Biological Synthesis of Low Cytotoxicity Silver Nanoparticles (AgNPs) by the Fungus Chaetomium thermophilum—Sustainable Nanotechnology

Fungal biotechnology research has rapidly increased as a result of the growing awareness of sustainable development and the pressing need to explore eco-friendly options. In the nanotechnology field, silver nanoparticles (AgNPs) are currently being studied for application in cancer therapy, tumour detection, drug delivery, and elsewhere. Therefore, synthesising nanoparticles (NPs) with low toxicity has become essential in the biomedical area. The fungus Chaetomium thermophilum (C. thermophilum) was here investigated—to the best of our knowledge, for the first time—for application in the production of AgNPs. Transmission electronic microscopy (TEM) images demonstrated a spherical AgNP shape, with an average size of 8.93 nm. Energy-dispersive X-ray spectrometry (EDX) confirmed the presence of elemental silver. A neutral red uptake (NRU) test evaluated the cytotoxicity of the AgNPs at different inhibitory concentrations (ICs). A half-maximal concentration (IC50 = 119.69 µg/mL) was used to predict a half-maximal lethal dose (LD50 = 624.31 mg/kg), indicating a Global Harmonized System of Classification and Labelling of Chemicals (GHS) acute toxicity estimate (ATE) classification category of 4. The fungus extract showed a non-toxic profile at the IC tested. Additionally, the interaction between the AgNPs and the Balb/c 3T3 NIH cells at an ultrastructural level resulted in preserved cells structures at non-toxic concentrations (IC20 = 91.77 µg/mL), demonstrating their potential as sustainable substitutes for physical and chemically made AgNPs. Nonetheless, at the IC50, the cytoplasm of the cells was damaged and mitochondrial morphological alteration was evident. This fact highlights the fact that dose-dependent phenomena are involved, as well as emphasising the importance of investigating NPs’ effects on mitochondria, as disruption to this organelle can impact health.


Introduction
Over-exploitation of natural resources and exponential human growth are the roots of modern social concerns [1]. In this context, sustainable consumption and production patterns, extensively described by the United Nations in the Sustainable Development Agenda as Goal 12, have inevitably become needed [2]; hence, the growing attention given Chemicals (GHS) category of 4 (300 mg/kg to 2000 mg/kg). Furthermore, preserved cell structures were observed following the interaction of AgNPs and Balb/c 3T3 NIH cells at an inhibitory concentration (IC) of IC 20 and with an evident mitochondrial morphological alteration at IC 50 . These results highlight the applicability of the fungi system as a source of bio-nanomaterials with low cytotoxicity, low cost, and less impact on the environment that may eventually lead to sustainable development in the green nanotechnology field.

Biological Synthesis and Characterisation of AgNPs 2.2.1. AgNP Synthesis
The AgNP synthesis activity level of the C. thermophilum cell-free extract was tested following the study by Alves and Murray [3]. Briefly, the cell-free extract was heated for 15 min at 90 • C; then, 127.40 mg/L silver nitrate (AgNO 3 , Sigma-Aldrich, St. Louis, MO, USA) was added for a total volume of 500 µL, and heating was continued for one hour at 90 • C. In the same reaction conditions, a reaction synthesis control was produced using ultrapure water and AgNO 3 .

AgNP Characterization
The resulting AgNPs were characterised based following the study by Alves and Murray [3]. Ultraviolet-visible spectrophotometry (UV-Vis, BioTek Synergy 4 Microplate Reader, Bad Friedrichshall, Germany) was first used to measure the localised surface plasmon resonance (LSPR) absorbance. AgNPs were scanned between 300 nm and 1000 nm, with 2 nm steps. Afterwards, transmission electron microscopy (TEM, JEOL JEM 2100 Field Emission Electron Microscope, Tokyo, Japan) was used to evaluate the shape and size distribution of the AgNPs. Samples were drop-coated onto Formvar carbon-coated copper grids with a 200 µm mesh size and dried over 24 h. Images were obtained using a JEOL JEM 2100 Field Emission Electron Microscope operated at 200 kV with a field emission electron gun equipped with a Gatan Ultrascan digital camera. An average of 200 NPs was recorded from several TEM images, with AxioVision Rel 4.8 software used to evaluate size distribution. Furthermore, the elemental composition was analysed using energy dispersive X-ray spectrometry (EDX, Hitachi 3000, Tokyo, Japan). Five microliters of the samples was drop-coated onto polished aluminium slides and dried in the oven at 60 • C for 1 h, thrice. A Hitachi 3000 electron microscope with EDX capability at 15 kV accelerating voltage and a working distance of 2 mm was used to obtain the ED spectra of the samples.

