEA, Universidad de la República, Uruguay), according to the National Uruguayan Legislation N° 18.611. Experimental protocols were approved by the Ethics Committee of Facultad de Medicina (Universidad de la República, Uruguay) and were given the approval numbers 070151‐000048‐22 and 070151‐000049‐22 (www.expe.edu.uy).

Female CD‐1 mice (8–12 weeks old) were bred and housed at the animal facilities of Instituto de Higiene (Universidad de la República, Uruguay). Mice were housed under conventional conditions and were fed ad libitum with sterilized food and water.

2.3. Mycosynthesis and Characterization of AgNPs

Fungal strains were cultivated on Potato Dextrose Agar at 28°C (Sanguiñedo et al. 2018). Two agar plugs per strain were transferred into liquid media (PDB, MEB, or MGYP), generating nine combinations of mycelium (Pch/Pe/Pa) and medium (PDB/MEB/MGYP). Cultures were incubated for 72 h at 28°C under agitation. Biomass was collected, washed and resuspended in sterile distilled water to obtain cell‐free filtrates after 96 h of incubation. Silver nanoparticles (AgNPs) were synthesized by mixing fungal filtrates with AgNO3 (final concentration 2.5 mM) and incubating the reaction mixtures in darkness. Formation of AgNPs was initially monitored by colour change and confirmed by UV–Vis spectroscopy through detection of the surface plasmon resonance peak. Reactions were stopped at 96 h, and AgNPs were purified by centrifugation, washed, sterile‐filtered (0.22 μm) and stored at 4°C (Data S1).

UV–Vis spectra (300–800 nm) were recorded to confirm AgNPs formation. Morphology was analyzed by high‐resolution transmission electron microscopy (TEM). Hydrodynamic diameter and ζ‐potential were determined by dynamic light scattering (DLS). Particle concentration was measured by nanoparticle tracking analysis (NTA). Protein content associated with AgNPs was quantified using a MicroBCA assay and normalized to nanoparticle concentration (Data S2).

2.4. In Vitro Biocompatibility Assessment

Haemolytic activity (HA) was evaluated by incubating a suspension of freshly isolated mouse erythrocytes (1%) with serial dilutions of AgNPs for 1 h at 37°C (Ratia et al. 2022). Haemoglobin release was quantified spectrophotometrically. Saline and SDS served as negative and positive controls, respectively. The HA50 value was defined as the concentration of AgNPs producing 50% haemolysis.

Cytotoxic activity (CA) was assessed using RAW264.7 murine macrophages and the MTT assay (Estevez et al. 2025). Cells were incubated with serial dilutions of AgNPs for 72 h, and final viability was calculated relative to untreated controls. The CA50 value was defined as the concentration of AgNPs reducing viability to 50% (Data S3).

2.5. In Vivo Acute Toxicity Assays

Acute toxicity assays (ATA) were performed in female CD‐1 mice following the subcutaneous (s.c.) administration of AgNPs (Ratia et al. 2022). In the first screening, mice received a 10× intended dose (2.0 × 1010 AgNPs/kg), and body weight and clinical signs were monitored for 14 consecutive days. A second independent assay evaluated selected AgNPs at a 200× intended dose (4.0 × 1011 AgNPs/kg). At Day 14, animals were euthanized and spleen, liver, and kidneys were collected and weighed. Humanitarian endpoints were predefined (≥ 15%) body weight loss or evident signs of general (dis)comfort (Neun et al. 2024) (Data S4).

2.6. Evaluation of the Adjuvant Activity of AgNPs

Adjuvant potential of AgNPs was assessed using ovalbumin (OVA) as a protein antigen model. Mice were immunized s.c. on Days 0 and 14 with either OVA alone, OVA plus Alum (positive control), or OVA plus AgNPs. Doses of AgNPs ranged from 1× (2.0 × 109 AgNPs/kg) to 100× (2.0 × 1011 AgNPs/kg) depending on the experiment. Sera were collected on Day 28 for antibody determination. Two independent immunization experiments were performed: a first screening assay for the nine AgNP formulations at a single dose and a second dose‐escalation study for selected AgNPs (Data S5).

2.7. Determination of Specific Antibodies

Serum OVA‐specific IgG and IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) were quantified by ELISA. Plates were coated with OVA, blocked and incubated with serially diluted sera. Bound antibodies were detected using isotype/subclass‐specific HRP‐conjugated secondary antibodies and TMB substrate. Absorbance was read at 450 nm (Mourglia‐Ettlin et al. 2016). An internal standard (OVA‐specific hyperimmune serum) was used for normalization (Data S6).

