Green synthesis of nanoparticles with extracellular and intracellular extracts of basidiomycetes

Fungi and culture conditions

Pleurotus ostreatus (Fr.) Kumm. HK-35 (oyster mushroom), Lentinus edodes (Berk.) Sing F-249 (shiitake), Grifola frondosa (Fr.) S.F. Gray 0917 (maitake), and Ganoderma lucidum (Curtis: Fr.) P. Karst 1315 (reishi) were obtained from the Basidiomycetes Culture Collection of Komarov Botanical Institute Russian Academy of Sciences, and from the Collection of Higher Basidial Fungi, Department of Mycology and Algology, Lomonosov Moscow State University. The fungi were maintained on beer-wort agar plates at 4 °C in the higher fungi collection of the Laboratory of Microbiology, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences.

The fungi were grown submerged in a synthetic medium of the following composition (g/L): d-glucose, 1; l-asparagine, 0.1; KH₂PO₄, 2; K₂HPO₄, 3; MgSO₄ × 7H₂O, 2.5; FeSO₄ × 7H₂O, 0.03 (PH 5.8). Growth was at 26 °C for 21 days in 100-mL flasks containing 50 mL of the medium. Morphogenetic structures were obtained by growing the fungi on a wood substrate in the laboratory under conditions closest to natural (Vetchinkina & Nikitina, 2007). The structures used were nonpigmented mycelium (NM), brown mycelial mat (BMM), primordia (PR), and fruiting bodies (FB).

Conditions for preparation of extracts

The culture liquid was separated from the mycelium by centrifugation and was filtered. For intracellular extracts, mycelia or individual morphological structures were separated from the culture medium, rinsed in distilled water, and mechanically ground at 18 °C in a porcelain mortar with quartz sand to destroy the cell envelope. Next, they were extracted with 20 mM Na/K-phosphate buffer (pH 6.0), centrifuged at 12,000 g for 20 min, and filtered. The supernatant liquids were dialyzed against water and were used in the experiments.

For bioreduction studies, the filtrates of the culture liquids and the aqueous intracellular extracts were incubated in the dark at room temperature with aqueous solutions of silver nitrate (AgNO₃, ≥99.0%), sodium selenite (Na₂SeO₃, ≥98.0%), sodium silicate (Na₂SiO₃, ≥98.0%) at 0.5 mM, and chloroauric acid (HAuCl₄, ASC reagent) at 50 µM final concentrations, respectively. All chemicals were from Sigma-Aldrich, St. Louis, MO, USA. Previously, we selected optimal concentrations of these compounds, at which intensive formation of stable nanoparticle colloids took place (Vetchinkina et al., 2017). Solutions of the compounds were added to each sample under sterile conditions. The incubation time needed for nanoparticles formation varied from several minutes to several hours, depending on the used compound and extract.

Phenol oxidase activity measurements

Enzyme activities were measured with a Specord M40 spectrophotometer (Carl Zeiss, Jena, Germany) in 1-cm path-length quartz cuvettes at 18 °C. Laccase activity was measured at 436 nm by the oxidation rate for 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Sigma-Aldrich, USA) (Slomczynski, Nakas & Tanenbaum, 1995). Tyrosinase activity was measured at 475 nm by the oxidation rate for 3-(3,4-dihydroxyphenyl)-l-alanine (l-DOPA; Serva, Germany) (Pomerantz & Murthy, 1974). Mn-peroxidase activity was measured at 468 nm by the oxidation rate for 2,6-dimethoxyphenol (DMOP; Sigma) (Paszczynski, Crawford & Huynh, 1988). Protein was estimated by the Bradford method (Bradford, 1976).

X-ray fluorescence and X-ray diffraction analysis

The biologically formed nanoparticles were filtered through a Millipore membrane filter (pore size, 0.45 µm), resuspended in minimal distilled water, and air dried at 20 °C. The contents of Au, Ag, Se, and Si were analyzed with an ED 2000 energy-dispersive spectrometer (Oxford Instruments, Abingdon, UK) by using the basic parameters method included in the instrument’s software. The oxidation states of the elements were examined by X-ray diffraction analysis of nanoparticle suspensions. The analysis was conducted with a DRON-3.0 diffractometer (CuKα irradiation; wavelength, 1.54173 Å), by using the JCPDS powder diffraction database (USA 1987).

