Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2020 Aug 17;5(33):20983–20990. doi: 10.1021/acsomega.0c02455

Human Nontumorigenic Microglia Synthesize Strongly Fluorescent Au/Fe Nanoclusters, Retaining Bioavailability

Richard T Agans †,‡, Cayley E Dymond ‡, Rebecca E Jimenez §, Nile J Bunce §, Karima J Perry §, Richard L Salisbury †,‡, Saber M Hussain ‡,*, Raj K Gupta ∥, Shashi P Karna §,*
PMCID: PMC7450618  PMID: 32875234

Abstract

graphic file with name ao0c02455_0006.jpg

The ability for cells to self-synthesize metal-core nanoclusters (mcNCs) offers increased imaging and identification opportunities. To date, much work has been done illustrating the ability for human tumorigenic cell lines to synthesize mcNCs; however, this has not been illustrated for nontumorigenic cell lines. Here, we present the ability for human nontumorigenic microglial cells, which are the major immune cells in the central nervous system, to self-synthesize gold (Au) and iron (Fe) core nanoclusters, following exposures to metallic salts. We also show the ability for cells to internalize presynthesized Au and Fe mcNCs. Cellular fluorescence increased in most exposures and in a dose dependent manner in the case of Au salt. Scanning transmission electron microscopic imaging confirmed the presence of the metal within cells, while transmission electron microscopy images confirmed nanocluster structures and self-synthesis. Interestingly, self-synthesized nanoclusters were of similar size and internal structure as presynthesized mcNCs. Toxicity assessment of both salts and presynthesized NCs illustrated a lack of toxicity from Au salt and presynthesized NCs. However, Fe salt was generally more toxic and stressful to cells at similar concentrations.

Introduction

Nanoparticles (NPs) and nanoclusters (NCs) offer interesting avenues toward biodetection, sensing, labeling, and tissue imaging via intracellular reporting.1−3 Gold (Au) and iron (Fe) are prime materials for imaging applications as they possess unique and tunable plasmon resonance properties (gold) and unique magnetic properties, high signal-to-noise ratio, and increased spatial resolution (for iron), exhibit intense photoluminescence, high stability and biocompatibility, and do not suffer as easily from photobleaching and blinking as fluorescent dyes.1−6

Synthesis of nanoclusters in vitro has long been possible and can be achieved through multiple methods.7 These methods fall under two large synthesis categories: top-down and bottom-up. An example of top-down methodologies is “parent” nanoparticles that can be slowly degraded into clusters through core and surface etching where ions are chemically removed from the parent particle followed by protein stabilization.8−11 Bottom-up methodologies involve the building of nanoclusters from metal precursors12−14 in which a charged metallic salt is reduced to a neutral state before being protein stabilized. Regardless of methodology, it appears that in vitro synthesis of nanoclusters is tunable in the number of atoms making up the cluster (ranging from <10 to >100 atoms per cluster) and fluorescence/luminescence signal (from blue to near infrared).7,15

Larger nanoparticles have been long investigated for intracellular applications; however, there is a risk of accumulation within the cell and inhibition of normal cellular processes.4,16,17 The use of NCs promises to avoid this, especially if NC synthesis happens within cells. Protein-synthesized AuNCs in vitro have been shown to internalize into cervical, kidney, and fibroblast cell lines without negatively impacting cell viability while producing a strong green fluorescence localized to the cell nucleus.18 Wang et al. were able to image tumors in mice via AuNC fluorescence within and around tumorigenic tissue.19 Indeed, it has been previously shown that intracellular proteins and peptides help synthesize and stabilize NCs from their precursors.4,14,20,21 Although the biochemical mechanisms for cellular protein-mediated synthesis and stabilization of metal NCs are not yet known and fully understood, considerable advancements have been made in recent years. In situ-synthesized NCs are generally believed to be formed via reduction of metallic ions that have diffused through the cell membrane, which are then sequestered via proteins and peptides into sites of nucleation to create NCs.20−22 As such, any nonreduced ions are then free to diffuse out of the cell without any cellular interference, essentially being viewed as a “friend” via the presence of endogenous proteins.4,19

