The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of –35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

Abbreviations

AU: arbitrary unit
AuNPs: biologically produced gold nanoparticles
cryo-EM: cryo-electron microscopy
DLS: dynamic light scattering
DMEM: Dulbecco's Modified Eagle Medium
DMSO: dimethyl sulfoxide
EDS: energy dispersive X-ray spectroscopy
ELS: electrophoretic light scattering
FBS: fetal bovine serum
GF-AAS: graphite furnace atomic absorption spectroscopy
INF-γ: interferon gamma
LPS: lipopolysaccharide
MTT: 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NC: negative control
PBS: phosphate-buffered saline
PC: positive control
SDB: Sabouraud Dextrose Broth
TEM: transmission electron microscopy
TNF-α: tumor necrosis factor alpha

1 INTRODUCTION

Gold nanoparticles (AuNPs) less than 50 nm in diameter are widely used for targeted drug delivery because AuNPs are known to be the most biocompatible nanoparticles available.¹ They have a large surface providing space to attach various molecules used in targeted cancer therapy.^2-4 Even if the AuNP–drug conjugates do not contain a specific ligand for cancer cells targeting in vivo, they accumulate in the tumor due to the high blood circulation and low lymphatic drainage in the tumor area.⁵

In biomedical applications of AuNPs, nanoparticles produced biological way are inherently more favorable than those produced chemical way. The biological approach, a new method to produce AuNPs engages various microorganisms such as fungi and bacteria, as well as plant extracts.^6-8 The AuNP production by microorganisms employs its intracellular or extracellular reducing agents.⁹ In both variants, an unknown material of the microorganism, which is mostly protein,¹⁰ adsorbs to the surface of AuNPs and stabilizes them.

Using AuNPs as drug delivery vehicles (targeted or nontargeted) requires good knowledge of the nanoparticles’ fate and accumulation in different cell types so any potentially harmful nano-bio interactions are limited. Despite several studies on nonbiologically produced AuNPs,^{11, 12} there are no studies on the uptake of biologically produced nontargeted AuNPs by tumor and nontumor cells. Therefore, we aimed to contribute to our research to understand the interaction between the biologically produced AuNPs and three various cell types (tumor-derived cells, nontumor fibroblasts, and macrophages).

In this study, we prepared AuNPs by the biological method using Fusarium oxysporum and obtained 15 nm small negatively charged polygonal AuNPs coated with capping agents specific for the AuNP producent. Produced AuNPs of the same size, charge, and shape were evaluated for their immediate toxicity, uptake, and basic cellular responses (stress fibers reorganization and cytokine secretion) in three cell lines of different origin.

2 MATERIALS AND METHODS

2.1 AuNPs production

F. oxysporum (CCF 3732) was cultured for 1 week in a sabouraud dextrose broth (SDB) at 30°C under shaking conditions (150 rpm). The culture was centrifuged at 8000 rcf for 10 min, and the supernatant was used to prepare AuNPs. A molar HAuCl₄⋅3H₂O (Sigma– Aldrich, Prague, Czech Republic) in ddH₂O was prepared, and 100 µL of this stock solution was added to 100 mL of supernatant, and the pH of the dispersion was adjusted to pH 7. This flask and a flask of SDB containing the same concentration of HAuCl₄⋅3H₂O as control were incubated overnight at 37°C in a shaker incubator (200 rpm).^{13, 14}

2.2 Characterization of AuNPs

The first sign of AuNPs production is a color change of the dispersion from yellow to red. After this first check, AuNPs were washed with ddH₂O and centrifuged three times at 22,000 rcf for 30 min. The pellet of AuNPs was dispersed in ddH₂O with pH 7,^{7, 15} and the pellet was tested for AuNP-specific spectra. For spectrophotometric analysis, we used Infinite 200 PRO UV-visible spectrophotometer (Tecan, Männedorf, Switzerland), ddH₂O as a blank, and wavelengths between 400 and 700 nm. The maximum absorption peak of AuNPs should be between 500 and 550 nm, proving the presence of AuNPs.^16-18