2.8. Statistical Analyses

General data processing and statistical analyses were performed using GraphPad Prism software (version 8.0.2, GraphPad Inc., San Diego, USA). Group outliers identified by the ROUT method (Q = 1%) were routinely removed. Differences in non‐parametric values were evaluated through the Mann–Whitney U‐test (p‐value < 0.05).

2.9. Faecal Microbiota Analysis

Faecal samples were collected at Day 28 from immunized mice (100× dose) and controls. Three pooled samples per group (n = 3) were generated and stored at −80°C (Data S7, Table S1). DNA was extracted from pooled faeces and the V3–V4 region of the bacterial 16S rRNA gene was amplified and sequenced using Illumina MiSeq technology. Amplicon sequence variants (ASVs) were inferred using the DADA2 pipeline (Callahan et al. 2016; Roldán et al. 2022) (Data S7).

Sequence analyses were performed in R using Phyloseq (McMurdie and Holmes 2013). Non‐bacterial sequences were removed and data were transformed into relative abundance. Alpha diversity was evaluated using observed ASVs, Shannon and Simpson indices. Beta diversity was analyzed using Bray–Curtis distances and visualized by multidimensional scaling (MDS). Group differences were tested using Wilcoxon tests and PERMANOVA. Differential abundance analyses were performed using DESeq2 with a significance threshold of α = 0.05 DESeq2 (Love et al. 2014) (Data S7). The datasets generated for this study can be found in the NCBI, BioProject ID PRJNA1471718.

3. Results

3.1. Mycosynthesis and Characterization of AgNPs

Extracellular mycosyntheses were performed using mycelia from three local fungal strains capable of producing AgNPs: P. chrysosporium (Pch), P. expansum (Pe) and P. atropurpurascens (Pa), each grown in three culture media: PDB, MEB and MGYP to maximize AgNPs diversity.

The formation of AgNPs was visually monitored through progressive colour changes (translucent to reddish/brownish) (Figure 1A) and confirmed by UV–Vis spectroscopy (300–800 nm) assessing the characteristic surface plasmon resonance (SPR) peak at ~440 nm (Figure 1B). Nine different AgNPs were obtained, six—those derived from MEB and MGYP cultures—reported here for the first time.

FIGURE 1.

FIGURE 1

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Mycosynthesis of AgNPs. Mycosyntheses were performed using the mycelium of three fungal species (Pch, Pe and Pa) grown in different culture media (PDB, MEB and MGYP). Daily pictures were taken to report the translucent to reddish/brownish colour switch of duplicate solutions (A). UV–Vis absorption spectra for each AgNPs were recorded at the end of mycosyntheses (B).

Transmission Electron Microscopy analyses revealed mostly spherical nanoparticles (Figure 2), while DLS showed hydrodynamic sizes ranging from 7 to 78 nm with acceptable PDI values (Table 1). While size varied depending on fungal strain and growth medium, ζ‐potential values were similar for all AgNPs (−16.9 to −22.9 mV), suggesting good colloidal stability (Table 1).

FIGURE 2.

FIGURE 2

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Morphology of AgNPs. Mycosynthesized AgNPs were analyzed through Transmission Electron Microscopy.

TABLE 1.

Physicochemical characterization of AgNPs. Results are shown as mean values, and when applicable, SD is also shown.

AgNPs Parameter
DLS Size (nm) PDI ƺ‐potential (mV) Proteins in capping (fg/NP) Productivity (NPs/g biomass)
PchPDB 12 ± 2 0.5 −20.9 ± 4.3 5.6 ± 0.9 1010
PchMEB 25 ± 5 0.3 −18.5 ± 6.6 4.3 ± 2.4 108
PchMGYP 39 ± 5 0.5 −19.5 ± 4.6 125.3 ± 7.9 108
PePDB 7 ± 1 0.5 −16.9 ± 3.2 19.0 ± 0.8 1010
PeMEB 78 ± 20 0.4 −19.3 ± 3.4 45.4 ± 2.2 109
PeMGYP 16 ± 2 0.5 −20.6 ± 4.4 36.6 ± 5.4 109
PaPDB 23 ± 4 0.3 −21.8 ± 5.7 3.7 ± 0.7 1011
PaMEB 14 ± 2 0.3 −22.9 ± 5.5 14.5 ± 0.1 1010
PaMGYP 24 ± 4 0.3 −21.4 ± 6.8 11.0 ± 2.9 1010
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Nanoparticle concentration was determined by NTA. The estimation of capping protein content ranged from 3.7 to 125.3 fg/AgNP, varying with both strain and medium and tending to be higher in Pe‐derived AgNPs (Table 1). Productivity (AgNPs per g of fungal biomass) also varied markedly, mainly with fungal strain: Pch showed the lowest productivity and Pa the highest. Within each strain, PDB‐based syntheses were the most productive. Thus, PchMEB corresponded to the least productive condition (6.5 × 108 AgNPs/g), whereas PaPDB yielded the highest value (2.3 × 1011 AgNPs/g), representing more than a 350‐fold difference among them (Table 1).