Cytotoxicity Evaluation Profile in Mammalian Cell
The cytotoxicity evaluation of the C. thermophilum AgNPs and of the C. thermophilum cell-free extract was based on the Organisation for Economic Co-operation and Development (OECD) guidelines from the Environment, Health and Safety Publications Series on Testing and Assessment No. 129 guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic toxicity tests [20]. In brief, the cells were plated into the inner wells of 96-well tissue culture microtiter plates at a density of 2.5 × 10 3 cells (100 µL/well); the outer wells were filled with culture medium and then cultivated for 24 h at 37 • C, 90% humidity, and 5.0% CO 2 /air. After 24 h of incubation, the culture medium was removed. The AgNPs were diluted immediately prior to use with a solution of 4.5 g/L DMEM, 4 mM L-Glutamine (Gibco Invitrogen, Carlsbad, CA, USA), 100 IU/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), according to ICCVAM recommendations [31]. Eight serial dilutions of AgNPs ranging from 31.03 µg/mL to 460.32 µg/mL were prepared. For the AgNP serial dilution, the log-factor of 1.47 was used. Eight serial dilutions of the C. thermophilum cell-free extract ranging from 0.38 µg/mL to 1200 µg/mL were also prepared, with a serial dilution log factor of 3.16. Plates were incubated for 48 h at 37 • C, 90% humidity, and 5.0% CO 2 /air. After this period, Balb/c 3T3 NIH cells were stained with a neutral red (NR) medium composed of 25 µg/mL NR dye (Sigma-Aldrich, St. Louis, MO, USA). NR was extracted from cells using 250 µL/well of NR desorb solution (freshly prepared with 49 parts water, 50 parts ethanol, and 1 part glacial acetic acid, all bought from Sigma-Aldrich, St. Louis, MO, USA) over a period of 20 min in a shaker protected from light [32,33]. The optical density of the samples was measured at a wavelength of 540 nm using a Multi-Modo Synergy H1 (Biotek, Winooski, Vermont, EUA) spectrophotometer reader.

Evaluation of Balb/c 3T3 NIH Cells and AgNP Interaction by TEM
Balb/c 3T3 NIH were plated into 6-well tissue culture microtiter plates at a density of 7.5 × 104 cells/well in 3 mL of the routine medium and cultivated for 24 h at 37 • C, 90% humidity, and 5.0% CO 2 /air. After this period, the routine medium was discharged and 1.5 mL of a fresh one was added. The cells were submitted to three treatments: a control (cells without AgNPs), a non-toxic concentration (IC 20  After washing, the samples were dehydrated using a graded acetone series (30%, 50%, 70%, 90%, and 100%) and embedded using EMBed 812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections of each sample were obtained using a Leica EM UC6 ultramicrotome (Leica, Wetzlar, Germany). The samples were contrast-stained with 5% uranyl acetate (Sigma-Aldrich, St. Louis, MO, USA) for 30 min and with lead citrate (Sigma-Aldrich, St. Louis, MO, USA) for 5 min, then analysed using a JEOL JEM1400-Plus TEM (JEOL, Tokyo, Japan) [30,33].