The size and composition of the AuNPs were determined by transmission electron microscopy (TEM, JEOL JEM -F 200) and energy dispersive x-ray spectroscopy (EDS). Two microliter of the sample was placed on a glow discharged Cu 300 mesh quantifoil™ 2/1 grid, after blotting the grid was plunged into a liquid ethane at −183°C, stored under liquid nitrogen, and analyzed by TEM. The instrument was operated at 200 kV and equipped with Cold FEG, TVIPS XF416 CMOS camera, and JED 2300 x-ray spectrometer (JEOL, Freising, Germany).¹⁹

The size distribution and zeta potential of the sample were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) using the Zetasizer Ultra instrument (Malvern Panalytical, Malvern, UK). The sample was placed in an ultra-low volume ZEN2112 quartz cuvette and a DTS1070 Zeta cell with folded capillaries for size and zeta potential analysis, respectively. ddH₂O served as the dispersant, the refractive index was 0.18, the absorbance was 3.43, and the temperature was 25°C.^{7, 14, 20, 21}

2.3 Cell viability assay

AuNPs dispersion was sterilized by the Tyndallization method,⁹ and the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay was performed using 4T1 (ATCC CRL −2539), NIH/3T3 (ATCC CRL −1658) and RAW264.7 (ATCC TIB −71) as cancerous, noncancerous, and phagocytotic cell lines, respectively. 4T1 cells were cultured in Roswell Park Memorial Institute Medium 1640 (RPMI-1640, Sigma Aldrich, Prague, Czech Republic) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 50 mg/L gentamicin (Sandoz, Prague, Czech Republic), 4.5 g/L glucose and 1.1% sodium pyruvate (Sigma Aldrich, Prague, Czech Republic). NIH/3T3 and RAW264.7 cells were cultured in the same working medium consisting of Dulbecco's modified Eagle medium (Sigma Aldrich, Prague, Czech Republic) with 10% FBS (Gibco, Waltham, MA, USA), and 50 mg/L gentamicin (Sandoz, Novartis Company, Prague, Czech Republic). 2 × 10⁵ cells/mL of each cell line (passage 16–18) were cultured in two rows of a 96-well tissue culture plate (JETBiofil, Guangzhou, China).

After overnight incubation at 37°C in the presence of 5% CO₂, the 80% confluent cells were challenged with ½ concentration of AuNPs obtained by titration, and the control wells contained only the cells. Four plates were cultured and treated in the same way and at two different intervals (8 and 24 h) the viability of cells was analyzed by the MTT method. For this purpose, 20 µL of 5 mg/mL MTT (EMD Millipore, CA, USA) in phosphate-buffered saline (PBS) was added to all wells and after 4 h of incubation, the media were replaced with 100 µL of dimethyl sulfoxide (DMSO; Sigma Aldrich, Prague, Czech Republic) to solve the formazan crystals and mixed by pipetting. Using a Tecan spectrophotometer with the test absorbance at 570 nm and the reference wavelength at 630 nm, the cell survival rate percent was calculated as (A − B)/(C − B) × 100 (where A is the absorbance of the test compound, B is the absorbance of blank (i.e., DMSO) and C is the absorbance of control).²²

Tests were repeated three times, and differences between the groups were compared using the analysis of variance (ANOVA) online tool (https://astatsa.com/OneWay_Anova_with_TukeyHSD) and a p-value of ≤ 0.05 was considered as significant.

2.4 Cell culture preparation for Au content analysis

Cells and their supernatants were analyzed for gold content by graphite furnace atomic absorption spectroscopy (GF-AAS) and visible spectrophotometry, respectively. The cells and supernatants were prepared as follows: 1 mL of 4 × 10⁵ cells/mL of each cell line was placed into five cell culture plates (47 mm in diameter), and 10 µL of AuNPs dispersion (at the nontoxic dose determined from the MTT assay) was added to each plate. The plates were gently shaken for a few seconds and incubated under standard conditions. One plate from each group was selected at different time intervals (5 min, 1 h, 3 h, 24 h, and 48 h). Cells were detached from the plate with a cell scraper, centrifuged at 2000 rpm for 5 min, culture medium was poured into a microtube, cell pellets were washed three times with PBS, and stored at −80°C before experiments.^{11, 23} The experiments were repeated three times.