3.2. Biocompatibility Assessment of AgNPs

Biocompatibility of AgNPs was assessed in vitro and in vivo to ensure safety. In vitro HA and CA activities were first assessed to compare AgNPs toxicities. HA was evaluated using freshly isolated mouse erythrocytes exposed to increasing AgNPs concentrations to calculate HA50 values. Marked differences were observed among mycosynthesized AgNPs suggesting differential erythrocyte sensitivity depending on the AgNP (Figure 3A). HA50 values ranged from 6.5 × 107 AgNPs/mL (PchMEB) to > 1.0 × 109 AgNPs/mL (PePDB, PeMGYP and PaPDB). Although HA50 values varied with fungal strain and culture medium, Pch‐ and Pe‐derived AgNPs were overall the most and least haemolytic, respectively (Table 2).

FIGURE 3.

FIGURE 3

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In vitro biocompatibility of AgNPs. In vitro biocompatibility profiles of mycosynthesized AgNPs were assessed through determination of (A) haemolytic activity (HA) and (B) cytotoxicity. For HA assays, the haemoglobin release of freshly obtained mouse erythrocytes was determined after incubation with increasing amounts of AgNPs, while cytotoxicity was assessed using the MTT assay and the mouse macrophage cell line RAW264.7. Results (mean ± SEM) are depicted in logarithmic scale after normalization with the corresponding controls. Dashed lines correspond to 50% HA or cell viability.

TABLE 2.

In vitro biocompatibility of AgNPs. HA50: Concentration of AgNPs inducing a 50% haemolysis on fresh murine erythrocytes. CA50: Concentration of AgNPs reducing viability of RAW264.7 cells to 50%.

AgNPs HA50 (NPs/mL) CA50 (NPs/mL)
PchPDB 7.0 × 107 1.5 × 108
PchMEB 6.5 × 107 3.0 × 107
PchMGYP 3.5 × 108 3.8 × 108
PePDB > 1.0 × 109 > 1.0 × 109
PeMEB 3.3 × 108 > 1.0 × 109
PeMGYP > 1.0 × 109 > 1.0 × 109
PaPDB > 1.0 × 109 > 1.0 × 109
PaMEB 5.9 × 108 3.8 × 108
PaMGYP 2.6 × 108 1.3 × 108
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CA was evaluated in RAW264.7 mouse macrophages using the MTT assay. Cells were exposed to increasing AgNPs concentrations and CA50 values were determined from post‐incubation viability. Variations in CA profiles suggested differential macrophages sensitivity depending on the AgNP type (Figure 3B). CA50 values ranged from 3.0 × 107 AgNPs/mL (PchMEB) to > 1.0 × 109 AgNPs/mL (PaPDB and the three Pe‐derived AgNPs). Despite strain‐ and medium‐dependent differences, Pe‐derived AgNPs were consistently the least cytotoxic (Table 2).

Because no in vitro‐in vivo toxicity correlations currently exist for AgNPs, acute toxicity assays (ATA) were performed in mice to evaluate in vivo safety. Considering their intended adjuvant use, subcutaneous (s.c.) administrations were performed. Based on previous reports using chemically synthesized AgNPs as vaccine adjuvants for s.c. administration (Xu et al. 2013), a working dose (1× dose) of 2.0 × 109 AgNPs/kg/dose was estimated. ATA were conducted in CD‐1 mice using a single 10× higher dose, followed by daily monitoring of weight and general signs of discomfort for 14 days. No mortality nor observable toxicity signs were detected, and weight‐gain curves showed no significant differences compared with controls (Figure 4).

FIGURE 4.

FIGURE 4

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In vivo biocompatibility of AgNPs. Acute toxicity assays were performed in mice after a single subcutaneous 10× dose of AgNPs. Mice were daily weighed for 14 consecutive days, and variations (%) to Day 0 are depicted (mean ± SEM). Statistical significance respect to the control group (saline) was assessed for each AgNPs using the Mann–Whitney test (*p < 0.05) applied to individual AUC values.