Statistical Analysis
All assays were run in triplicate. Data were expressed as means ± standard deviation (St. Dev). The software packages used to analyse the data generated in the characterisation process were Gen5, Microsoft Office Excel, and Quantax 70 Microanalysis. Cell viability based on optical density data was analysed in Microsoft Office Excel. Outliers were analysed with the statistics Grubbs test (available online at www.graphpad.com/ quickcalcs/Grubbs1.cfm, accessed on 12 June 2021). GraphPad Prism ® 5.0 was used to create a sigmoidal dose-response (variable slope) with four parameters, rearranged in the Hill function. IC 20 , IC 50 , and IC 80 were expressed graphically with mean and standard deviation. IC 50 was used to predict LD 50 using the formula: log LD 50 (mg/kg) = 0.372 log IC 50 (µg/mL) + 2.024 (R 2 = 0.325) [30][31][32][33]. Once the LD 50 was predicted, it was possible to classify the AgNPs according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification [34]. The acceptance criteria of the assay followed ICCVAM guidelines [31]. Sodium dodecyl sulphate (SDS, Sigma-Aldrich, St. Louis, MO, USA) was used as the control drug, and the acceptance criteria of the assay also followed the ICCVAM guidelines [31,35].

Production of Chaetomium thermophilum Cell-Free Extract
The fungus C. thermophilum grows with septate hyphae that develop initially with a lightly hyaline colour (glass-like) and change over time to olivaceous (brownish olive) with thicker septate walls [36]. Photos of the fungus growth in CTMA plates and of the cell-free extract were taken, as shown in Figure 1A,B, and the mycelia cells were analysed using a microscope (VWR, Dublin, Ireland) with 100× magnification ( Figure 1C).

Statistical Analysis
All assays were run in triplicate. Data were expressed as means ± standard deviation (St. Dev). The software packages used to analyse the data generated in the characterisation process were Gen5, Microsoft Office Excel, and Quantax 70 Microanalysis. Cell viability based on optical density data was analysed in Microsoft Office Excel. Outliers were analysed with the statistics Grubbs test (available online at www.graphpad.com/quickcalcs/Grubbs1.cfm, accessed on 12 June 2021). GraphPad Prism ® 5.0 was used to create a sigmoidal dose-response (variable slope) with four parameters, rearranged in the Hill function. IC20, IC50, and IC80 were expressed graphically with mean and standard deviation. IC50 was used to predict LD50 using the formula: log LD50 (mg/kg) = 0.372 log IC50 (μg/mL) + 2.024 (R 2 = 0.325) [30][31][32][33]. Once the LD50 was predicted, it was possible to classify the AgNPs according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification [34]. The acceptance criteria of the assay followed ICCVAM guidelines [31]. Sodium dodecyl sulphate (SDS, Sigma-Aldrich, St. Louis, MO, USA) was used as the control drug, and the acceptance criteria of the assay also followed the ICCVAM guidelines [31,35].

Production of Chaetomium thermophilum Cell-Free Extract
The fungus C. thermophilum grows with septate hyphae that develop initially with a lightly hyaline colour (glass-like) and change over time to olivaceous (brownish olive) with thicker septate walls [36]. Photos of the fungus growth in CTMA plates and of the cell-free extract were taken, as shown in Figure 1A,B, and the mycelia cells were analysed using a microscope (VWR, Dublin, Ireland) with 100× magnification ( Figure 1C).

AgNP Characterisation
The LSPR absorbance in the wavelength region of 380 nm to 435 nm indicates AgNP synthesis. The UV-Vis spectrophotometry spectrum of the AgNPs displayed a maximum wavelength of 405 ± 1.15 nm, with a correspondent maximum absorbance value of 1.250 ± 0.01, demonstrating a different pattern from the spectrum of the synthesis reaction control (Figure 2). The AgNO 3 control solution did not present an absorbance peak in the relevant UV-Vis region.