2.5 Estimate of intracellular Au content

GF-AAS was used to determine the amounts of Au internalized to different cells. A series of calibration standards containing 0, 2, 4, 10, 30, and 100 ng mL⁻¹ Au were prepared. The Analytik Jena ContrAA600 instrument with the Zeeman background correction was operated with a xenon lamp at a current of 13 A, a slit width of 0.7 nm (L), and a wavelength of 242.8 nm and a permanent Ir modification of graphite furnace. Prior to analysis, each sample was quantitatively transferred to a 15-mL polypropylene tube containing freshly prepared 2 mL aqua regia (concentrated HNO₃ plus HCl in a ratio of 1:3 v/v) and allowed to react at room temperature for 6 h to dissolve the gold. After that, the content of Au was determined with appropriate dilution.²⁴

2.6 Analysis of noninternalized AuNP by visible spectrometry

The supernatants were analyzed by the Tecan spectrophotometer mentioned above for the presence of uninternalized AuNPs. The decrease in the AuNP-specific maximum absorbance peaks in tested supernatants over time indicated penetration of AuNPs into the cells. Spectra were measured between 400 and 700 nm with the AuNP-specific peak reaching a maximum between 500 and 550 nm. Adequate media was used as a blank.

2.7 Analysis of cytokines secreted into the cell supernatant

Cell supernatants collected above were also used for the evaluation of AuNP-mediated cytokine release by macrophages. The LEGENDplex kit (BioLegend, Prague, Czech Republic) and BD™ LSRII flow cytometer (BD Life Sciences, Franklin Lakes, NJ) were used to analyze AuNP-mediated macrophage activation. We focused on the detection of inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (INF-γ) released by RAW264.7 cells in response to AuNP interaction. Positive control samples were obtained by incubation of 1 mL of 4 × 10⁵ cells/mL RAW264.7 cells with 1 µL of commercial lipopolysaccharide (LPS; 10 µg/mL, Sigma Aldrich, Prague, Czech Republic) at different time intervals (5 min, 1 h, 3 h, 24 h, and 48 h). ddH₂O was used as a negative control and added to the cells in the same manner as LPS or AuNPs. Supernatants were obtained by centrifugation (at 2000 rcf for 5 min) and stored at −80°C until analyses.²⁵ We used the LEGENDplex kit according to the manufacturer´s instructions. The fluorescent signal from the used beads (allophycocyanin, APC channel, 660 nm emission) and the signal from the biotinylated detection antibody (phycoerythin, PE channel, 575–585 nm emission) were obtained and data were analyzed using FlowJo v9 software (BD Life Sciences, Franklin Lakes, NJ).

2.8 Actin reorganization analysis by confocal microscopy

For confocal microscopy, NIH/3T3, 4T1, and RAW264.7 cells in the exponential growth phase were detached from cell culture plates with a cell scraper and counted. Ten milliliter of AuNPs (at the nontoxic dose determined from the MTT assay) was added to 1 mL of each cell line (1 × 10⁵ cells/ml). Cells were then seeded on a 35-mm plate with a 20-mm glass bottom (Cellvis, Ontario, Canada) and incubated for 24 and 48 h. After each interval, plates were fixed with 4% paraformaldehyde, permeabilized with 0.1 % Triton X-100, and stained with phalloidin conjugate of F-actin (CytoPainter F-actin Staining Kit, Abcam, Cambridge, UK). Hoechst33342 (1 µg/mL, Invitrogen, Waltham, MA, USA) counterstain was used for nuclei visualization. The excitation/emission parameters used for the detection of fluorescent signals were Hoechst 405/461 nm and phalloidin 550/572 nm. One-plane imaging with the highest intensity of the nucleus channel and Olympus FluoView 2.0 software were used to acquire images.²⁶ Acquired images were subsequently analyzed using the online software Fiji, and the image processing was optimized to detect multiple parameters describing changes in F-actin organization.²⁷ In particular, nuclei were counted using the “Nuclei” script (detailed in online Appendix 1). Actin signal intensity was evaluated using the “Actin intensity” script (see online Appendix 2). The number, size, and density of filaments were evaluated according to the modified protocol of Horzum et al. 2014²⁸ (online Appendix 3). Filament number and density were calculated per cell. Filament length represented the average size within the entire area studied. The linear feature detection algorithm was used to quantify actin filament length/area and actin filament number and intensity/cell by averaging the intensity of pixels within filaments.

