TY - JOUR
AU - de Souza, Ana Olívia
AB - Abstract Silver nanoparticles (AgNPs) are widely incorporated into different hygiene, personal care, and healthcare products. However, few studies have been undertaken to determine the effects of biogenic AgNPs on human health. The effect of biosynthesized AgNPs using the fungus Aspergillus tubingensis culture was evaluated on human umbilical vein endothelial cells (HUVECs), normal human fibroblasts (FN1), human hepatoma cells (HEPG2) and a Galleria mellonella model. HUVECs were more susceptible to biogenic AgNPs than normal fibroblasts FN1 and intense cytotoxicity was observed only for very high concentrations at and above 2.5 μM for both cells. Normal human fibroblasts FN1 exposed to AgNPs for 24 h showed viability of 98.83 ± 8.40% and 94.86 ± 5.50% for 1.25 and 2.5 μM, respectively. At 5 and 10 μM, related to the control, an increase in cell viability was observed being 112.66 ± 9.94% and 117.86 ± 8.86%, respectively. Similar results were obtained for treatment for 48 and 72 h. At 1.25, 2.5, 5 and 10 μM of AgNPs, at 24 h, HUVECs showed 51.34 ± 7.47%, 27.01 ± 5.77%, 26.00 ± 3.03% and 27.64 ± 5.85% of viability, respectively. No alteration in cell distribution among different cycle phases was observed after HUVEC and normal fibroblast FN1 exposure to AgNPs from 0.01 to 1 μM for 24, 48 and 72 h. Based on the clonogenic assay, nanoparticles successfully inhibited HEPG2 cell proliferation when exposed to concentrations up to 1 μM. In addition to that, AgNPs did not induce senescence and no morphological alteration was observed by scanning electron microscopy on the endothelial cells. In the larvae of the wax moth, Galleria mellonella, a model for toxicity, AgNPs showed no significant effects, which corroborates to the safety of their use in mammalian cells. These results demonstrate that the use of A. tubingensis AgNPs is a promising biotechnological approach and these AgNPs can be applied in several biomedical situations. Graphical Abstract Open in new tabDownload slide Biogenic silver nanoparticles are ecofriendly and offers valuable possibilities of applications in several industrial areas. Introduction Different synthesis methods of silver nanoparticles (AgNPs) are being widely described in the literature. However, chemical and physical syntheses are always considered hazardous and costly and can promote interference in biomedical functions.1 As an ecological approach, biogenic AgNPs have been gaining special attention due to their prolonged stability and wide biocompatibility and applications. Bacteria, fungi, actinomycetes, yeasts, algae and plants are the main organisms that have been described as biogenic AgNP producers.2–4 AgNP biosynthesis using fungi is an ideal method mainly due to the capability of these organisms to grow faster, with high tolerance and easy metal bioaccumulation.5 Besides this, fungi produce some enzymes that are directly involved in AgNP stabilization and capping4,6 providing unique properties for these biological nanomaterials that can be applied in different industrial areas.2,3 Several studies highlight fungal biogenic AgNP antimicrobial activities.7,8 According to Birla et al.9 the antimicrobial activities can be improved when AgNPs are associated with an antibiotic, corroborating their potential medical application. In addition, biogenic AgNPs have also shown activity against cutaneous leishmaniasis,10 viruses such as human immunodeficiency virus type 1 (HIV-1), and Herpex sp. and antitumoral properties.11 AgNPs exhibited significant antibacterial activity against the Gram-positive and negative clinical pathogens including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Recently, Saravanakumar and Wang12 reported that the biogenic AgNPs of Trichoderma atroviride presented significant antimicrobial activities against E. coli, P. aeruginosa and S. aureus, and human breast cancer cell (MDA-MB-231) death at an inhibitory concentration of 16.5 μg mL−1 (IC50). Mohanta et al.13 observed high biocompatibility between biogenic Ganoderma sessiliforme AgNPs and normal murine fibroblast L-929 which showed IC50 of 256.6 ± 0.92 μg mL−1. Paradoxally, these AgNPs showed good anticarcinogenic activity against breast cancer cell lines MCF-7 and MDA-MB-231 with IC50 of 6.62 ± 0.05 μg mL−1 and 8.06 ± 0.01 μg mL−1, respectively. AgNPs obtained from Penicillium italicum with size distribution in the range of 32–100 nm, by the MTT assay, showed cytotoxicity concentration-dependence in tumoral cells MCF-7, and after 24 h of incubation with 5 and 80 μg mL−1, the viability was 84 and 13%, respectively.14 Hajebi et al.15 evaluated the