AgNP Characterisation
The LSPR absorbance in the wavelength region of 380 nm to 435 nm indicates AgNP synthesis. The UV-Vis spectrophotometry spectrum of the AgNPs displayed a maximum wavelength of 405 ± 1.15 nm, with a correspondent maximum absorbance value of 1.250 ± 0.01, demonstrating a different pattern from the spectrum of the synthesis reaction control ( Figure 2). The AgNO3 control solution did not present an absorbance peak in the relevant UV-Vis region.   Size distribution, with the measurement obtained using ImageJ software, with 8.93 ± 2.29 nm average size.

AgNP Characterisation
The LSPR absorbance in the wavelength region of 380 nm to 435 nm indicates AgNP synthesis. The UV-Vis spectrophotometry spectrum of the AgNPs displayed a maximum wavelength of 405 ± 1.15 nm, with a correspondent maximum absorbance value of 1.250 ± 0.01, demonstrating a different pattern from the spectrum of the synthesis reaction control ( Figure 2). The AgNO3 control solution did not present an absorbance peak in the relevant UV-Vis region.   Size distribution, with the measurement obtained using ImageJ software, with 8.93 ± 2.29 nm average size.

NRU Cytotoxicity Evaluation
The cytotoxicity of the AgNPs synthesised using C. thermophilum cell-free extract was evaluated using the NRU following OECD guidelines. AgNP concentrations ranging from 31.03 µg/mL to 460.32 µg/mL (dilution log factor: 1.47) were tested. Dose-response curves were obtained using GraphPad Prism ® and Excel software (Figure 4). The average IC values were as follows: IC 20 = 91.77 ± 24.24 µg/mL, IC 50 = 119.69 ± 21.15 µg/mL, and IC 80 = 144.92 ± 23.22 µg/mL. The IC 50 was used to predict an LD 50 value of 624.31 ± 41.87 mg/kg, suggesting that the AgNPs synthesised might belong to GHS category 4 with regard to inducing acute toxicity ( Figure 5 and Table 1). The cytotoxicity of the AgNPs synthesised using C. thermophilum cell-free extract was evaluated using the NRU following OECD guidelines. AgNP concentrations ranging from 31.03 µ g/mL to 460.32 µ g/mL (dilution log factor: 1.47) were tested. Dose-response curves were obtained using GraphPad Prism ® and Excel software (Figure 4). The average IC values were as follows: IC20 = 91.77 ± 24.24 µ g/mL, IC50 = 119.69 ± 21.15 µ g/mL, and IC80 = 144.92 ± 23.22 µ g/mL. The IC50 was used to predict an LD50 value of 624.31 ± 41.87 mg/kg, suggesting that the AgNPs synthesised might belong to GHS category 4 with regard to inducing acute toxicity ( Figure 5 and Table 1).    The cytotoxicity of the AgNPs synthesised using C. thermophilum cell-free extract was evaluated using the NRU following OECD guidelines. AgNP concentrations ranging from 31.03 µ g/mL to 460.32 µ g/mL (dilution log factor: 1.47) were tested. Dose-response curves were obtained using GraphPad Prism ® and Excel software (Figure 4). The average IC values were as follows: IC20 = 91.77 ± 24.24 µ g/mL, IC50 = 119.69 ± 21.15 µ g/mL, and IC80 = 144.92 ± 23.22 µ g/mL. The IC50 was used to predict an LD50 value of 624.31 ± 41.87 mg/kg, suggesting that the AgNPs synthesised might belong to GHS category 4 with regard to inducing acute toxicity ( Figure 5 and Table 1).     Additionally, the cytotoxicity of the C. thermophilum cell-free extract was analysed to evaluate potential toxicity derived from fungal metabolites. Concentrations ranging from 0.38 µg/mL to 1200 µg/mL (dilution log factor: 3.16) were tested. At 1200 µg/mL, the highest IC tested for the extract (2.5 times higher than the maximum AgNP concentration tested), a maximum of 50% of the cells were killed (Figure 6.). Thus, the IC 50 and, consequently, the LD 50 were not statistically calculated, nor was the GHS acute toxicity classification, as this was based on the LD 50 values. Hence, the extract cytotoxicity was beyond the evaluated cytotoxicity range of this test. Therefore, the C. thermophilum cell-free extract was effectively non-toxic at the IC tested. Additionally, the cytotoxicity of the C. thermophilum cell-free extract was analysed to evaluate potential toxicity derived from fungal metabolites. Concentrations ranging from 0.38 µ g/mL to 1200 µ g/mL (dilution log factor: 3.16) were tested. At 1200 µ g/mL, the highest IC tested for the extract (2.5 times higher than the maximum AgNP concentration tested), a maximum of 50% of the cells were killed (Figure 6.). Thus, the IC50 and, consequently, the LD50 were not statistically calculated, nor was the GHS acute toxicity classification, as this was based on the LD50 values. Hence, the extract cytotoxicity was beyond the evaluated cytotoxicity range of this test. Therefore, the C. thermophilum cell-free extract was effectively non-toxic at the IC tested.