Overall, AgNP toxicity varied mostly with the fungal strain used for mycosynthesis, with Pe‐derived AgNPs showing the lowest toxicity regardless the culture medium used. AgNPs also appeared more haemolytic than cytotoxic, consistent with the known erythrocyte membrane properties. Nevertheless, in vivo results demonstrated that all mycosynthesized AgNPs were safe for s.c. administration in mice, at least up to a 10× dose.

3.3. Screening of the Adjuvant Activity of AgNPs

To evaluate the adjuvant potential of the nine mycosynthesized AgNPs, mice were immunized with OVA mixed with AgNPs (1× dose). Unadjuvanted (OVA in saline) and positive (OVA in Alum) controls were included. Mice received s.c. priming (Day 0) and booster (Day 14) injections, and sera were collected on Day 28.

OVA‐specific IgG levels measured by ELISA showed that six AgNPs significantly modulated the antibody response compared with the unadjuvanted group (Figure 5). PePDB and the three Pa‐derived AgNPs increased IgG levels, indicating adjuvant‐like activity. In terms of median values, Alum induced a 130‐fold increase, whereas AgNPs enhanced OVA‐specific IgG from 6‐fold (PePDB, PaPDB, PaMGYP) to 9‐fold (PaMEB). In contrast, PeMGYP—and PchMGYP to a lesser extent (p = 0.056)—impaired the response, showing immunosuppressive‐like effects, with 0.2‐fold and 0.4‐fold decreases, respectively.

FIGURE 5.

FIGURE 5

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Screening of AgNPs immunomodulating activity. Groups of mice (n = 5/group) were immunized with OVA (20 μg/dose) mixed with each AgNPs (1× dose). Unadjuvanted (OVA in saline) and positive (OVA in Alum) control groups were included. Mice received s.c. priming (Day 0) and booster (Day 14) inoculations, sera were collected at Day 28, and the OVA‐specific IgG response was evaluated by ELISA. Identified outliers were removed (1 value in the saline group from the experimental setting containing PchPDB, PePDB and all Pa‐derived AgNPs, and 1 value within each group containing Pa‐derived AgNPs). Final results are depicted as box‐and‐whiskers, and statistical significance respect to the unadjuvanted group (saline) was assessed using the Mann–Whitney test (*p < 0.05).

Induced IgG subclass profiles were also analyzed for AgNPs with adjuvant‐like activity (Figure 6). Unlike the mixed IgG response induced by Alum, AgNPs strongly polarized the antibody response towards IgG2a. Only PaMEB additionally induced IgG1 and IgG3, while IgG2b was not induced by any AgNPs.

FIGURE 6.

FIGURE 6

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Characterization of the IgG polarization induced by AgNPs. OVA‐specific IgG subclass responses (IgG1, IgG2a, IgG2b and IgG3) were evaluated by ELISA. Serum samples (n = 5/group) from unadjuvanted (OVA in saline) and positive (OVA in Alum) control groups, as well as from mice immunized with OVA (20 μg/dose) mixed with PePDB or Pa‐derived AgNPs (1× dose) were analyzed. Identified outliers were removed: IgG1 (1 value in PaMEB group), IgG2a (1 value in both the saline and PePDB groups), IgG2b (1 value in PaPDB group) and IgG3 (1 value in PaMEB group). Final results are depicted as box‐and‐whiskers, and statistical significance with respect to the unadjuvanted group (saline) was assessed using the Mann–Whitney test (*p < 0.05).

Overall, immunomodulatory effects of AgNPs depended on both fungal strain and culture medium, although Pa‐derived AgNPs consistently showed adjuvant activity regardless of the growth medium used. Moreover, AgNPs with adjuvant activity strongly biased the antibody response towards IgG2a, a subclass of outstanding immunological relevance.

3.4. AgNPs Exhibit Dose‐Dependent Adjuvant Activity

To determine whether AgNP adjuvant activity is dose‐dependent, a comparative study was performed using PaPDB (adjuvant‐active at 1× dose) and PchPDB (inactive at 1× dose). Biocompatibility at higher doses was confirmed through ATA using a 200× dose. No mortality, toxicity signs, or significant differences in body weight and organ weights (spleen, kidneys, liver) were observed compared with controls (Figure S1).

Mice were then immunized with OVA plus PaPDB or PchPDB at 1×, 6×, 60× and 100× doses. PaPDB significantly enhanced OVA‐specific IgG at 1 × −60× doses (median fold changes: 6.2–7.9), but lost its adjuvant activity at 100× (median fold changes: 2.5) (Figure 7A). In contrast, PchPDB showed no adjuvant effect at 1× and 6× (median fold changes: 1.1 and 3.1, respectively), but displayed significant immunoenhancement at 60× and 100× doses (median fold changes: 15.1 and 16.3, respectively) (Figure 7B). These results indicate dose‐dependent immunostimulation, which may be lost at very high doses and suggest potentially saturable IgG enhancement by AgNPs, since they induced ≤ 20‐fold increases, whereas Alum reached ~100‐fold increments.