cytotoxic, proapoptotic, antiangiogenic and antioxidant property effects of rapeseed flower pollen AgNPs (RFP/Ag-NPs) with a size of 24 nm, and found that these biosynthesized nanoparticles suppress cancer cells MDA-MB-231 and MCF-7 with IC50 of 3 and 2 μg mL−1. However, above 12.5 μg mL−1 these AgNPs have no inhibitory effect on normal human skin fibroblast HDF. These data are interesting and show selectivity or specificity of these nanoparticles by using tumoral cells. White et al.16 exposed NIH3T3 fibroblasts to monodisperse Allium sativum (garlic extract) AgNPs with a size of 4–6 nm. In comparison with the control, the nanomaterial effect was not significantly different; however, these nanoparticles induced proliferation of vascular smooth muscle cells (VSMCs). Furthermore, Boroumand et al.17 reviewed the application of fungi and yeast in the green synthesis of inorganic nanoparticles and their cytotoxicity. In this revision the authors highlighted the study by Hsin et al.18 which considers that AgNPs induce apoptosis in NIH3T3 fibroblasts via a mitochondria-mediated mechanism of release of cytochrome C into the cytosol and the translocation of Bax to mitochondria. Biological AgNPs obtained using a crude extract of Metarhizium robertsii were non-cytotoxic to L929 fibroblasts and the pulmonary epithelial A549 cell line19 which after 24 h presented IC50 of 25.5 and 22.6 μg mL−1, respectively. Similarly, mouse embryo fibroblast cells exposed to different concentrations (1.5 to 200 μg ml−1) of lyophilized biogenic AgNPs of Phanerochaete chrysosporium (MTCC-787) presented no significant toxicity up to 12.5 μg mL−1, even after 72 h of exposure.20 In a recent study developed by our group21 biogenic AgNPs from the Bionectria ochroleuca fungus were incorporated into cotton and polyester fabrics. Both fabrics presented antimicrobial activity for S. aureus, E. coli, Candida albicans, Candida glabrata, and Candida parapsilosis. In addition to that, the biological nanomaterial did not present toxicity in a set of experiments using Galleria mellonella larvae indicating a potential biotechnological application. Furthermore, our group showed the biosynthesis of biogenic antimicrobial AgNPs using the mangrove fungus Aspergillus tubingensis,4 and the secreted proteins involved in the proteic capping layer in which the fungal proteins were covalently bound mainly through S–Ag bonds due to cysteine residues (HS-) with few N–Ag bonds from H2N-groups were described.6 Besides that, the effect of the A. tubingensis AgNPs on the aerobic heterotrophs soil microorganisms, rice seeds (Oryza sativa) and zebrafish (Danio rerio) was studied. Compared to AgNO3, these AgNPs were less harmful for soil microbiota, and on rice seeds, displayed a dose-dependent inhibitory effect on germination and on their subsequent growth and development. The rice seed germination was inhibited by 30, 69 and 80% for 0.01, 0.1 and 0.5 mM AgNPs, respectively. After 24 h, AgNPs at 0.2 mM induced no mortality of the zebrafish D. rerio.22 Considering the relevant antimicrobial effect of these latter AgNPs and all the relevant context and questions regarding metallic nanoparticles, the aim of the present study was to evaluate the biogenic A. tubingensis AgNP effects and interaction with mammalian cell metabolism such as in human umbilical vein endothelial cells (HUVECs), normal human fibroblasts (FN1), HEPG2 cells and larvae of Galleria mellonella. Materials and methods Materials, cells and fungus strain Penicillin, streptomycin, inactivated fetal bovine serum (FBS) and RPMI 1640 medium were purchased from Cultilab (Campinas, SP, Brazil). Potato dextrose agar (PDA) and potato dextrose (PD) broth were both purchased from Himedia (India). AgNO3 was purchased from Merck and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) from Sigma. All the other chemical reagents were of analytical grade or in specific cases, as described in the text. The endophytic fungus A. tubingensis was isolated from Rizhophora mangle as previously reported in ref. 4 and deposited at ‘Embrapa Recursos Genéticos e Biotecnologia – Collection of Microorganisms for Biocontrol of Plant, Pathogens and Weeds’ (Brasília, DF, Brazil) under the number CEN1066, and at Oswaldo Cruz Institute Collection (Rio de Janeiro, RJ, Brazil) under the number IOC 4684. HUVECs were obtained from ATCC CRL-1730/HUVEC and HEPG2 0291 from “Banco de Células do Rio de Janeiro” (BCRJ) (Rio de Janeiro, RJ, Brazil). Normal human fibroblasts (FN1) were isolated in 2006 from eyelid blepharoplasty performed at the University of São Paulo Medical School under the ethics committee approval, guidelines