AgNPs-Balb/c 3T3 NIH Cells Interaction
The interaction between the AgNPs and the Balb/c 3T3 NIH cells was evaluated at an ultrastructural level in three different scenarios: (1) a control in which the cells were not exposed to the AgNPs, (2) a non-cytotoxic AgNP concentration (IC20), and (3) the IC50. Compared to the control ( Figure 7A,B), most cells had a preserved ultrastructure at the IC20 ( Figure 7C,D). Hence, no evidence of cell alteration at the ultrastructural level was detected. However, at the IC50 ( Figure 7E,F), the cell cytoplasm was damaged, and mitochondrial morphological alteration was evident.

AgNPs-Balb/c 3T3 NIH Cells Interaction
The interaction between the AgNPs and the Balb/c 3T3 NIH cells was evaluated at an ultrastructural level in three different scenarios: (1) a control in which the cells were not exposed to the AgNPs, (2) a non-cytotoxic AgNP concentration (IC 20 ), and (3) the IC 50 . Compared to the control ( Figure 7A,B), most cells had a preserved ultrastructure at the IC 20 ( Figure 7C,D). Hence, no evidence of cell alteration at the ultrastructural level was detected. However, at the IC 50 ( Figure 7E,F), the cell cytoplasm was damaged, and mitochondrial morphological alteration was evident. (C) Balb/c 3T3 NIH cells exposed to a non-toxic AgNP concentration (IC20 = 91.77 ± 24.24 µg/mL), demonstrating no significant cell damage or alteration at the ultrastructural level. (D) Mitochondria organelle of Balb/c 3T3 NIH cells exposed to a non-toxic AgNP concentration (IC20). (E) Balb/c 3T3 NIH cells exposed to an IC50 (119.69 ± 21.15 µg/mL), showing that the cell cytoplasm was damaged. (F) Mitochondria organelle of Balb/c 3T3 NIH cells exposed to an IC50 with an evident morphological alteration.

Discussion
Research involving silver nanostructures has expanded rapidly due to their promising applications within the biomedicine and biotechnology fields. However, despite their exceptional physicochemical properties, concerns related to environmental toxicity and health-related hazards exist [16,17]. Therefore, efforts have been made in order to develop eco-friendly approaches to synthesising AgNPs. Overall, the advantage of the biological (C) Balb/c 3T3 NIH cells exposed to a non-toxic AgNP concentration (IC 20 = 91.77 ± 24.24 µg/mL), demonstrating no significant cell damage or alteration at the ultrastructural level. (D) Mitochondria organelle of Balb/c 3T3 NIH cells exposed to a non-toxic AgNP concentration (IC 20 ). (E) Balb/c 3T3 NIH cells exposed to an IC 50 (119.69 ± 21.15 µg/mL), showing that the cell cytoplasm was damaged. (F) Mitochondria organelle of Balb/c 3T3 NIH cells exposed to an IC 50 with an evident morphological alteration.