FIGURE 7.

FIGURE 7

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Dose‐dependent adjuvant activity of AgNPs. Groups of mice (n = 5–7/group) were immunized with OVA (20 μg/dose) mixed at 1×, 6×, 60× and 100× doses of PaPDB (A) or PchPDB (B), following previously described schemes. Unadjuvanted (OVA in saline) and positive (OVA in Alum) control groups were included. Samples obtained at Day 28 were characterized by ELISA regarding levels of OVA‐specific IgG and subclasses (IgG1, IgG2a, IgG2b and IgG3), and individual values were normalized to the corresponding unadjuvanted group (saline). Dashed lines are shown at the median value of the unadjuvanted and positive control groups. Identified outliers were removed: From (A) 1 value in IgG group 1×, 1 value in IgG2a group 6×, 1 value in IgG2b group 1× and 1 value in IgG3 group 100×; from (B) 2 values in IgG2a group 100× and 2 values in IgG2b group 60×. Final results are depicted as box‐and‐whiskers, and statistical significance with respect to the unadjuvanted group (saline) was assessed using the Mann–Whitney test (*p < 0.05).

Profiles of IgG subclasses also revealed dose‐dependent polarizations. For PaPDB, higher doses (6 × −100×) significantly induced IgG2b and IgG3 (median fold changes: 1.7–3.3 and 6.1–11.5, respectively), while IgG2a induction declined from ~50‐fold inductions at 1× doses to near control levels at higher doses (median fold changes: 2.6–0.5). Consistently, IgG1 levels were not induced by PaPDB regardless of the dose used. Conversely, PchPDB significantly increased all IgG subclasses at higher doses (Figure 7B).

Overall, mycosynthesized AgNPs displayed dose‐dependent and functionally versatile immunostimulatory activity, affecting both overall adjuvant efficacy and IgG subclass polarization.

3.5. Distal Effects of AgNPs on the Faecal Microbiota

To evaluate potential effects of adjuvants on intestinal microbiota, faecal microbiota analyses were performed in mice immunized with OVA formulated with Alum, PaPDB, or PchPDB, plus a saline control. To maximize potential effects, AgNPs administered at 100× doses were evaluated. Faecal samples collected on Day 28 were pooled by groups, DNA was extracted, and 16S rRNA gene sequencing was performed using the Illumina MiSeq platform.

Sequencing generated 1,084,690 reads grouped into 1020 ASVs, and rarefaction curves confirmed sufficient sequencing depth. Alpha diversity indices showed no significant differences among groups. Observed richness ranged from 632 ASVs in saline to 716 and 755 ASVs in Alum and PchPDB, respectively. Shannon index values ranged from 4.52 (PaPDB) to 4.77 (saline), and Simpson values from 0.97 to 0.98, indicating minimal variation in richness and evenness. Beta diversity separated saline from Alum/AgNP groups, although PERMANOVA detected no significant differences (p > 0.05), with a marginal trend (p = 0.1).

Taxonomic analyses identified 10 phyla, dominated by Firmicutes (18.4%–83.7%) and Bacteroidota (13.3%–73.9%) (Figure S2). AgNP treatments reduced Firmicutes and increased Bacteroidota, while minor phyla such as Actinobacteriota reached up to 11.9% compared with < 1% in controls. Accordingly, the Firmicutes/Bacteroidota ratio was higher in saline (3.07 ± 2.29) than in Alum (0.48 ± 0.06), PchPDB (0.84 ± 0.26) and PaPDB (0.33 ± 0.09), indicating a shift towards Bacteroidota under adjuvant exposure.

Differential abundance analysis (DESeq2) revealed treatment‐specific changes: PaPDB increased Atopobiaceae, Coriobacteriaceae and Eggerthellaceae and decreased Lactobacillaceae; Alum increased Christensenellaceae and Marinifilaceae; and PchPDB increased Mycoplasmataceae and reduced Ruminococcaceae (Figure 8). Shared responses included increases in Muribaculaceae and decreases in Rikenellaceae, along with shifts in Lachnospiraceae, Oscillospiraceae and Erysipelatoclostridiaceae. Overall, similarly to Alum, AgNPs modulated the gut microbiota composition, affecting both dominant and low abundant taxa in treatment‐specific patterns.