and regulations, and patient consent (Comissão de Ética para Análise de Projetos de Pesquisa do HCFMUSP – CAPpesq project # 921/06). Following that, the fibroblasts FN1 were established in culture by Prof. Durvanei A. Maria and used for posterior assays, such as those in this study. Fungus and silver nanoparticle biosynthesis A. tubingensis was previously grown in PDA for 3–4 days, and colonies were used for biogenic AgNP biosynthesis. Details regarding the AgNP biosynthesis and physical properties were previously published by our group.4 Briefly, the formation of biosynthesized AgNPs was initially confirmed by UV-Visible spectrophotometry that demonstrated a plasmon band at 440 nm. The AgNP spherical shape form was determined by transmission electron microscopy (TEM). In addition to that, the nanomaterial presented average in size 35 ± 10 nm, polydispersity index 0.337, zeta potential 7.8 and stability over 90 days.4 Prior to any experimental procedure, the AgNPs were sonicated for 10 minutes and the experiments are subsequently performed. Cell culture Endothelial cells HUVECs (ATCC® CRL-1730™) and normal human fibroblasts (FN1) were grown in RPMI-1640 medium, pH 7.0, supplemented with 10% of inactivated FBS and 1% of antibiotics (10 000 IU mL−1 of penicillin and 10 mg mL−1 of streptomycin), and the culture flasks were incubated at 37 °C/5% CO2 under a humidified atmosphere. After confluence reaching approximately 90%, the cells were trypsinized and neutralized with FBS and the suspensions were adjusted with RPMI-1640 medium serum free, in accordance with each assay. For the assays, the cells were seeded on microplates, incubated overnight at 37 °C/5% CO2 under a humidified atmosphere and then treated following respective protocols. The immortalized cell line HEPG2 consisting of human liver carcinoma cells was cultured with DMEM medium supplemented with FBS and antibiotics under the same conditions above described. Evaluation of cell viability Cell viability was assayed by the colorimetric assay of MTT as previously described.11 MTT is reduced by intracellular dehydrogenases of viable living cells that lead to the formation of purple formazan crystals, insoluble in aqueous solutions. After dissolution in organic solvents, the absorbance was measured at 570 nm in a spectrophotometer. Endothelial cells and fibroblasts were plated at a density of 1 × 105 cells per well, in a 96-well flat-bottom microplate and after overnight incubation were exposed for 24, 48 and 72 h to AgNPs at concentrations ranging from 0.1 until 10 μM in at least four replicates. AgNPs were diluted in RPMI 1640 medium without FBS or antibiotics and the vehicle was used as a form of control to untreated cells. Assays were performed at least three times and the cell viability was expressed by the percentage of viable cells in comparison with the control considered as 100% of viability. The concentration of AgNPs that inhibited cell growth by 50% was defined as the inhibitory concentration (IC50). Analysis of the cell cycle phases by flow cytometry HUVECs and FN1 seeded in a 6-well microplate (1 × 105 cells per well) after overnight incubation at 37 °C/5% CO2 were treated with AgNPs at concentrations of 0.01, 0.1 and 1 μM for 24, 48 and 72 h, in six replicates. AgNPs were diluted in RPMI 1640 medium without FBS or antibiotics and the vehicle was used for untreated cells as the control. After treatments, the cells were harvested and fixed in 70% cold ethanol (with RNase 0.01%) at −20 °C overnight. Cells were thawed, centrifuged for 5 min at 2000g/4 °C and suspended in 200 μL of buffer (185 μL FACs buffer, 10 μL Triton-X 0.1%, and 3–5 μg of propidium iodide). The analysis was performed by flow cytometry (BD FACSCalibur, USA) collecting 10 000 events for each sample and the DNA content in the cell cycle phases (sub-G1, G0/G1, S, and G2/M). Cell cycle phase distribution was analysed using the Cell-Fit ModFit LT 3.2 software (BD), and data acquisition was gated to exclude cell doublets. Data are presented in percentage. Senescence assay by cytochemical staining for SA-β-galactosidases Cytochemical staining in situ for senescence associated β-galactosidase (SA-β-gal) was performed as previously described.23 HUVECs and FN1 at 1 × 105 cell per well in a 12-well microplate were treated with AgNPs in three replicates, at 0.01, 0.1 and 1 μM, for 24, 48 and 72 h. Briefly, after treatments cells were fixed with 0.6 mL of buffer (Sigma code F1797) and incubated with β-galactosidase substrate staining solution (kit Sigma code CS0030) at 37 °C, for 12 h. Staining solution was removed and the cells covered with glycerol at 70% were kept in