Discussion
Research involving silver nanostructures has expanded rapidly due to their promising applications within the biomedicine and biotechnology fields. However, despite their exceptional physicochemical properties, concerns related to environmental toxicity and health-related hazards exist [16,17]. Therefore, efforts have been made in order to develop eco-friendly approaches to synthesising AgNPs. Overall, the advantage of the biological route involves the process simplicity, the use of non-hazardous reducing and stabilising reagents, and comparatively low production costs [4,5].
This study showed that the fungus C. thermophilum cell-free extract can biologically synthesise spherically shaped AgNPs with an average size of 8.93 ± 2.29 nm. The genus Chaetomium has been previously investigated with regard to NP synthesis. The Chaetomium species C. globosum and C. cupreum were used to produce copolymer NPs with bioactive compounds from their crude methanol extract [37]. Furthermore, the species C. globosum was used in iron NP production [38]. To the extent of our knowledge, the species C. thermophilum has not been yet investigated for NP synthesis.
C. thermophilum is a moderately thermophilic fungus with slow growth at 35 • C but with optimum growth temperatures in the 40 to 55 • C range [36]. Its genome sequence was first described by Amlacher et al. [39]. It has since been used as a model organism system for biophysical research. Recently, this fungus was involved in high-temperature protein adaptation studies that demonstrated that protein glycosylation and deglycosylation are the mechanisms that allow their thermophily [40]. Furthermore, different authors have investigated this species' enzymes in the breakdown of lignocellulose for biofuel-renewable biomass, as well as for their potential to degrade cellulosic waste [18,41].
In vitro cytotoxicity tests are used to determine the toxicity of substances in cell lines as an alternative to direct animal testing. In vitro systems are relevant models when investigating the common toxicity mechanisms of AgNPs because they are cost-effective and allow direct assessment of NPs, providing valuable data for the screening of toxicity [12]. The advantages relate to the costs, the time required to obtain the final results, and ethical issues [42]. The NRU assay is an example of an in vitro cytotoxicity test. The OECD published a guideline document (No. 129 from the Environment, Health and Safety Publications Series on Testing and Assessment) for this test in 2010. The cell lines Balb/c 3T3 (murine fibroblast) or NHK (normal human keratinocytes) are used to measure toxicity as a concentration-dependent reduction of the chemical NR cell uptake after the substance's test exposure [20]. Obtaining cytotoxicity results with a sensitive cell line, such as Balb/c 3T3, is a valuable screening approach. However, additional tests must be taken, not only to assess toxicity mechanisms but also to assess the effectiveness in other models of biomedical relevance.
The GHS establishes that the acute toxic estimate (ATE) via the oral exposure route can be used to classify substances into five categories based on the LD 50 values. A lower LD 50 (mg/kg) is an indicator of greater toxicity [34]. This study successfully employed the OECD-based methodology (Figure 4). The NRU cytotoxicity assay was used to calculate the value of the IC 50 (119.69 ± 21.15 µg/mL) of the biological AgNPs produced by the fungus C. thermophilum and to predict an LD 50 value of 624.31 ± 41.87 mg/kg. Hence, this indicates a GHS ATE classification as category 4 ( Figure 5). For contextual reference, the NRU assay control drug, sodium dodecyl sulphate (SDS), which is used in cleaning and hygiene products, has a rat oral LD 50 of 977 mg/kg, also fitting into class 4 (300 mg/kg to 2000 mg/kg) [43].
The AgNPs biologically synthesised by the fungus C. thermophilum were significantly less toxic than chemically synthesised AgNPs, which have reported IC 50 values ranging from 2.20 µg/mL to 10 µg/mL, indicating higher toxicity [22,23,44,45]. Importantly, they were also found to be 4 to 30 times less toxic than other AgNPs synthesised with different species (Table 2) [45][46][47]. It is essential to highlight that AgNPs are currently being explored for applications in cancer therapy, tumour detection, drug delivery, wound dressing, and elsewhere [46,[48][49][50][51][52]. Therefore, synthesising NPs with lower toxicity, as demonstrated in this study, may lead to an expansion in the range of applications in the biomedical field. Although multiple species of fungi have been claimed to synthesise AgNPs, some of these species are pathogenic. For example, F. oxysporum was reported to synthesise welldispersed, spherically shaped AgNPs with sizes ranging from 5 nm to 13 nm. However, this species is responsible for soil-borne diseases that are extremely difficult to control, affecting food plants such as tomatoes, bananas, and onions [8,46]. Another example is the fungus A. fumigatus, described as responsible for the extracellular synthesis of extremely small (0.68 nm) cube-shaped AgNPs [15]. Nonetheless, this genus is also responsible for a parenchymal lung disease called aspergillosis [56].