FIGURE 8.

FIGURE 8

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Distal effects of AgNPs on faecal microbiota. Groups of mice were immunized with OVA (20 μg/dose) mixed at 100× doses of PaPDB or PchPDB, following previously described schemes. Unadjuvanted (saline) and positive (OVA in Alum) control groups were also included. Pooled faecal samples obtained at Day 28, and DNA was extracted for microbiota studies (n = 3 pools per group). Venn diagrams based on DeSeq2 analyses highlight key differences found in abundance of bacterial taxa among samples from mice treated with a PaPDB (red), PchPDB (blue), or Alum (yellow), all referred to the unadjuvanted control group (saline). Arrows indicate significant changes in the relative abundance of specific ASVs respect to the control group (↑: Increase, ↓: Decrease).

4. Discussion

A systematic comparative study was conducted to evaluate the in vivo safety, adjuvant activity and microbiota impact of biogenic AgNPs obtained by mycosynthesis. To maximize nanoparticle diversity, nine AgNPs were synthesized using three fungal strains—P. chrysosporium, P. expansum and P. atropurpurascens—previously reported as efficient AgNP producers (Sanguiñedo et al. 2018), each grown in three media (PDB, MEB, MGYP). Six AgNPs produced from MEB and MGYP cultures are reported here for the first time. Despite large productivity variations, Pa‐ and PDB‐derived syntheses were consistently the most productive (Table 1), highlighting their biotechnological relevance.

All AgNPs were purified, sterilized and physicochemically characterized. As previously reported for these fungi (Sanguiñedo et al. 2018), nanoparticles were spherical (Figure 2), while size depended on both fungal strain and culture medium (Table 1). Although ζ‐potential values were similar, protein capping content varied according to strain and medium, with generally higher values in Pe‐derived AgNPs (Table 1). These parameters influence colloidal stability (Sidhu et al. 2022). The organic capping—containing fungal proteins—was previously shown to be stably attached to the AgNP core (Sanguiñedo et al. 2018). Culture medium composition influences filtrate composition and final nanoparticle properties (Guilger‐Casagrande et al. 2021). Since capping composition affects stability and bioactivity (Chugh et al. 2021; Zaki et al. 2022; Herrera Pérez et al. 2024), proteomic analyses of filtrates and AgNP capping are ongoing.

Biomolecular capping may also reduce toxicity compared with chemically produced AgNPs (Wypij et al. 2020; Spagnoletti et al. 2021; Durán et al. 2023). Biocompatibility was assessed in vitro (haemolysis, cytotoxicity) and in vivo (ATA). Results varied depending on strain and medium (Figure 3); notably, PchPDB cytotoxicity agreed with previous findings in THP‐1 cells (Estevez et al. 2025). Pe‐derived AgNPs showed the lowest haemolytic and cytotoxic activities (Table 2), possibly associated with their higher protein content (Table 1). Despite these differences, all AgNPs were safe in vivo for s.c. administration up to a 10× dose (Figure 4), and PchPDB and PaPDB up to a 200× dose (Figure S1).

Nanostructured systems are increasingly explored as vaccine adjuvants (Elhassan Taha et al. 2024; Petrov et al. 2025; Li, Wu, et al. 2025), yet AgNPs remain poorly studied in this context (Xu et al. 2013; Sanchez‐Guzman et al. 2019), and biogenic AgNPs are largely unexplored. Indeed, only four studies currently exist: two using plant‐derived AgNPs (Asgary et al. 2016; Jabbar Sekhi and Abbas Aboud AL‐Samarraae 2023) and two using Fusarium oxysporum AgNPs (Buchhorn de Freitas et al. 2024; de Freitas et al. 2025). These fungal nanoparticles were spherical, negatively charged (−35 mV) and ~80 nm in diameter (Figueiredo et al. 2019), consistent with our results (Figure 2, Table 1).

Adjuvant activity of AgNPs was evaluated following NIH Nanotechnology Characterization Laboratory guidelines (Neun et al. 2024), determining OVA‐specific antibody responses after the s.c. immunization of mice (prime/boost scheme) with a mixture of OVA and AgNPs (1× dose). Six AgNPs significantly modulated IgG responses: PeMGYP and PchMGYP showed immunosuppressive‐like effects, while PePDB and all Pa‐derived AgNPs displayed adjuvant‐like activity (Figure 5). This functional diversity supports a strong association between AgNP bioactivity and fungal strain/culture medium. Pa‐derived AgNPs showed consistent adjuvant activity regardless the growth medium used, highlighting the fungal strain relevance in the screening of nanoparticle bioactivities. Immunosuppressant AgNPs may also have biomedical relevance (Hegde et al. 2025).

The adjuvant activity of AgNPs polarized the antibody response towards IgG2a, unlike the mixed response induced by Alum (Figure 6). Adjuvant‐induced immune profiles depend greatly on their mechanism of action (Turley and Lavelle 2022; Ben‐Akiva et al. 2025), allowing the induction of different immunity patterns for a single antigen (Visciano et al. 2012; Voutssas‐Lara et al. 2021). Notably, IgG2a is particularly valuable due to its broad effector functions (Bruhns 2012; Vidarsson et al. 2014; Nimmerjahn and Ravetch 2021). In contrast, F. oxysporum‐derived AgNPs mainly induced IgG1 responses (Buchhorn de Freitas et al. 2024; de Freitas et al. 2025), further emphasizing the importance of the fungal strain in determining immunological outcomes. While mechanistic insights were not explored in detail in this study, they will be addressed in the near future by focusing specifically on AgNPs that exhibit adjuvant activity and promote antibody response polarization towards the IgG2a isotype.

Dose‐dependent activity of adjuvants allows for the optimization of efficacy while minimizing adverse side effects. Dose‐response experiments with PaPDB and PchPDB (6 × −100× doses) showed clear dose‐dependent immunostimulation (Figure 7), with potential saturation at higher doses. Antibody polarization was also shown to be dose‐dependent, altering specific IgG subclass production depending on the AgNPs (Figure 7). These results suggest strong functional versatility of AgNPs and potential antigen dose‐sparing effects, as reported for particulate vaccines (Bansal et al. 2022).

Because adjuvants may affect other physiological systems, their impact on the gut microbiota was also evaluated. Microbiota‐vaccine interactions are bidirectional: microbiota composition influences vaccine responses, while vaccines can alter microbial communities (Gioula and Exindari 2025). Faecal microbiota analyses from mice immunized with OVA plus Alum, PchPDB, or PaPDB showed minimal disruption in alpha and beta diversity, consistent with NP‐based vaccines (Bowser et al. 2024) and differing from oral AgNP exposure (Van den Brule et al. 2015), highlighting the importance of the administration route (Campos et al. 2022).

Ratios of Firmicutes‐to‐Bacteroidota were higher in saline controls and lower in adjuvant‐treated groups, indicating shifts towards Bacteroidota usually associated with metabolic stress and immunomodulation (Shin et al. 2015; Li et al. 2023). Similar systemic effects after s.c. administration of AgNPs have been reported (Roy and Trinchieri 2017). Unlike silica NPs, exposure to AgNPs increased Actinobacteriota—especially for PaPDB—suggesting selective nanomaterial pressures (Campos et al. 2022).

Differential abundance analyses revealed that both Alum and AgNPs modulated gut microbiota composition, affecting dominant and rare taxa. Changes in Lachnospiraceae, Oscillospiraceae, Muribaculaceae, and Rikenellaceae indicated sensitivity of core microbiota members to adjuvant exposure (Figure 8). PaPDB increased Eggerthellaceae, Atopobiaceae and Coriobacteriaceae families linked to mucin degradation, lipid/steroid metabolism and anaerobic community functions (Just 2017; Ravcheev and Thiele 2017; Watanabe et al. 2021; Little et al. 2024; Viehof et al. 2024)‐ while reducing Lactobacillaceae potentially affecting probiotic functions (Walter and O'Toole 2023). PchPDB increased Mycoplasmataceae and reduced Ruminococcaceae, patterns associated with stress and loss of metabolic contributors (Biagi et al. 2016; Agranyoni et al. 2021; Su et al. 2022), whereas Alum enriched Christensenellaceae and Marinifilaceae, families linked to metabolic regulation (Cheng et al. 2022; Akbuğa‐Schön et al. 2024).

Overall, both Alum and mycosynthesized AgNPs modulated gut microbiota composition even after s.c. administration. Further mechanistic studies on selective taxa enrichment—such as Actinobacteriota‐ together with longitudinal metagenomic and metabolomic analyses will be essential to understand interactions between AgNP‐based adjuvants and microbial communities. It is important to note that, because faecal samples were pooled prior to sequencing, the observed microbiota shifts should be considered exploratory and interpreted with caution. While pooling reduced the influence of inter‐individual variability and facilitated the identification of treatment‐associated community‐level patterns, it also precluded the assessment of animal‐to‐animal variation.

5. Conclusions

In summary, the herein reported results strongly support mycosynthesized AgNPs as novel biotechnological platforms for the development of safe and functionally diverse vaccine adjuvants. Our findings showed that mycosynthesized AgNPs display interesting dose‐dependent immunoenhancement activities while exhibiting outstanding functional versatilities regarding IgG subclass polarization. Moreover, selected AgNPs were shown to exert distal effects on gut microbiota composition, both in a similar way to classical Alum as well as following AgNPs‐specific patterns; highlighting the importance of considering host‐microbiota interactions during the assessment of novel nanomaterial‐based therapies. Our results constitute an important contribution to the growing field of microbiota research at the cross‐intersection with translational immunology and applied nanotechnology.

Author Contributions

Diego M. Roldán: investigation, methodology, writing – original draft, writing – review and editing. Ana Clara Muniz‐Lagos: investigation, methodology, writing – original draft, writing – review and editing. Gustavo Mourglia‐Ettlin: writing – review and editing, resources, supervision, writing – original draft, conceptualization, project administration. Paula Arbildi: investigation, methodology, writing – review and editing. Silvana Alborés: conceptualization, writing – original draft, writing – review and editing, project administration, supervision, resources.

Funding

This research was supported by scholarship Agencia Nacional de Investigación e Innovación (ANII, Uruguay), grant code POS NAC 2022 1 173738 and of CSIC‐ Iniciación a la Investigación (Uruguay), grant code 22420230100070UD. The authors also acknowledge general financial support from Universidad de la República and Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) (Uruguay).

Disclosure

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: Mycosynthesis of AgNPs.

Data S2: Characterization of AgNPs.

Data S3: In Vitro Biocompatibility Assays.

Data S4: In vivo Acute Toxicity.

Data S5: Evaluation of the adjuvant activity of AgNPs.

Data S6: Determination of specific antibodies.

Data S7: Fecal microbiota analysis.

Figure S1: In vivo biocompatibility of PchPDB and PaPDB. Acute toxicity assays were performed in mice (n = 5–6/group) after a single sub‐cutaneous 200× dose of AgNPs (4.0 × 1011 NPs/kg). Mice were daily weighted and variations to Day 0 are depicted as % values (mean ± SEM). All mice were euthanized at Day 14, and the weight of vital organs (livers, kidneys and spleens) are shown as box‐and‐whiskers. Statistical significance respect to the control group (saline) was assessed for each AgNPs using the Mann–Whitney test (*p < 0.05). For weight gaining curves, individual AUC values were compared.

Figure S2: Bacterial diversity based on 16S rRNA gene analysis in faecal samples of mice across different experimental conditions. Heatmap showing percentages of relative abundance of the 10 phyla present in the samples under different experimental conditions (n = 3/group: Alum, PaPDB, PchPDB and Saline).

Table S1: Sample names and corresponding experimental groups for faecal microbiome sequencing in mice. DNA pool of each sample was obtained from two different mice.

MBT2-19-e70411-s001.docx (5.1MB, docx)

Contributor Information

Gustavo Mourglia‐Ettlin, Email: gmourglia@higiene.edu.uy.

Silvana Alborés, Email: salbores@fq.edu.uy.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1: Mycosynthesis of AgNPs.

Data S2: Characterization of AgNPs.

Data S3: In Vitro Biocompatibility Assays.

Data S4: In vivo Acute Toxicity.

Data S5: Evaluation of the adjuvant activity of AgNPs.

Data S6: Determination of specific antibodies.

Data S7: Fecal microbiota analysis.

Figure S1: In vivo biocompatibility of PchPDB and PaPDB. Acute toxicity assays were performed in mice (n = 5–6/group) after a single sub‐cutaneous 200× dose of AgNPs (4.0 × 1011 NPs/kg). Mice were daily weighted and variations to Day 0 are depicted as % values (mean ± SEM). All mice were euthanized at Day 14, and the weight of vital organs (livers, kidneys and spleens) are shown as box‐and‐whiskers. Statistical significance respect to the control group (saline) was assessed for each AgNPs using the Mann–Whitney test (*p < 0.05). For weight gaining curves, individual AUC values were compared.

Figure S2: Bacterial diversity based on 16S rRNA gene analysis in faecal samples of mice across different experimental conditions. Heatmap showing percentages of relative abundance of the 10 phyla present in the samples under different experimental conditions (n = 3/group: Alum, PaPDB, PchPDB and Saline).

Table S1: Sample names and corresponding experimental groups for faecal microbiome sequencing in mice. DNA pool of each sample was obtained from two different mice.

MBT2-19-e70411-s001.docx (5.1MB, docx)

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Articles from Microbial Biotechnology are provided here courtesy of Wiley

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