a refrigerator until analysis. Stained cultures were observed using an inverted light microscope (Olympus CKX41) at 200× magnification, and a digital camera (Sony DSC 300) was used to record digital micrographs of three representative fields for each well. The SA-β-gal positive and negative cells were determined by counting the number of blue cells (positive) and uncoloured cells (negative) under bright field illumination. The percentage of senescent cells was determined for each treatment in comparison with untreated cells (control). Scanning electron microscopy Endothelial cells HUVECs at 2 × 105 cells per mL were distributed on sterile coverslips in 24-well microplates and incubated overnight. Cells were then exposed to 500 μL of 0.01 to 100 μM of AgNPs at 37 °C/5% CO2 for 24, 48 and 72 h, in two replicates. Control cells received only RPMI-1640 medium without FBS and antibiotics. Following treatments, the cells were fixed with paraformaldehyde at 4% and glutaraldehyde at 2.5% in 0.1 M cacodylate buffer (pH 7.2) for 4 h.24 After the fixation, the cells were washed three times with the same buffer for 15 min, dehydrated in a graded series of ethanol, and then subjected to critical point drying with CO2 (Leica CPD 030). Samples were covered with a gold film and examined with a FEI QUANTA 250 scanning electron microscope at an accelerating voltage of 10 kV. Images were obtained by secondary electron analysis25 and analyses were performed considering modifications or ultrastructure arrangements in the cell morphology. Clonogenic assay Clonogenic assay is an in vitro cell survival assay that evaluates all modalities of cell death based on the ability of a single cell to grow into a colony. The crystal violet method was applied to perform clonogenic assay following previous publications.26,27 Briefly, in a 24-well microplate, HEPG2 cells seeded at 7.5 × 104 cells per well were incubated overnight at 37 °C/5% CO2. Cells were exposed to 500 μL of AgNPs at 0.01, 0.1 and 1 μM diluted in DMEM (containing FBS and antibiotics) for 24 h, in triplicate. Following that, each well was washed with PBS and the cells were collected after trypsinization. In the next step, cells were counted and suspended in supplemented DMEM and seeded in a 6-well microplate (1 × 103 and 2 × 103 cells per well). Cells were grown for 1–3 weeks under the same conditions, until the colony control turned visible. Formed colonies were fixed and stained with crystal violet. Colonies were counted using a stereomicroscope and the data were analyzed as follows: PE (Plating efficiency) = (Number of colonies formed/Number of cells plated) × 100% and SF (surviving fraction) = (colonies formed after treatment/Number of cells plated) × PE. Assays were performed in four replicates in two independent assays and clonogenicity was expressed by the percentage of colonies formed in comparison with untreated cells used as the control. Statistical analysis was performed by one-way ANOVA/Dunnett's multiple comparison tests. Toxicity test in the Galleria mellonella larvae model The caterpillar larvae or wax worm of G. mellonella was used as a model for toxicity assay.28 Briefly, AgNPs were inoculated in the hindmost larvae proleg using a 50 μL 22s gauge gas-tight Hamilton syringe with 10 μL of AgNPs at 0.05, 0.25, 0.5 and 1 mM. Controls consisted of non-inoculated larvae to measure the effects of the incubation procedure and inoculated larvae with PBS to verify any potential lethal effects of the injection process. For each treatment, ten larvae were inoculated per experiment. Following injection, larvae were incubated at 28 °C or 37 °C and their survival was monitored daily up to 120 h. The death of the larvae was assessed daily through visual inspection of the lack of movement and blackening process of larvae. Data are presented as mean with SD of two independent experiments. A Kaplan–Meier survival plot with a log-rank (Mantel-Cox) test and Bonferroni correction (GraphPad Prism 5) was used to compare larval survival within the G. mellonella infection model. Results and discussion Cell viability Through the colorimetric MTT assay it was observed that endothelial cells HUVECs were more susceptible to the AgNP effect than the normal fibroblasts FN1, and also that cell viability was dose dependent (Fig. 1). Fig. 1 Open in new tabDownload slide Cellular viability of HUVECs and normal human fibroblasts (FN1) treated with AgNPs from 0.1 to 10 μM at 24, 48 and 72 h. The control refers to the untreated group corresponding to 100% of cell viability. Results are expressed by media ± SD of three experiments in sextuplicates. Statistical analysis by