3 RESULTS AND DISCUSSION

3.1 AuNPs production and characterization

The color of the supernatant containing AuNPs (crimson) changed in contrast to the negative control (yellow) as shown in Figure 1 (top right corner). The spectra of the AuNPs had a maximum absorption peak at 530 nm (Figure 1).

Details are in the caption following the image — **FIGURE 1**
Open in figure viewer PowerPoint

Spectrum of produced AuNPs with maximum absorbance at 530 nm. The color change pointing out AuNPs production is shown, the left flask is a control supernatant, and the right flask is a supernatant after AuNPs production.

The obtained AuNPs had spherical and polygonal shapes and sizes of about 15 nm as shown by TEM (Figure 2A,B). EDS analysis showed that the chemical composition of the electron-dense spots was Au (Figure 2C,D).

The zeta potential and size distribution of AuNPs were determined using the Zetasizer, and the test was performed in five replicates. The results showed that the size of AuNPs was 57.8 nm ± 17.43, and the zeta potential was −35.8 mV ± 1.17. The DLS data showed that the obtained size was different from the size measured by TEM since TEM indicates the actual NP size and DLS indicates the average hydrodynamic diameter of the nanoparticles.^{14, 29}

3.2 Cell viability assay

The MTT assay was performed using 4T1, NIH/3T3, and RAW264.7 cells as cancerous, noncancerous, and phagocytotic cell lines. Cells were incubated with different concentrations of AuNPs (from 0.12 µg of AuNPs per well to 61.1 µg of AuNPs per well), and their viability was analyzed at four different intervals. The results showed that the same percentage of AuNPs in different cells produced different toxic effects at different time intervals. According to the formula (see Section 2), the calculated percentages of cell viability for 4T1, NIH/3T3, and RAW264.7 cells after incubation with the highest amounts of AuNPs (61.1 ± 1.3 µg per well) at different intervals are shown in Figure 3.

The results showed that the cells were able to grow in the presence of AuNPs, starting with the first well of each row. The calculated percentage of cell viability for RAW264.7, NIH/3T3, and 4T1 cells after 8 h was 93 ± 2.9%, 95 ± 1.2%, and 78 ± 8.83 %, respectively (Figure 3). After 24 h, the viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The data are consistent with our previous study on the toxicity of the biologically produced AuNPs showing the toxicity of the AuNPs was low and was dependent on the AuNP load.⁹ The viability of the 4T1 cell line was lower compared to the others, which might be due to the smaller size with the highest loading and accumulation of AuNPs within the cells as shown below. Since the AuNPs were not remarkably toxic even in the highest concentration tested, we used that amount (61.1 µg of AuNPs per 100 µL) in all the subsequent experiments.

The statistical analysis, ANOVA with post hoc Tukey HSD test, showed that there was no significant difference between the incubation time (8 and 24 h) in each cell line. There was a significant difference in viability between 4T1 and NIH/3T3 cells after 8 h of incubation with AuNPs (p < 0.05).

3.3 Uninternalized AuNP content in culture supernatants

The cell supernatants collected after incubation of cells with AuNPs (with several incubation times) were analyzed by spectrophotometer and maximum absorption peaks indicating AuNPs were evaluated regarding their position and intensity.

As shown in Figure 4, the maximum absorbance peak of AuNPs in the supernatant of each cell line decreased with time, indicating that the AuNPs were captured by the cells. Overall, the highest amounts of AuNPs in the cell supernatant were found in the NIH/3T3 cell culture and the lowest in the 4T1 cell culture, demonstrating that the greatest amounts of AuNPs were located within 4T1 cells. It correlates with the results of the cell viability assay (especially after 24 h of incubation), in which the lowest number of viable cells was found in the 4T1 cell line. The higher uptake of AuNPs likely results in lower cell viability, that is, also in consistency with the former toxicity study.⁹

The shape of the AuNP absorption peak is different in each supernatant. It can be due to the formation of a protein corona on the surface of the AuNPs after incubation with the cell-specific culture media. The culture medium for NIH/3T3 and RAW264.7 differed from the 4T1 media. Usually, FBS is involved in protein corona formation, but the amounts of FBS were the same for both types of culture media, so we expect the involvement of other media-specific proteins and amino acids. We have already shown that the nature of the hard protein corona varies depending on the different serum concentrations.¹⁴ The surface corona of AuNP can also affect the stability and endocytosis of AuNPs. Therefore, analysis of the hard protein corona of the biologically produced AuNPs in different media will be considered in our future study.