Transmission electron microscopy (TEM) and selected-area electron diffraction (SAED) analysis

All nanoparticles were examined by negative-contrasting TEM. To this end, the material was mounted on nickel grids coated with 1% formvar in dichloroethane. A Libra 120 electron microscope (Carl Zeiss, Germany) operating at 80 keV was used to take photomicrographs. The size, shape, and relative number of electron-dense nanoparticles were evaluated from the TEM images. For SAED analysis, samples were mounted on nickel grids coated with 1% formvar in dichloroethane. The analysis was conducted with a Libra 120 TEM instrument operated at 120 keV (camera length, 360 mm). Both analyses were done at the Simbioz Center for the Collective Use of Research Equipment in the Field of Physical–Chemical Biology and Nanobiotechnology at the Russian Academy of Sciences’ Institute of Biochemistry and Physiology of Plants and Microorganisms.

UV spectroscopy

This was used to examine the optical properties of the nanoparticles. Absorption spectra were measured with a Specord 250 UV–vis spectrophotometer (Analytik Jena, Germany) at 190–1100 nm.

Zeta-potential measurements

The ζ-potential of the nanoparticles was measured with a Zetasizer Nano ZS instrument (Malvern, UK).

Assay for cytotoxicity of Au and Ag nanoparticles

The cytotoxicity of the Au and Ag nanoparticles was evaluated with a standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Niks & Otto, 1990), with minor modifications. HeLa (human breast cancer) cells and Vero (kidney epithelial cells extracted from an African green monkey) cells were obtained from the Institute of Cytology, Russian Academy of Sciences, and were grown in complete Dulbecco’s modified Eagle’s medium (DMEM; Biolot, Russia) containing 10% fetal bovine serum (Biolot), 100 µg/mL of penicillin, and 100 µg/mL of streptomycin (both from Sigma-Aldrich, USA). Au and Ag nanoparticles were formed by 30-min incubation of HAuCl₄ or AgNO₃ with L. edodes extracellular extracts at 25 °C. The suspension was centrifuged for 15 min at 15,000 g and the pellet was resuspended in DMEM to a final nanoparticle concentration of 100 µg/mL.

The cells were grown in 25 cm² tissue culture flasks in a water-jacketed incubator at 37 °C in a humidified atmosphere of 5% CO₂. Twenty-four hours before the assay, the cells were seeded in 96-well plates (about 20,000 cells per well). Then, the cell medium was refreshed and double dilutions of the nanoparticle solutions (1:10, v/v) were added to each well (starting concentration, 100 µg/mL). The controls were cells grown without nanoparticles. After incubation for 24 and 48 h, the cell medium was removed, and 100 µL of 5 mg/mL of MTT (Sigma-Aldrich) in 0.01 M phosphate-buffered saline (PBS, pH 7.4; Biolot) was added to each well. The plates were incubated at 37 °C for 1 h in the dark in a humidified atmosphere of 5% CO₂. After that, the supernatant liquid was decanted, 100 µL of dimethyl sulfoxide (C₂H₆OS; Reachim, Russia) was added to each well, and the plates were incubated at 37 °C for 15 min to resuspend the precipitate of formazan. Absorbance was read at 490 nm with a Spark 10M microplate spectrophotometer (Tecan, Austria). The respiratory activity of the cells grown in a particle-free medium was taken as 100%. Each experiment was at least triplicated, and the standard deviation was used to estimate the error. Before and after the nanoparticles were added, the cell monolayer was directly observed in the bright-field mode by using a Leica DMI3000 B inverted microscope equipped with a Leica 420 D CCD camera (Leica Microsystems, Germany; provided by the Simbioz Center for the Collective Use of Research Equipment in the Field of Physical–Chemical Biology and Nanobiotechnology at the Russian Academy of Sciences’ Institute of Biochemistry and Physiology of Plants and Microorganisms).

Statistics

There were five independent experiments, each having no less than five replications. Data were processed with Excel software (Microsoft Corp., Redmond, WA, USA).