one-way ANOVA/Dunnett's multiple comparison test (among treatments). Significantly different from the untreated control: *P < 0.05; **P < 0.01 and ***P < 0.001. A similar result was observed for the biological AgNPs produced by the fungus Cunninghamella echinulata on Vero cells (African green monkey kidney cells),29 and for DMSA/Ag2S QDs on V79 cells at 400–2000 μg mL−1 in which the viability was dose-dependent and reduced with the increasing concentration of the nanoparticles.30 On fibroblasts FN1, the AgNPs did not show cytotoxicity during treatment for 24 h and the viability was 98.83 ± 8.40% and 94.86 ± 5.50% for 1.25 and 2.5 μM, respectively. At 5 and 10 μM, an increase in cell viability related to the control was observed, being 112.66 ± 9.94% and 117.86 ± 8.86%, respectively (Fig. 1). For these cell lines, treatments higher than 0.2 μM of AgNPs during 72 h induced cytoxicity statistically significant, probably due to the long period of exposition. At 1.25, 2.5, 5 and 10 μM of AgNPs, at 24 h, HUVECs showed 51.34 ± 7.47%, 27.01 ± 5.77%, 26.00 ± 3.03% and 27.64 ± 5.85% of viability, respectively. Similar results were obtained for treatment during 48 and 72 h. Even for a longer time of exposure, at 1.25 μM of AgNPs, the cell viability was 62.22 ± 18.75% and 80.01 ± 17.60% for treatments for 48 and 72 h, respectively. Interestingly in 72 h, AgNPs at 0.1 μM were not cytotoxic to HUVECs and a significant increase in the cell proliferation was observed. The increase of cell viability observed by MTT assay for both cells treated at 0.1 μM for 72 h, although statistically significant, was only around 30%, which could be due to the increase of the metabolic activity of the cells or due to an increase of the cell number induced by the nanoparticles in low concentrations. However, at higher concentrations this effect is not observed, which could be due to a metabolic pathway control. AshaRani et al.31 proposed that nanoparticle toxicity is multifactorial, where the size, shape, surface functionalization and potential to release the corresponding metal ions could play pivotal roles. These properties affect NP passage across cell membranes, distribution, and toxicity31–35 and are the main factors that determine how the NPs are taken up and the intake efficiency.35 Furthermore, there are four distinct endocytosis pathways, namely phagocytosis, macropinocytosis, and clathrin-mediated and caveolin-mediated endocytosis, that could be considered as the mechanisms for the internalization of NPs into HUVECs or other ECs in nanomedicine and nanotoxicology studies.36 Although HUVECs are not as phagocytic as macrophages, they can still engulf certain types of NPs through phagocytosis or macropinocytosis. In general, non-phagocytic cells preferentially take up spherical 20–50 nm NPs.37 The MTT assay also measures cell viability based on endocytosis, a fundamental feature of most living cells. However, various factors affecting the endocytosis of MTT, the exocytosis of MTT formazan, and cellular MTT reductase activity can influence the reduction of MTT.38,39 This may have occurred with the AgNP treatment at lower concentration. Furthermore, several factors can interfere with chemical cytotoxicity including concentration of chemical agents, length of exposure, cell density and cell type. The duration of exposure (T) and drug concentration (C) are related, although C × T is not always a slant. Longer exposure can increase sensitivity beyond that predicted by C × T due to cell cycle effects and cumulative effect from the agents.38 Hondroulis et al.40 performed a set of experiments to characterize the cytotoxicity of AgNPs of 10 and 100 nm on CCL-153 and RTgill-W1 fibroblast cell exposure. The authors observed that smaller nanoparticles with a size of 10 nm showed stronger cytotoxicity. Smaller AgNPs have the ability to enter into cells more easily, thereby decreasing the cell attachment and thus lowering resistance values. A previous report described that NPs of various sizes and chemical compositions are shown to preferentially localize in mitochondria,41 induce major structural damage, and contribute to oxidative stress.20 AshaRani et al.31 observed by TEM analysis, the presence of AgNPs inside the mitochondria and nucleus, implicating their direct involvement in the mitochondrial toxicity and DNA damage. The HUVECs were exposed to NPs including gold (Au), platinum (Pt), silica (SiO2), titanium dioxide (TiO2), ferric oxide (Fe2O3), and oxidized multi-walled carbon nanotubes, with different surface chemistry and size distribution.42 The exposure to NPs at non-cytotoxic concentration induced the upregulation of