3.4 Release of cytokines TNF-α and INF-γ as a response to AuNPs

Internalization or even just interaction of bare and functionalized nanomaterial potentially triggers the cellular response. If the nanomaterial is meant to be further used in multicellular organisms, we need to consider the response of immune cells, particularly phagocytizing cells. Our experimental setup thus involved macrophage cell line RAW264.7 as a representative phagocytizing cell and we tested secretion of inflammatory cytokines TNF-α and INF-γ as macrophage characteristic response to AuNPs. Since the cytokine level varies with time, we tested five different time points as shown in Figure 5.³⁰

We found increased secretion of TNF-α and INF-γ by RAW264.7 cells after their incubation with AuNPs and the observations were cytokine-specific and time-dependent. The positive signal pointing out an increased level of cytokine was evaluated based on the cytokine pattern in positive control (PC) that represented cells stimulated with LPS (inducer of inflammatory cytokine production³⁰ for 48 h). Cells stimulated with PBS served as negative control (NC) for correct gating of fluorescent signal. The RAW264.7 incubation with AuNPs resulted in the release of INF-γ with a maximum after 1 h of incubation. The increased INF-γ level was also then observed to a lesser extent after 3, 24, and 48 h of incubation (Figure 5B). Regarding TNF-α, we found a remarkable increase in cytokine release after 1 h incubation of RAW264.7 with AuNPs and the higher level of TNF-α persisted till the end of the experimental period after 2 days (Figure 5B). The significance of cytokine increase was calculated by ANOVA and subsequent post hoc Tukey test. The increased levels of both cytokines, TNF-α and INF-γ, observed in cells stimulated with AuNPs for 1, 3, 24, and 48 h were equally significant with p-values < 0.01 (compared to PBS—stimulated cells; negative control). That increase was as high as cytokine increase after LPS stimulation (p-value < 0.01 when compared to PBS-stimulated cells). The difference between PBS-stimulated cells and cells stimulated with AuNPs for 5 min was not significant (p-values for TNF-α and INF-γ were 0.899 and 0.493, respectively). The intensity of production of proinflammatory cytokines after LPS stimulation of RAW264.7 cells depends on the amount, time, presence of co-stimulators, chain length, and hydrophilic part of LPS.³⁰ Thus, we observed different maximum values of TNF-α and INF-γ (different maximum percentages of fluorescent signal). Importantly, the maximum concentrations are consistent in both LPS and AuNP-stimulated samples. We suggest the intensity of cytokine release triggered by AuNPs is executed similarly to LPS stimulation. The quick decrease of IFN-γ level after it reached maximum release peak in 1 h proposes transient activation of the cytokine. Transient cytokine activation induced by conventional, nonbiological, AuNPs was also found by Khan et al., who suggested that the AuNPs are conceivably immune compatible.³¹ Khan et al. analyzed the changes in cytokine levels based on mRNA expression and thus the transient change was apparent faster.³¹ In our setup, we analyzed the secreted proteins and thus the changes in cytokine expression would reflect slower.

3.5 Analysis of the intracellular Au content

Each cell line was incubated with an equal starting amount of AuNPs (61.1 ± 1.3 µg), and the amount of intracellular gold (i.e., AuNPs) was determined with GF-AAS in different time intervals (Figure 6). Total Au content in the samples was determined on three different days, and average values from all three experiments are summarized in Table 1.

TABLE 1. Au content determined by GF-AAS in different cell lines at different time intervals. The percentage of internalization shows the content of AuNPs found inside the cells with respect to the starting amount.