Characterization of Au and Ag nanoparticles reduced with intra- and extracellular extracts

TEM showed that depending on the extract type and on the compound reduced, the nanoparticles made by different fungi differed widely in shapes and sizes. The reduction of HAuCl₄ with extracellular extracts yielded sufficiently homogeneous, mostly spherical particles of 2 to 20 nm diameter (Figs. 1A–1D). With G. lucidum and G. frondosa, the particles were larger, less homogeneous, and often stuck together in aggregates (Figs. 1C and 1D). The Au particles obtained with intracellular extracts were sevenfold larger than those obtained with extracellular extracts. Some particles, of hexagonal, tetragonal, and triangular shape, were even larger (50–100 nm; Figs. 1E–1H). The synthesized Au nanoparticles had a plasmon resonance peak at 550 nm. The ζ-potentials of the colloidal Au ranged from −15 to −27 mV. The colloids of Au nanoparticles were stable (except that of G. lucidum), and the particles did not aggregate or flocculate for 90 days at 4 °C.

In the case of reduction of AgNO₃ with extracellular extracts, the particles formed were large, irregular, and aggregated (Figs. 2A–2D). The ζ-potentials of the colloidal Ag solutions ranged from –9 to –12 mV. With mycelial extracts, very small homogeneous particles formed that had diameters of 1 to 5 nm (Figs. 2E–2H). The ζ-potential of these particles was 20 mV. The colloids were stable, and the particles did not aggregate or flocculate for 30 days at 4 °C. UV spectroscopy of a suspension of Ag nanoparticles in the culture liquid showed a major absorption peak at 370 nm.

Effect of phenol oxidase activity of extracts on biodegradation of metals and metalloid compounds

The extracts were examined for the activity of Mn-peroxidases, laccases, and tyrosinases. The activity of L. edodes Mn-peroxidases in the extracellular extracts was fivefold higher than that in the extracts of G. lucidum and was sevenfold higher than that in P. ostreatus (Fig. 3A). Total laccase activity in L. edodes was 4.5-fold greater than that in P. ostreatus and was 3–3.5-fold greater than that in G. lucidum and in G. frondosa. Tyrosinase activity in submerged fungal cultures was relatively low, but it was somewhat higher in L. edodes than in the other fungi. The intracellular extracts of the submerged cultures gave a similar picture, and the most active enzymes were those of L. edodes (Fig. 3B). The ability to reduce HAuCl₄ and AgNO₃ to Au and Ag was directly proportional to enzyme activity. The biofabrication of Se and Si nanoparticles did not depend on phenol oxidase activity.

Characterization of Se and Si nanoparticles obtained with intra- and extracellular extracts

The Se particles formed with the fungal extracts had a more or less regular spherical shape (Fig. 4). In L. edodes (Figs. 4A and 4E) and in P. ostreatus (Figs. 4B and 4F), the differences between the use of extracellular and intracellular extracts were small and the particle diameter ranged from 50 to 150 nm. Intracellular extracts of G. lucidum (Fig. 4G) and G. frondosa (Fig. 4H) yielded larger-diameter particles (about 200–300 nm), and their extracellular extracts yielded nanospheres of 20 to 50 nm, often aggregated (Figs. 4C and 4D). The synthesized Se nanoparticles had a plasmon resonance peak at 350 nm. The ζ-potentials of the colloidal Se ranged from −12 to −18 mV.

Si nanoparticles were detected when Na₂SiO₃ was incubated with extracellular extracts (Fig. 5). In L. edodes and in G. lucidum, the particles were larger and did not aggregate (Figs. 5A and 5B), whereas in P. ostreatus and in G. frondosa, they were very small and stuck together in aggregates (Figs. 5C and 5D). The ζ-potentials of the colloidal Si ranged from −10 to −15 mV.

X-ray fluorescence and X-ray diffraction analysis of nanoparticles

To confirm that the resultant nanoparticles were indeed Au, Ag, Se, and Si, we examined them by X-ray fluorescence. The spectra showed intense emission lines typical of these elements. The presence of Au was determined by the lines at 9.713 (Lα1), 11.443 (Lβ1), 11.585 (Lβ2), 10.308 (Ln), and 13.382 (Ly1) keV (Fig. 6A); the presence of Ag, by the lines at 22 (Lα1) and 25 (Lβ1) keV (Fig. 6B); the presence of Se, by the lines at 11.22 (Lα1) and 12.49 (Lβ1) keV (Fig. 6C); and the presence of Si, by the line at 1.75 (Lα1) keV (Fig. 6D). The elements were detected in all the fungi tested.