intracellular ROS43 and increased the antioxidant (CAT) activity at the same time, which led to the downregulation of VE-cadherin. Formation of gaps was observed between the cells resulting in endothelial leakiness which could compromise the integrity of endothelial barriers and induce risk of vascular diseases. Klingberg et al.44 showed that cellular uptake of 80 nm AuNPs by HUVECs is proportional to the nanoparticle concentration in the media. The uptake by endothelial cells occurs mainly by clathrin-mediated endocytosis. The adverse effects caused by NPs are generally attributed to the direct destruction of the cell membrane and organelles or binding to biomacromolecules to alter their structure and function. Therefore, the ways by which NPs are taken up, the cellular distribution and how NPs are excreted were the basis of understanding the cytotoxicity of NPs. Although it is known that the interaction between NPs and cells, and the internalization and distribution of NPs in cells are closely related to the toxicological mechanism of NPs, the NP cytotoxicity mechanism is still unclear. HUVECs that cover the lumen of blood vessels have been considered as a general model to assess the toxicity of NPs.45 There are two prevailing paradigms on how NPs traverse across endothelial barriers.36 One is a transcellular process which is energetically costly and slow, and the other is a paracellular transport process with crossing cell barriers by diffusion between cells through the cell–cell junctions. AgNPs which can lead to intracellular Ag accumulation have been shown to induce cytotoxicity of HUVECs as they reduced mitochondrial viability and membrane integrity and inhibited proliferation as well as increased lysosomal destabilization and apoptosis.43–48 In this study, the AgNPs were spherical and with sizes around 35 ± 10 nm (TEM), which could allow their intracellular accumulation. So, the previous studies regarding the uptakes and mitochondria localization of NPs contribute for the explanation of the AgNP effect on HUVECs and normal fibroblasts FN1. Probably, only at higher concentrations there was an accumulation of AgNPs in this organelle affecting the mitochondrial enzymatic process in both cell lines analysed. Colorimetric MTT assay is based on the enzymatic cleavage of tetrazolium salts to formazans by the cellular mitochondrial dehydrogenases present in viable cells, and consequently cytotoxicity was detected for AgNPs at higher concentrations due to the poor enzymatic activity. The external layer of A. tubingensis AgNPs applied in this study is involved by a proteic capping through mainly covalent S–Ag bonds due to cysteine residues (HS-), and few N–Ag bonds from H2N-groups.6 This characteristic could be preventing release of high concentration of Ag+ from the NP core to the cells. The coating material preventing cytotoxicity was already described by several authors.31,48 Cell cycle phases by flow cytometry For the cell cycle phases, for HUVEC (Fig. 2) and FN1 (Fig. 3) treated with AgNPs at 0.01, 0.1 and 1 μM for 24, 48 and 72 h, there was no significant statistical difference in the cell percentage distribution among the different cell phases in comparison with the control group. Fig. 2 Open in new tabDownload slide Cell cycle analysis of HUVECs treated with silver nanoparticles from 0.01 to 1 μM at 24, 48 and 72 h. The bars represent the proportions of (A) G0/G1 quiescent cells, (B) G2/M proliferative cells, (C) in phase S synthesis, and (D) debris in sub-G1 (fragmented DNA). Data represent mean ± SEM from at least two experiments with six replicates for each concentration. Statistical analysis by one-way ANOVA/Dunnett's multiple comparison test showed no statistical difference among the control and treatments. Fig. 3 Open in new tabDownload slide Cell cycle analysis of normal human fibroblasts (FN1) treated with silver nanoparticles from 0.01 to 1 μM at 24, 48 and 72 h. The bars represent the proportions of (A) G0/G1 quiescent cells, (B) G2/M proliferative cells, (C) in phase S synthesis, and (D) debris in sub-G1 (fragmented DNA). Data represent mean ± SEM from at least two experiments with six replicates for each concentration. Statistical analysis by one-way ANOVA/Dunnett's multiple comparison test showed no statistical difference among the control and treatments. Cell cycle phase analysis by flow cytometry analysis showed no alteration in cell distribution among the different cycle phases after exposure to biogenic AgNPs. This result is very interesting and different from those described in the literature for colloidal synthetic AgNPs.41,49–54 Recent studies have shown that