Time/ Cells	m Au (µg)	SD (µg)	Internalization (%)	m Au (µg)	SD (µg)	Internalization (%)	m Au (µg)	SD (µg)	Internalization (%)
	NIH/3T3			4T1			RAW264.7
5 min	0.23	± 0.00	0.4	0.87	± 0.02	1.4	1.46	± 0.02	2.4
1 h	0.96	± 0.02	1.6	1.31	± 0.02	2.1	2.96	± 0.03	4.8
3 h	3.97	± 0.00	6.5	3.34	± 0.01	5.5	2.32	± 0.03	3.8
24 h	6.98	± 0.14	11.4	43.3	± 0.50	70.9	29.8	± 1.00	48.8
48 h	21.1	± 0.50	34.5	49.0	± 0.60	80.2	34.6	± 2.30	56.6

Abbreviations: m, the total mass of Au in the samples; SD: standard deviation.

Based on the results of the GF-AAS analyses, AuNPs were able to quickly interact and enter into all the cell types. With time, the accumulation of gold increased, and the maximum mass of Au was determined in all cell lines after 48 h. We are aware that there is likely a portion of AuNPs that are not internalized yet but firmly adhered to the cell surface, especially in the early time points. Even though we expected RAW264.7 to internalize the highest amount of Au because of their phagocytotic properties, we found the highest amount of Au internalized into 4T1 cancer cells instead (as observed after a longer incubation time of 24 and 48 h). The lowest amount of Au that was internalized into NIH/3T3, normal immortalized fibroblasts, as expected. There are many factors that can affect the internalization of nanoparticles, from the size, surface charge, and shape of the nanoparticles to the growth phase, shape, and type of the cells. In our study, we used the same entry amount of AuNPs possessing equal characteristics and we employed cells in their exponential growth phase. Omitting the phagocytic cells, our result shows that the cancer cells (4T1) internalized remarkably higher amounts of AuNPs than the noncancer cells (NIH/3T3). It may be due to the faster growth rate of cancer cells. Additionally, in contrast to the RAW264.7, the 4T1 cells are smaller which means (considering the same cell number at the beginning of the experiment) a larger cell surface/AuNPs uptake area.

3.6 Actin reorganization in response to AuNPs interaction

Reorganization of intracellular actin filaments is a general cell response to stress signals including those after the interaction of the cell with a nanoparticle. In order to analyze actin reorganization as a complex process, several parameters such as the number of filaments and their average length and brightness, were determined as a function of actin density. Several macros and plug-ins were tested within the Image J / Fiji platform and compared with data from neurite outgrowth, Skeletonization, or filament detector.²⁷ However, here we found a high number of false positives, inappropriate filament labeling, or poor reproducibility due to the varying quality of the biological sample (e.g., heterogeneous cell density and uniform staining across different cell lines and with/without treatment). Finally, we used a more complex protocol from Horzum et al.,²⁸ which we adapted to our type of biological samples. This approach revealed several different parameters characterizing actin reorganization that are summarized in Table 2 for each cell line with respect to the presence of AuNP and incubation length.

TABLE 2. Summary of the different parameters characterizing the reorganization of F-actin filaments after exposure of different cell types to AuNPs. AU stands for the arbitrary unit.

Cell type	Time (h)	AuNPs	Filament density/cell (AU)	Actin signal intensity/cell (AU)	Filament count/cell	Filament size (nm)	Filament area (AU)
Fibroblast nontumor (NIH/3T3)	24	+	238.3 ± 77.8	284.9	2.03 ± 0.65	154.3 ± 9.3	310.8 ± 90.5
	24	–	300.7 ± 146.8	450.6	2.92 ± 0.57	160 ± 11.5	467.6 ± 97.7
	48	+	848.3 ± 150	391.4	1.9 ± 0.5	189 ± 12	362.9 ± 100
	48	–	169.8 ± 30.9	3290.9	2.5 ± 0.6	191.7 ± 7	476.4 ± 98.7
Breast tumor (4T1)	24	+	347.5 ± 173	264.8	2.09 ± 0.3	121.7 ± 10.1	253.8 ± 40.7
	24	–	274.9 ± 90.2	327.5	2.5 ± 2.9	186 ± 30.1	462.8 ± 30.3
	48	+	202.8 ± 26.6	295.1	1.8 ± 0.1	169.7 ± 34	309.3 ± 77.6
	48	–	309.8 ± 65.9	307.1	1.6 ± 0.3	240.3 ± 36.7	386.6 ± 24.4
Macrophage (RAW264.7)	24	+	168.8±56.2	357.4	3.5 ± 0.7	145.8 ± 11.6	508.9 ± 73.8
	24	–	165.8 ± 45.9	273.8	2.2 ± 0.1	135 ± 5.6	302.1 ± 16.6
	48	+	154.7 ± 2.2	340.1	4.2 ± 0.7	136.7 ± 3.2	573.7 ± 88.9
	48	–	195.4 ± 82.2	432.8	3.9 ± 1.9	149.7 ± 3.1	581.4 ± 277.5