The oxidation states of the Au, Ag, Se, and Si in nanoparticle suspensions were examined by X-ray diffraction analysis. The metals were fully reduced to the elementary state, and there were no signals from either HAuCl₄ or AgNO₃ (Figs. 7A and 7B). There were four signals belonging to the major reflex of Au (interplanar distances, 1.230, 1.441, 2.030, and 2.350 Å  [4–784]) and four signals corresponding to the major reflex of Ag (interplanar distances, 1.232, 1.446, 2.049, and 2.356 Å [4–783]). Also found were seven minor signals belonging to the major reflex of Se (1.768, 1.993, 2.014, 2.060, 2.171, 2.990, and 3.750 Å  [6–0362]) and two signals from SeO₂ (2.529 and 4.171 Å [22–1314]). In the samples with Si nanoparticles, there were minor signals belonging to two silicon structures, one with three reflexes (1.630, 1.918, and 3.140 Å [5–565]) and the other with two reflexes (1.957 and 2.280 Å [35–1158]). Also present were five signals that were assigned with high confidence to the major reflex of quartz SiO₂ (2.228, 2.280, 2.460, 3.351, and 4.230 Å [5-0490]).

TEM and SAED analysis

Diffraction patterns of the nanoparticles were obtained by TEM and by SAED analysis. Figure 8 shows the ring diffraction patterns from pure Au (Fig. 8A) and Ag (Fig. 8B) particles with crystalline structure. The Se particles were shown by SAED analysis to be noncrystalline (Fig. 8C). SAED analysis of the Si structures by using individual particle ensembles showed diffused ring patterns typical of amorphous materials, in line with the amorphous nature of the formed Si superstructures (Fig. 8D). There also were diffraction signals from crystalline Si, indicating that Na₂SiO₃ transformation was accompanied by the synthesis of Si nanoparticles with crystalline structure (Figs. 8E, 8F).

Nanoparticle synthesis with intracellular extracts from different stages of fungal development

With extracts of nonpigmented mycelia, the synthesized Au particles had an irregular spherical shape and a size of 2–50 nm. Also formed were a small number of nanotriangles (50–80 nm) and hexagons (20–90 nm) (Fig. 9A). With pigmented mycelia, 10–40-nm spherical particles and many triangles (up to 100 nm in size) were formed (Fig. 9B). In extracts of brown mycelial mats, we found homogeneous, mostly spherical particles of 5 to 10 nm in size (Fig. 9C). Extracts of primordia reduced HAuCl₄ to irregular particles about 10–30 nm in diameter, most of which were aggregated (Fig. 9D).

Nanoparticles of Ag were smaller and were homogeneous (Figs. 9E–9H). Extracts of nonpigmented mycelia and brown mycelial mats produced a stable suspension of 1 to 10-nm particles (Figs. 9E and 9G). By contrast, the particles formed with extracts of pigmented mycelia were larger (5–20 nm) (Fig. 9F). A slightly different picture was observed for extracts of L. edodes primordia; alongside small, homogeneous nanoparticles (2–5 nm), there were many 10 to 20-nm particles stuck together in aggregates (Fig. 9H). With extracts of L. edodes fruiting bodies, there were many small spherical nanoparticles of 10 to 15 nm (Fig. 10A). With G. lucidum, the particles were large (50–100 nm) and mostly cubic and rectangular (Fig. 10B). Incubation of HAuCl₄ with fruiting body extracts of L. edodes yielded inhomogeneous particles, among them many large irregular spheres of up to 170 nm, some triangles of 60–90 nm, and some irregular spheres of 15 to 50 nm (Fig. 10C). Extracts from G. lucidum synthesized mostly spheres of 30–50 nm (Fig. 10D).

Assay for cytotoxicity of nanoparticles

The toxicity of Au and Ag nanoparticles to HeLa and Vero cells was evaluated with a standard MTT assay. The average decrease in cell respiratory activity of both Vero (Figs. 11A, 11B) and HeLa (Figs. 12A, 12B) cells, as compared to the control, was about 20% within 24 and 48 h with all Au particle concentrations tested (1–100 µg/mL). This indicated that the cell toxicity had a non-dose–response character. Ag nanoparticles were strongly toxic at as low as 3.75 µg/mL (HeLa) and 8.5 µg/mL (Vero) after being incubated with the cells for 24 and 48 h (Figs. 11 and 12). The presence of unattached dead cells indicated partly or totally disrupted cell monolayer (Figs. 11C–11H and Figs. 12C–12H).