synthetic AgNPs can promote cell cycle arrest to the G1 phase and be blocking the S phase in macrophages RAW264.7, followed by apoptosis.49,50 Furthermore, colloidal synthetic AgNPs can also induce DNA damage and cell cycle arrest to the G2/M phase in epithelial cells from the human proximal tubule HK-2 – NRF2 knockdown.41 These effects can be due to AgNP interaction with DNA molecules.50 Senescence and cell morphology by scanning electron microscopy The histochemical dye by β-galactosidase associated with senescence is widely accepted as an important biomarker for senescent cells with super expression of acidic lysosomal β-galactosidase.29 Usually, senescent cells lose their proliferative potential with morphological alteration such as an increase in size and multinucleated or irregular nucleus with chromatin reorganization. Normal fibroblasts FN1 and HUVECs exposed to AgNPs until 1 μM for 72 h showed normal morphological characteristics such as in the control group that had received only culture media (Fig. 4). In comparison with the control group there was no difference in senescence induction in cells treated with AgNPs. In Fig. 4, it is possible to observe that for 48 h and 72 h of exposure, both cells present membrane alteration probably due to the cytotoxicity that was also observed by MTT assay. Fig. 4 Open in new tabDownload slide Illustration of senescent-associated β-galactosidase (SA-β-gal) activity staining of non-senescent (uncoloured cells) and senescent (blue) normal human fibroblasts (FN1) and HUVECs (bar 50 μm) under bright field illumination. This senescence result is very important since it is indicating no cellular modification due to AgNP exposition. A similar morphological characteristic was also observed by scanning electron microscopy (SEM) on HUVECs until 10 μM (Fig. 5). Fig. 5 Open in new tabDownload slide Scanning electron microscopy for HUVECs exposed to 0.01, 0.1, 1, and 10 μM of AgNPs. Samples were covered with a gold film and examined with a FEI QUANTA 250 SEM an accelerating voltage of 10 kV. Control cells received only RPMI-1640 medium and the inset is showing the magnitude of cells at 300 μm. In the control and treated cells until 1 μM of AgNPs there is cell–cell interaction through the microvillus (highlighted). Proliferating cells are indicated by white arrows for AgNPs at 0.01 and 0.1 μM. Images were obtained from secondary electron analysis. For 10 μM of AgNPs it is possible to observe cytoplasmatic retraction and reduction of cell numbers. Also, there was presence of nanoparticle aggregates in some cells (red arrows). By scanning electron microscopy (SEM) it is possible to observe that endothelial cells HUVECs from the control group showed fibroblastoid morphology with cytoplasmatic prolongation and cell–cell interactions with the presence of several microvilli on their surface. This same effect was observed for AgNP treatment at non-cytotoxic concentrations. The treatment with 0.01, 0.1 and 1 μM of AgNPs did not induce alterations in the cell morphology and no signs of toxicity were observed. As expected due to the high concentration, for 10 μM there was an important alteration in the cell morphology with cytoplasm retraction and the presence of nanoparticle aggregates. In addition to that, it was possible to observe reduction of the cell density. The AgNP ability to mediate cellular morphological alterations in presence of high concentrations and cytotoxicity induction has been demonstrated in the literature. Studies showed that AgNPs induced cellular death on RAW264.7 and that nanoparticles were released to the culture media sometime after cellular death.49 Clonogenicity The biogenic AgNP treatment induced an increase in the proliferative ability of the HEPG2 cell to form clones, except when the AgNP concentration was 0.1 μM (Fig. 6 and 7). However, by statistical analysis there was no difference in the results among the three different concentrations evaluated and the untreated cells. Fig. 4 illustrates the clonogenic assay. Fig. 6 Open in new tabDownload slide Illustration of the clonogenic cell survival on HEPG2 treated with AgNPs at 0.01, 0.1 and 1 μM for 24 h. Untreated cells were used as the control. Fig. 7 Open in new tabDownload slide Clonogenic cell survival on HEPG2 treated with AgNPs at 0.01, 0.1 and 1 μM for 24 h. Untreated cells were used as the control. Data represent mean ± SEM from two experiments with four replicates for each concentration. Statistical analysis by one-way ANOVA/Dunnett's multiple comparison test showed no statistical difference among the control and treatments. Satapathy et al.47 observed in their experiments a