The results showed that AuNPs prevented cell growth of mouse fibroblasts (NIH/3T3 cells) exposed to AuNPs for 48 h (Figure 7A, NIH/3T3). Fibroblasts with and without exposure to AuNPs showed a similar growth pattern in the first 24 h of incubation, which could be due to the lower amounts of AuNPs in the cells (11.4%). However, after 48 h, the cultures differed significantly, which could be due to the higher amounts of AuNPs within the cells (34.5%). Visual observation is consistent with the fact that in contrast to control cells, cells exposed to AuNPs showed a remarkable loss of filament density, but filament intensity per cell increased (Table 2 and Supplementary image 1c and e). The NIH/3T3 cells possessed a similar number of filaments of the same size. Thus, a lower number of filaments expressing a higher amount of actin per cell could indicate that the cells aggregate and form compact colonies in response to stress conditions.³² In this situation, the cells were alive but had stopped growing due to the stress.

In contrast to fibroblasts, only minor changes in growth and confluence were observed in tumor culture (4T1 cells) (Figure 7C, 4T1 cells). The tumor cells show a slight increase in F-actin filament density after 48 h of exposure to AuNPs, which was due to the high amount of AuNPs accumulating in the cells over time, while the unexposed cells show a slight decrease in filament density over the same time period (Table 2 and Supplementary image 1c). On the other hand, a smaller average size of actin filaments was observed in the cells exposed to AuNPs, which could be due to suppressed growth. The slight growth suppression can also be seen in the lower confluence of the cells (Figure 7C), again the cells are alive with a high concentration of AuNPs.

In macrophage culture (RAW264.7 cells), changes in filament parameters were observed after a shorter incubation of 24 h (Table 2 and Supplementary image 1a and d). Here, a higher filament number corresponding to a larger filament area per cell was observed, which was due to the high percentage of AuNPs within the cells (48.8%) and a proinflammatory response, indicating a stress-like response of AuNP-exposed cells after seeding in contrast to control cells. Visually, the confluence of the control culture appeared to be higher than the confluence of the cells exposed to AuNPs (Figure 7B). After 48 h, the cultures behaved the same which was due to a slight increase in AuNPs concentration in cells (56.6%) and control of the release of the proinflammatory cytokines. In conclusion, AuNPs affected the reorganization of F-actin filaments and probably triggered a stress response. Therefore, we suggest that the next step should focus on evaluating markers of the stress response, such as reactive oxygen species production.

The current observation supports our previous study on the toxicity of AuNPs produced by F. oxysporum in human fibroblasts.⁹ Here we found changes in cell morphology without any apparent toxicity detected by the MTT assay. Observed morphological changes were likely to result from the F-actin reorganization, similar to the current study.

AuNPs are known as the most biocompatible and safest type of metal-based nanoparticles. There are several studies on nonbiologically produced AuNPs showing that the cellular uptake of AuNPs and the extent of cellular damage depend on the dose, size, and shape of AuNPs and exposure time.³³ For example, it was shown that cell penetration of AuNPs with sizes of 45 and 13 nm occurred via clathrin-mediated endocytosis and phagocytosis, respectively.³⁴ In addition, the changes in cellular activities were transient and the functional effect on cells could be reversed after the removal of AuNPs.³⁴ For nonbiological AuNPs, it was reported that larger cells exhibited higher cellular uptake overall but simultaneously, the average uptake per unit area of cells was actually lower.³⁵ On the other hand, our study shows that the total nanoparticle uptake of biologically produced AuNPs depends primarily on a cell type since we detected the highest cellular internalization of AuNPs in cancer cells that were the smallest cells we tested.