While fungi produce a large variety of secondary metabolites, which may be critical for NP synthesis and stabilisation, they are also known to produce harmful self-preservation chemicals, including mycotoxins. In small concentrations, these low-molecular-weight compounds can be toxic to vertebrates and other animal groups [57]. Hence, fungal extracts also require toxicity evaluation for any proposed biological applications of NPs synthesised using fungi.
Furthermore, this study evaluated the interaction between the AgNPs and the Balb/c 3T3 NIH cells at an ultrastructural level (Figure 7). The different mechanisms by which AgNPs induce cell death include Ag ion release, disruption of cell membrane integrity, oxidative stress, protein or deoxyribonucleic acid (DNA) damage, generation of reactive oxygen species, and apoptotic cell death [59,60]. The ultrastructural damage caused by NP-cell interaction is often identified using TEM.
The TEM study demonstrated that most cells had preserved ultrastructures at the IC 20 (91.77 ± 24.24 µg/mL, Figure 7C,D). However, at the IC 50 (119.69 ± 21.15 µg/mL), the cell cytoplasm was damaged ( Figure 7E). The mitochondrial morphological alteration was evident in the swelling of the inner membrane ( Figure 7F). Similar results were found after analysing the interaction between mitochondria and chemically synthesised AgNPs. For example, a significant decrease in the mitochondrial membrane potential, adenosine diphosphate (ADP)-induced depolarisation, and respiratory control ratio were reported in rat liver mitochondria exposed to 40 nm and 80 nm of AgNPs. The function impairment was mainly attributed to changes in the membrane permeability [25]. Furthermore, exposure of AgNPs and titanium NPs to rat liver mitochondria was also demonstrated to lower the respiratory control ratio and induce mitochondrial swelling [61]. Additionally, the interaction of adult Wistar rats with chemically produced AgNPs (10 ± 4 nm) at a low dosage (0.2 mg/kg b.w.) resulted in mitochondrial swelling and cristolysis (damage of cristae) caused by silver nano-granules in the brain [24].
Mitochondria are cytoplasmic, double-membrane-bound organelles known to play an essential role in cellular energy production and to participate in calcium signalling, cell growth, differentiation, and death [62]. Moreover, mitochondria dysfunction has been related to many diseases, including neurodegenerative disorders; Huntington's, Parkinson's, and Alzheimer's diseases; epilepsy; schizophrenia; and Leigh syndrome [63][64][65]. Thus, it is essential to investigate the effects of NPs on mitochondria at a structural level, as disruption of this organelle can result in health effects.

Conclusions
As sustainable development awareness has grown, there has been a shift in focus to the use of biological sources. In the nanotechnology field, fungi have been investigated for the production of AgNPs in an environmentally friendly manner. This study reported the successful biological synthesis of AgNPs using the fungus C. thermophilum and is, to the best of our knowledge, the first time this fungus has been studied with regard to NPs production. Furthermore, it demonstrated low AgNP cytotoxicity towards a reference mammalian cell line, Balb/c 3T3 NIH. Additionally, the interaction of the AgNPs with this cell line showed preserved cell structures at non-toxic IC 20 . Therefore, the biologically synthesised AgNPs described in this study have potential as sustainable substitutes for physically and chemically made AgNPs; hence, they are a step in the right direction in achieving sustainable development in the nanotechnology field.  Acknowledgments: All authors wish to acknowledge Shannon Applied Biotechnology Centre (Shan-nonABC) and Rede de Plataformas Tecnológicas da FIOCRUZ for the use of their facilities to conduct this research.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.