significant reduction in the number of colonies formed from human colon carcinoma cells HCT116 treated with plant derived silver nanoparticles (LC50 = 450 nM). In contrast, no appreciable reduction in colony formation was observed in HCT116 p53−/− up to 1 μM. Souza et al.54 reported the reduction of the clonogenic capacity of CHO-K1 and CHO-XRS5 cells exposed to AgNPs at 5 μg mL−1. CHO-K1 cells treated with AgNPs of 10 nm presented themselves as the most susceptible. By clonogecity assay, it was observed that the proliferative capacity of HeLa and HaCaT cells was reduced as the commercial AgNP concentration increased (0.05–20 mg L−1).55 HaCaT cells presented higher capability to form colonies in comparison with HeLa cells. Swanner et al.56 showed by multiple metrics including measurements of mitochondrial function (MTT assay), DNA replication (BrdU incorporation), and long-term proliferative potential by clonogenic assay that synthetic AgNPs capped with polyvinylpyrrolidone (PVP, 0.2 wt%) were highly cytotoxic toward triple-negative breast cancer (TNBC) cells at doses that have little effect on nontumorigenic breast cells or cells derived from liver, kidney, or monocyte lineages. According to these authors, this result provides a clear rationale for further development of AgNP-based therapeutics for TNBC, which opens an important new direction for the biomedical use of AgNPs. Toxicity test in G. mellonella larvae The G. mellonella model is a simple and inexpensive alternative method for the rapid evaluation of toxicity of different groups of compounds in vivo and can therefore serve as an additional pre-screening experiment to lower the number of drug candidates proceeding to tests in mammalian models regardless of ethical objections, since the larvae of wax moth have a complex innate immune system that share similarities to vertebrates.57,58 Herein we tested an environmental temperature (28 °C) and a clinical and human temperature (37 °C) to verify if the effects of AgNPs could be temperature-dependent which would provide further understanding of nanoparticle toxicity in the G. mellonella model (Fig. 8).59 There was a high percentage of G. mellonella larvae survival, even after long time of exposition AgNPs, indicating low toxicity. Similar results were observed for Bionectria ochroleuca AgNPs with sizes falling in the range of 8–21 nm where a very low toxicity was detected in this infection model.21 Fig. 8 Open in new tabDownload slide Illustration of G. mellonella larvae survival at 28 or 37 °C after injection with 10 μL of AgNPs at 0.05, 0.25, 0.5 or 1 mM (100%). Controls were injected with PBS or even not injected at all (untreated). The survival was monitored daily during 120 h. Results showed that at the end of the experiment performed at 28 °C, 80% of the larvae survived with 0.25 and 1 mM of AgNPs, whereas 90% survived with 0.05 and 0.5 mM, such as seen in the PBS treatment (Fig. 9). At 37 °C, there was 80% of the larvae survival with 0.05 and 0.25 mM of AgNPs, with the early deaths being observed as from the 96th h. In the treatments with 0.5 and 1 mM there was 90% of survival, more than with lower doses. For control groups, both at 28 and 37 °C, there was 100% of larvae survival. Regardless of the temperature assayed, the overall toxicity of AgNPs towards G. mellonella was not statistically different among treatments (log-rank test and Bonferroni correction; P < 0.05). Fig. 9 Open in new tabDownload slide Survival of G. mellonella larvae maintained at 28 and 37 °C after injection with 10 μL of AgNPs at 0.05, 0.25, 0.5 or 1 mM (100%). Controls were injected with PBS or even not injected at all (untreated). The survival was daily monitored during 120 h. Conclusions The cytotoxic effect of biogenic AgNPs with an average size of 35 ± 10 nm was detected only for high concentrations at and above 2.5 μM for HUVEC and FN1 cell lines. Flow cytometry analysis indicated no alterations in cell distribution. Based on the clonogenic assay, NPs successfully inhibited HEPG2 cell proliferation when exposed to concentrations down to 1 μM. AgNPs did not induce senescence, morphological alteration and toxicity to G. mellonella larvae. 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TI - Biogenic Aspergillus tubingensis silver nanoparticles’ in vitro effects on human umbilical vein endothelial cells, normal human fibroblasts, HEPG2, and Galleria mellonella
JF - Toxicology Research
DO - 10.1039/c9tx00091g
DA - 2019-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/biogenic-aspergillus-tubingensis-silver-nanoparticles-in-vitro-effects-LC04aJ80TY
SP - 789
EP - 801
VL - 8
IS - 6
DP - DeepDyve
ER -