Even though we showed that biological AuNPs are safe and suitable for drug delivery,^{15, 36} the intracellular effects of internalized AuNPs are still unclear. Moreover, most of the findings on the cellular effects of nonbiologically produced AuNPs relate to a short experimental period of 2–12 h.^{37, 38} The recent study attempted to understand the effect of AuNPs on the first cellular response to nanoparticles, represented by actin filament reorganization up to 48 h, revealing the long-term effect on cell function.

The main results of our study are (1) the biologically produced AuNPs had dose and time-dependent toxicity to the tested cells, (2) the biologically produced AuNPs penetrated all types of tested cells and the uptake depended on cell type and exposure time: cancer cells internalized remarkably more AuNPs than normal fibroblasts and the longer exposure time increased the amount of internalized AuNPs, (3) AuNPs induced time-dependent secretion of proinflammatory cytokines by macrophage cells (RAW264.7), and (4) biological AuNPs affected the reorganization of F-actin filaments with respect to cell type and exposure time.

Although the highest amount of AuNPs was detected in the cancer cell line, only minor changes in growth and viability were observed. Normal fibroblast cells (NIH/3T3) with the lowest amounts of internalized AuNPs showed unchanged viability, but there were apparent changes in the F-actin organization. This unfavorable effect of bare, nonfunctionalized AuNPs on normal fibroblasts can be avoided by proper functionalization and can trigger no remarkable organ damage.³⁵

4 CONCLUSIONS

n conclusion, although there are many studies on the uptake of nonbiologically produced AuNPs with different sizes and shapes,^39-42 this is the first report on the uptake of 15 nm biological AuNPs by different mammalian cell types. This study showed that the biologically produced AuNPs can penetrate all cell types and reorganize actin while exhibiting low and time-dependent toxicity. The cellular uptake of biological AuNPs and the extent of F-actin reorganization depend on the exposure time and cell type. The highest and lowest amounts of AuNPs were found in the cancer 4T1 and normal fibroblast NIH/3T3 cell lines, respectively. The AuNPs triggered the secretion of TNF-α and IFN-γ in RAW264.7 cells. The cytokine secretion is likely transient as seen from quickly decreased levels of IFN-γ after the maximum release peak, but it has to be further tested. However, using the biological AuNPs as drug carriers probably requires particular functionalization to avoid unintended immune activation as seen in the conventional AuNPs. Further understanding of the uptake mechanism of biologically produced AuNPs will be useful to evaluate the efficacy of this type of nanoparticles for therapeutic applications.

5 ACKNOWLEDGMENTS

This work was supported by OP JAK—MSCA Fellowships CZ (Institute of Microbiology of the CAS, v. v. i.) No. CZ.02.01.01/00/22_010/0002357. We thank CMS-Biocev (“Biophysical techniques, Crystallization, Diffraction, Structural mass spectrometry”) of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042) and CZ.02.1.01/0.0/0.0/18_046/0015974. We would also like to thank Miroslav Kolařík, the head of the Laboratory of Fungal Genetics and Metabolism, for his help with fungal cultivation. We acknowledge the Institute of Analytical Chemistry of CAS, v.v.i. supported by Institutional Research Plan No. RVO: 68081715. We acknowledge the Electron Microscopy Core Facility, IMG CAS, Prague, Czech Republic, supported by MEYS CR (LM2023050 Czech-BioImaging) and ERDF (Project CZ.02.1.01/0.0/0.0/18_046/0016045) for their support with obtaining scientific data presented in this article. The authors thank the Core of Cytometry and Microscopy, Institute of Microbiology CAS for their support with microscopy measurements and the Technical University of Liberec for assistance with image analysis.

CONFLICT OF INTEREST STATEMENT

All the authors state no conflict of interest.

Open Research

DATA AVAILABILITY STATEMENT

All data is presented in the manuscript or available from the corresponding author.

Supporting Information

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Citing Literature

Volume71, Issue4

August 2024

Pages 766-778

Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells