Green‑Synthesized Protein‑Coated Gold Nanoparticles from Tricholoma crassum: Antimicrobial, Apoptotic, and Gene‑Delivery Potentials

Abstract

We report the first green synthesis of protein‑coated gold nanoparticles (AuNPs) using the edible mycorrhizal fungus Tricholoma crassum. The AuNPs, 5–25 nm in diameter and of various shapes, form within one hour at 28 °C. UV–vis spectroscopy shows a characteristic SPR peak at 552 nm, shifting with reaction time and pH. Detailed characterization by SEM, TEM, AFM, XRD, and DLS confirms monodispersity and a natural 40 kDa protein corona. The particles exhibit potent antimicrobial activity against Gram‑negative, Gram‑positive, and fungal pathogens, including multi‑drug‑resistant strains, and inhibit spore germination. Comet assays reveal dose‑dependent apoptotic induction in plant cells, while the 40 kDa protein coat facilitates efficient gene delivery to Sarcoma 180 cells, evidenced by GFP expression. Hemolysis tests show negligible blood toxicity below 20 µl mL⁻¹, indicating a safe therapeutic window. Large‑scale production in fermenters is feasible, positioning these AuNPs as versatile agents for antimicrobial, anticancer, and gene‑delivery applications.

Background

The rapid adoption of nanoparticles in agriculture, medicine, and consumer products has heightened the need to understand their safety profile. Green synthesis, which employs living organisms or their enzymes, offers an eco‑friendly, cost‑effective route that eliminates hazardous chemicals. Filamentous fungi, with their high enzyme secretion, are particularly adept at producing extracellular metal nanoparticles. While several fungi have yielded gold nanoparticles (AuNPs), few reports describe natural protein coats on these particles, which are critical for colloidal stability and functionalization. Gold nanoparticles are attractive for therapeutic use because they do not elicit resistance and can be engineered to target tumor cells via the enhanced permeation and retention (EPR) effect. Protein‑capped AuNPs enhance cellular uptake and provide docking sites for drugs or genes, obviating the need for additional chemical capping steps. Nonetheless, biocompatibility, cytotoxicity, and aggregation risks must be carefully evaluated before clinical application.

Methods

Fungi, Bacteria, and Plant Growth Conditions

Tricholoma crassum was cultivated for nanoparticle synthesis. Antimicrobial tests used E. coli (DH5α), A. tumefaciens (LBA4404), and their multi‑drug‑resistant derivatives, as well as the plant pathogens M. oryzae and A. solani. Tomato and tobacco seedlings were grown under controlled conditions (28 ± 1 °C, 16:8 h light/dark, 50 µmol m⁻² s⁻¹).

Synthesis of AuNPs

Mycelial mats were incubated in potato dextrose broth (PDB) for 7 days at 28 °C. After 24, 48, or 72 h, 1 g of mycelium was shaken with 10 mL deionized water for 24 h at 50 RPM. The filtrate (pH 5.2) was mixed with 1 mM HAuCl₄ and stirred at 28 °C in the dark for 1 h. By adjusting the filtrate pH (3.5–9) and reaction temperature (0–100 °C), we tuned particle size and shape. Variations in filtrate volume (×0.5–×2) and Au³⁺ concentration (0.5–2 mM) further refined the synthesis.

Characterization Techniques

• UV–Vis spectroscopy (450–750 nm) for SPR analysis.
• SEM, TEM, and AFM for morphology and size distribution.
• XRD for crystalline phase confirmation.
• DLS for hydrodynamic size.
• SDS‑PAGE for protein corona identification.

Antimicrobial and Antifungal Assays

Disc diffusion and growth‑curve assays quantified inhibition against bacterial and fungal strains. Fungal spore germination was assessed with varying AuNP concentrations. The method involved standard incubation and microscopic observation.

Apoptosis Assessment

Comet assays on tobacco and tomato leaf nuclei measured DNA fragmentation after AuNP exposure. Different concentrations (0–30 % v/v) and incubation times (15–30 min) were tested.

Gene Delivery into Sarcoma 180 Cells

Plasmid pCAMBIA1302 (GFP marker) was complexed with AuNPs and incubated with Sarcoma 180 cells. GFP expression confirmed successful transfection. Hemolysis assays with human erythrocytes evaluated blood compatibility.

Results and Discussion

AuNP Formation and Spectroscopic Features

The transition from pale yellow to violet within 1 h indicates AuNP formation. UV–Vis spectra show a SPR peak at 552 nm for 24‑h filtrate, shifting redward with longer incubation and blueward with higher pH. Optimal synthesis occurs at 28 °C with 1 mM HAuCl₄, yielding monodisperse particles stable for >30 days.

Effect of Synthesis Parameters

Increasing filtrate volume or Au³⁺ concentration elevates color intensity and absorbance, with a slight blue shift at ×2 filtrate. Higher temperatures (75–100 °C) produce larger, less stable particles. Light exposure enhances particle size and broadens the SPR peak.

Morphology and Size Distribution

SEM and TEM reveal 5–22 nm AuNPs in shapes ranging from spheres and rhomboids to hexagons and triangles. AFM confirms sub‑10 nm heights, and XRD confirms face‑centered cubic gold with dominant {111} diffraction at 38.23°.

Antimicrobial Activity

AuNPs inhibited growth of E. coli, A. tumefaciens, and M. oryzae with dose‑dependent inhibition zones. Multi‑drug‑resistant strains were also suppressed, indicating broad‑spectrum efficacy. Growth‑curve analyses showed delayed log phase and reduced cell density upon AuNP treatment.

Fungal Spore Germination

Increasing AuNP concentrations progressively reduced spore germination and germ‑tube length of A. solani, demonstrating antifungal potency.

Apoptogenic Properties

Comet assays revealed significant DNA damage at >20 % AuNP exposure, while 5–10 % caused negligible apoptosis. Thus, a therapeutic window exists for antimicrobial or gene‑delivery applications without cytotoxicity.

Protein Corona Identification

SDS‑PAGE of boiled nanoparticles shows a single 40 kDa band identical to the extracellular protein from T. crassum. The protein coat stabilizes particles and provides binding sites for DNA.

Gene Delivery and Hemolysis

AuNP–plasmid complexes achieved GFP expression in Sarcoma 180 cells, whereas naked plasmid did not. Hemolysis remained below 8 % at concentrations up to 20 µl mL⁻¹, confirming low blood toxicity.

Conclusions

Edible mycorrhizal fungus Tricholoma crassum enables rapid, green synthesis of protein‑coated AuNPs with diverse shapes and robust antimicrobial, apoptotic, and gene‑delivery properties. The natural 40 kDa protein corona ensures colloidal stability and functionalization, while hemolysis data support safe therapeutic use. Large‑scale production in fermenters is feasible, offering a scalable platform for antimicrobial, anticancer, and nanomedicine applications.