To date, in situ synthesis of NCs has been carried out primarily in tumorigenic cell models.4,19 In situ-synthesized NCs have been reported to produce fluorescence in both the green and near-IR frequencies and are not necessarily stabilized spontaneously in cases requiring additional steps to produce the final NC product.19,20,23 Previous reports attributed increased levels of reactive oxygen and nitrogen species (ROS and RNS, respectively) to the synthesis of NCs because of their reducing capability. However, in a subsequent study, we demonstrated the formation of fluorescent AuNCs in nontumorigenic mouse microglia in a non-ROS-dependent mechanism.4 Microglia are a major component of the central nervous system’s immune system, offering first response to exogenous drugs as well as providing immune regulations of other glial cells, namely, astrocytes and oligodendrocytes.24 Therefore, understanding the interaction, uptake, and response of glial cells to nanoscale materials, such as the fluorescent metal nanoclusters, is critical in developing nano-biotechnologies for future brain studies.

Building upon our previous work,4 here we demonstrate that nontumorigenic human microglia (Hmic) are capable of synthesizing AuNCs and FeNCs with strong fluorescence in a similar ROS-independent manner. We further demonstrate the ability of Hmics to uptake presynthesized fluorescent AuNCs and FeNCs as well and the range in which Au and Fe salts and presynthesized NCs become cytotoxic or induce cellular stress. To the best of our knowledge, this is the first application of in situ AuNC and FeNC synthesis and presynthesized AuNC and FeNC treatment in a human nontumorigenic neuronal cell model. This work continues to support the potential benefits of fluorescent metal NCs to neurological research and further directs the need for determination of biochemical mechanism of in situ NC synthesis.

Results

In Situ NC Synthesis and Internalization of Presynthesized NCs

Synthesis of nanoclusters within cells was verified by TEM of cellular protein isolates following 24 h of exposure to 10 μM salt. Nanoclusters were confirmed as the dark lattice containing spots against a light background and were approximately 5 nm in diameter (Figure 1). Individual spacing between gold atoms was measured at 0.22 nm ± 0.03, which agrees with previously measured AuNC lattice distances.4,18,19 Nanoclusters were also evident following FeCl3 exposures (Figure S1), with a diameter less than 5 nm and lattice spacing at 0.21 nm ± 0.02. Similarly, TEM images revealed identifiable Au/Fe NCs within cellular protein isolates (Figure S1) with a measured interlattice spacing of 0.24 nm ± 0.05 and 0.25 nm ± 0.04 for Au and Fe, respectively. It is important to point out that due to its low standard electrode potential, the Fe atoms could be easily oxidized in an aqueous medium to form Fe2O3/Fe3O4. However, we were unable to clearly identify the iron oxide formation. Interestingly, shape and interlattice distances between self-synthesized and presynthesized nanoclusters were quite similar.

Figure 1.

Figure 1

Open in a new tab

Transmission electron microscopy of cell following exposure to HAuCl4. Clusters were verified by measuring the distance between lattices (yellow lines).

Cellular Uptake

In order to verify that cells uptake Au and Fe, STEM was used to visualize cells following exposures of 24 h to 10 μM of either HAuCl4, FeNC, or AuNC, while EDS was utilized to verify presence of metallic elements. Gold and iron aggregates were identified in cells following exposures to HAuCl4 and FeCl3 (Figure 2A–C and Figure S2), with corresponding peaks identified in the spectral analyses (Figure 2D and Figure S2). Similarly, exposure to presynthesized Au and Fe NCs also resulted in aggregation of the corresponding metals within cells (Figures S3 and S4) with correct peaks designated by the spectral analyses (Figures S3 and S4).

Figure 2.

Figure 2

Open in a new tab

Scanning transmission electron microscopy of the cell following HAuCl4 exposure. (A) Gold was visible as bright white against the dark background (red arrow). (B–D) Gold appeared sequestered within vesicles and was verified through EDX analysis.

Cellular Fluorescence

The uptake of HAuCl4 and presynthesized nanoclusters is believed to increase cellular fluorescence through either stabilization and synthesis of metal nanoclusters (following HAuCl4 or FeCl3 exposure) or accumulation of presynthesized nanoclusters within the cell. Confocal microscopy analysis confirmed increased cellular fluorescence 48 h post exposure (Figure 3 and Figures S5 and S6). Specifically, exposure to HAuCl4 resulted in a dose-dependent augmentation of cellular fluorescence compared to nontreated cells, while addition of presynthesized nanoclusters presented an opposite trend with lower concentrations promoting a greater fluorescence signal. Interestingly, the addition of FeCl3 did not appear to consistently change cellular autofluorescence (Table S1). Significant differences in fluorescence were determined for both low dose presynthesized nanoclusters and 100 μM HAuCl4 treatments (p < 0.001).

Figure 3.

Figure 3

Open in a new tab

Cellular fluorescence following 48 h of exposures to gold(III) chloride. (A) Nontreated cells exhibited low-level autofluorescence. (B, C) HAuCl4 exposure at 10 and 100 μM produces a dose-dependent increase in fluorescence.

Cytotoxicity of Metallic Salts and Presynthesized Nanoclusters

The toxicity of HAuCl4, FeCl3, and Au/Fe NCs was determined following exposure of cells to 10, 25, 50, 100, or 1000 μM HAuCl4 or 10, 100 nM, 1, 10, or 100 μM Au/FeNCs for 4, 8, 12, 24, 48, and 72 h. HAuCl4 exposures lower than 100 μM did not produce any change in cell viability compared to controls; however, at 100 and 1000 μM cells experience a significant drop in viability (Figure 4A). Specifically, 1000 μM exposures resulted in a 17% decrease in viability within 4 h (70.98 vs. 87.87% for nontreated p < 0.0001), with 29% of the population falling within an apoptotic subpopulation (28.92%, p < 0.0001) (Figure S7A). After 8 h following exposures, these cells dropped to 13.76% in viability (versus 88.52% for nontreated, p < 0.0001). The apoptotic population within the 100 μM decreased between 4 and 24 h, while the death population substantially increased in the first 8 h (Figure 4A and Figures S7A and S8A). Within 12 h, only 2.76% of cells remained viable, which was significantly different from all other exposures (nontreated: 94.28%, 10 μM: 94.18%, 25 μM: 90.51%, 50 μM: 91.53%, and 100 μM: 81.61%). Cells exposed to 10 μM HAuCl4 did not exhibit such an increase in cell death as those in the 100 μM group; however, they did possess a higher apoptotic population than the other exposure groups (Figure 4A and Figure S7A). Exposure to FeCl3 imposed a significant loss in viability, compared to controls, as early as 4 h post treatment all the way out through 72 h (Figure 4B, p-values < 0.0001). Interestingly, lower concentrations of FeCl3 appeared to rebound in viability beyond 24 h (10 μM: 27.27% at 24 h vs. 63.66–66.27% at 48 and 72 h; 25 μM: 31.96% at 24 h vs. 52.91–56.30% at 48–72 h), while higher concentrations saw a similar rebound beyond 48 h (50 μM: 8.62% at 48 h vs. 57.25% at 72 h; 100 μM: 6.87% at 48 h vs. 47.15% at 72 h). General apoptotic trends do not support a concentration versus time dependency; however, they present unique profiles within each measured time point and support consistent stress following FeCl3 exposure (Figure S7B). Furthermore, cells that were captured within the dead sample fraction were inversely correlated with viable cells. At lower concentrations, FeCl3 had greatest impact on cell death within the first 24 h of exposure (10 μM: 41.48% peak at 24 h; 25 μM: 42.57% peak at 8 h) followed by either drastic (10 μM: 6.73% at 48 h) or a steady and incremental reduction over time (25 μM: 32.18, 25.95, and 10.79% at 12–48 h) (Figure S8B). Higher concentrations still exhibited large dead populations at 48 h but began to show signs of reduction by 72 h (50 μM: 62.09 vs. 10.07%, 100 μM: 57.94 vs. 11.74%, and 1000 μM: 81.61 vs. 76.75%) (Figure S8B).

Figure 4.

Figure 4

Open in a new tab

Cell viability following exposure to metal salts and nanoclusters. (A) HAuCl4 concentrations below 100 μm were not immediately toxic, while higher concentrations induced a loss of viability in a dose-dependent manner. (B) FeCl3 concentrations were quite toxic beginning at 4 h. (C) Gold nanoclusters produced a significant decrease in viability at all concentrations within 72 h. (D) Iron nanoclusters consistently reduced viability at higher concentrations. Viability is presented as percentage of total counted cells ± S.E.M. significance was calculated via two way ANOVA with Bonferroni multiple hypothesis correction analysis and presented against nontreated conditions within each time point at p < 0.05(*), 0.01(**), and 0.0001(‡).

Nanoclusters containing Au or Fe cores elicited variable responses with respect to cellular viability. AuNCs appeared to elicit a significant decrease in cell viability within 12 h at concentrations greater than 10 μM (nontreated: 90.17%, 10 μM: 84.30%, and 100 μM: 84.32%, p < 0.0001). After 24 h, cells treated with AuNCs below and above 10 μM were drastically less viable compared to nontreated cells (p < 0.01 to p < 0.05). Interestingly, after 72 h, all AuNC exposures resulted in substantial loss to cell viability (nontreated: 94.92%, 10 nM: 88.45%, 100 nM: 87.67%, 1 μM: 82.13%, 10 μM: 85.00%, and 100 μM: 78.4%; all p-values < 0.0001) (Figure 4C). The population of cells undergoing apoptosis significantly increased under high AuNC concentrations following 12 h (nontreated: 8.67%, 10 μM: 14.28%, and 100 μM: 13.95%, p < 0.0001) (Figure S7C). Similar to the viable cell population, 48 h of exposure saw variable responses between the different metals and exposure doses; however, all exposures were significantly greater in apoptotic cells compared to nontreated ones (all p-values <0.0001). Cells within the dead population followed trends seen in both viability and apoptotic fractions. However, little of the exposures were drastically different from the amount of dead cells in the nontreated setup, with the exception of 72 h of exposure (Figure 4C and Figure S8C).

Exposure to FeNCs resulted in a quick loss to viable cell population within 4 h. However, this loss appeared rescued within 8 h (4 h - nontreated: 91.92% and 100 μM: 81.08%, p < 0.001; 8 h - nontreated: 94.45% and 100 μM: 89.92%, p < 0.001). The only consistent response among the viable cell population was seen among cells exposed to 10 and 100 μM (Figure 4D). Exposure to 100 μM FeNCs resulted in a dose-dependent decrease in viability between 24 and 72 h (82.87, 81.03, and 73.27% for 24, 48, and 72 h, respectively), while greater than 90% of the nontreated population remained viable. Similarly, 10 μM exposures appeared to create a threshold response in cells with a larger decrease in loss of viability between 48 and 72 h, compared to the other time points (87.87 and 82.68% for 48 and 72 h versus range of 91–88% in other time points) (Figure 4D). The apoptotic cell fraction exhibited similar patterns to the viable population, however via increased fraction size, with 100 μM exposures creating an initial spike in apoptotic response, and then increasing in a dose-dependent manner between 8 and 72 h (9.22–25.88% over the time course). In all exposures, cells appear to exhibit a bimodal response depending on the concentration, yet eventually producing a concentration-dependent increase in the apoptotic fraction at 72 h (Figure S7D). Unlike HAuCl4 and FeCl3, AuNC and FeNC exposures did not appear to consistently and significantly induce cell death (Figure S8D).

Reactive Oxygen Species Synthesis and Cellular Stress

The degree of cellular stress via ROS generation was measured using H2DCFDA fluorescent probe. Following HAuCl4 exposure, there was a notable increase in the fluorescent signal within 4 h. However, the signal decreased by 6 h before beginning to increase again after 8 h (Figure 5). Following 8 h, the fluorescent signal for 1000 μM-treated cells continued to increase through 48 h, at which point there was a plateau in the signal for the exposure duration. Although all other exposures similarly resulted in an increased fluorescent signal, none of them reached a level of significance as in the case for 1000 μM. Cell exposure to FeCl3 resulted in a quick ROS response for a high concentration (1000 μM) within 4 h, while concentrations below this only resulted in observable increases in ROS production beginning around 12 h (Figure S9A). Within 24 h, high FeCl3 exposures produced a substantial dose-dependent increase in ROS when compared to controls (p < 0.001 for 1000 μM). Furthermore, while all exposures ultimately led to increased ROS, no significant differences were observed between exposures below 100 μM.

Figure 5.

Figure 5

Open in a new tab

Reactive oxygen species production following exposure to gold(III) chloride. Cells exhibited a bimodal ROS response to exposures, in combination with dose dependence. Significant ROS production was seen for 1000 μM exposure compared to nontreated samples. Values are presented as mean relative fluorescence signal ± S.E.M. Significance was calculated via two way ANOVA with Bonferroni multiple hypothesis correction analysis and presented against nontreated conditions within each time point at p < 0.05(*), 0.01(**), and 0.0001(‡).

Cell exposure to AuNCs did not result in a strong production of ROS (Figure S9B). No observable difference was recorded within the initial 8 h of exposure with a notable, concentration dependence however insignificant, increase in ROS production from 8 h throughout the exposure period. In contrast, FeNCs produced a quick increase in ROS production within the first 2 h of exposure, with a significant production of ROS at 100 μM concentration (p < 0.0001) compared to the nontreated group. In fact, within the first 8 h, there was a concentration-dependent ROS production similar to the later AuNC time points. Interestingly, while 100 μM FeNC exposure produced a quick increase in ROS production, this increase in RO species remained steady throughout the rest of the exposure period. Lower concentrations of FeNCs still maintained increased ROS production, but only 10 μM resulted in a substantial increase (Figure S9C) (p < 0.01, 6 h; p < 0.0001, 8–72 h). Following 24 h of exposure to 10 nM FeNCs, ROS production in cells was notably increased, more so than what was observed for 100 nM and 1 μM exposures.

Discussion

Cells exposed to gold salt at concentrations greater than 50 μM resulted in a significant loss in viability within the first 8 h. However, only cells exposed to 100 μM gold salt saw an increased apoptotic fraction when compared to all other treatments. Exposure to 1000 μM gold salt appeared to not only stress cells but also induce cell death in lieu of apoptotic driven response. Exposures to FeCl3 produced rapid ROS production only at higher concentrations yet exhibited increased dead populations in lieu of apoptosis. Presynthesized FeNCs exhibited early decreases in cell viability at a 100 μM concentration at 4 h. The cell viability appear to rebound at 8 h but then continued to decline through 72 h, becoming significantly decreased with 48 h of exposure. Exposure to 10 μM FeNCs did not produce a significant loss of cell viability until 72 h post exposure. Exposure to AuNCs did not produce any significant changes to cell viability until 72 h post exposure and at a high concentration, which is in disagreement with previous works highlighting toxicity of gold nanoparticles in the nanomolar-to-micromolar range.17,25,26 Such a difference in toxicity could be attributed to size, charge, or protein corona around the clusters versus particles and cell type as the majority of previous works have been attempted in tumorigenic cell models.25−27 Cells appeared to exhibit higher apoptosis following exposure to a high concentration of FeNCs early on and then present with a dose-dependent increase in apoptotic positive cells over the exposure time. Cells exposed to AuNCs did not exhibit a substantial apoptotic population. Regardless of the concentration of presynthesized nanoclusters exposed to cells, none induced a high degree of cell death, suggesting that under these conditions presynthesized nanoclusters and physiologically relevant concentrations of gold salt induce cell stress but are not overtly cytotoxic.

Reactive oxygen species production following exposure to gold salt was only significantly higher in cells treated with 1000 μM gold salt; however, this elicited an initial increase after the first 8 h following exposure. Similar to cell apoptosis response, ROS production followed a dose-dependent trend with 1000 μM, significantly increasing cellular ROS response, whereas the other gold salt concentrations did not. These results together with cell viability data appear to suggest that, with the exception of cells exposed to 1000 μM gold salt, cell apoptosis in response to gold salt exposure is independent of ROS production. Presynthesized FeNCs also induced a significant ROS response. All concentrations of FeNCs elicited an early and pronounced ROS response. However, only the higher concentrations (10 and 100 μM) maintained this response with 100 μM FeNC, plateauing within 6 h of exposure and 10 μM FeNC continuing to increase in response through the exposure time. Similarly, the low dose 1 nM FeNC produced a noticeable increase in ROS production, which could suggest threshold effects to Fe exposures. Exposure to AuNCs did not elicit much of a ROS response within the first 24 h of exposure but then exhibited a dose-dependent response through the end of the exposure time.

Uptake of Au and Fe was verified through STEM with energy dispersive X-ray spectroscopy (EDX) of cells following exposure. STEM imaging highlighted the presence of both Au and Fe in cells following interactions with HAuCl4, FeCl3, or presynthesized nanoclusters. In all exposures, Au appeared to be associated with or within intracellular vesicles, which could highlight active import/endocytotic pathways.28

Cellular fluorescence increased following exposure to both gold salt and presynthesized nanoclusters but not iron salt. Although nontreated cells produced a slight autofluorescence, exposure to gold salt produced an increase in cellular fluorescence that was positively correlated with the amount of gold salt encountered by cells. In a similar manner, cells exposed to presynthesized nanoclusters increased internal fluorescence; however, the fluorescence intensity was inverse to the concentration of nanoclusters administered following 24 h of exposure. Cellular fluorescence between iron and gold nanoclusters were similar across similar treatments, while fluorescence following gold salt treatment was similar at a lower concentration; however, it is significantly greater at high concentration treatments. Regardless of treatment, cellular fluorescence appeared to be localized within the cytoplasm and excluded from the cell nucleus, which is in agreement with the previous work in mouse microglia and liver tissues.4,19 The concomitant increase in cellular fluorescence could be attributed to activation of the microglia, which has been shown to result in an upregulation of glutamate production and secretion, which may also contribute to the difference in the nanocluster stabilization and presence across different exposures.29−31 Furthermore, fluorescence was visualized at 523 nm (green), which corresponds well with cell autofluorescence but has also been attributed to particular metal atoms (at the NC core) and coatings of synthesized NCs.2,3,28,32 Therefore, differences in protein stabilizers or ion amounts could result in lower fluorescence or perhaps alterations in emission wavelengths.2

Existence of nanoclusters was verified through TEM of intercellular protein fractions. Nanoclusters persisted as spherical entities with diameters less than 10 nm and interlattice spacing less than 0.5 nm. In all treatments, the positive identification of nanoclusters was possible. Interestingly, the distance between lattices was slightly different between the presynthesized and in situ-synthesized clusters, suggesting that different proteins may be involved in the stabilization and synthesis between in situ and in vitro methods.6,11,14,19−21

Conclusions

We show here for the first time the ability for human nontumorigenic neural cells to synthesize metal-core nanoclusters following exposure to metallic salts. We have also determined that cells are capable of internalizing presynthesized metal-core nanoclusters. Furthermore, we have illustrated that following exposure/uptake, cells can be imaged owing to an increase in cellular fluorescence. Finally, we have investigated the cytotoxic range of metallic salts and metal-core nanoclusters. Our success with in situ synthesis of nanoclusters in nontumorigenic cells contradicts other works in noncancerous cell lines and supports a unique biochemical mechanism for stabilization and synthesis in situ.11 Such a unique mechanism is theorized to lie within the glutamate synthesis pathway,4,11 which deserves further scrutiny in future works. The results presented here provide the first evidence of biocompatibility of low micromolar (10–100 μM) self-synthesized nanocluster with healthy non-neuronal brain cells. Glial cells are the first cells to respond to exogenous drugs and compounds and help regulate the immune systems of other important cells in the glial family in CNS.24 The fact that the mNCs do not induce reactivity, hyperproliferation, or other adverse responses in healthy human microglial (HMic) cells at a micromolar concentration, as shown in this study, offers a unique capability to probe and study glial cells using nanotechnology. The present work further expands the biomedical applications Au and Fe NCs to nontumorigenic human cells.

Materials and Methods

Cell Culture

Human microglial cells, immortalized with viral SV40 gene, were expanded in Dulbecco’s modified eagle’s growth medium (DMEM) (Gibco) containing 10% nonheat inactivated fetal bovine serum (NHI-FBS) (Gibco) and 1% penicillin/streptomycin (Sigma). Cells were maintained under a humidified atmosphere of 10% CO2 at 37 °C. For all experiments, cells were seeded into multiwell tissue culture plates at 40,000 cells per cm2. Once seeded, cells were incubated for 24 h to ensure that adherence and appropriate morphology were attained, at which point the medium was changed and experiments commenced.

Nanocluster Synthesis

The Au and Fe NCs were synthesized using metal salts (HAuCl4 and FeCl3) and egg white albumen (EW), which serves both as a reducing agent and a cluster scaffold. Chicken eggs were purchased from the local supermarket. The metal salts and sodium hydroxide (NaOH) were purchased from Sigma Aldrich. Stock solutions of salts and EW/H2O mixtures were made fresh for each experiment. Gold nanoclusters were synthesized following the protocol described by Li et al.33 The synthesis protocol and preliminary results on the structure and photophysical properties of the EW-synthesized Au and Fe NCs have been reported earlier.34,35 Briefly, aqueous solution of HAuCl4 of different concentrations, namely, 0.1, 0.5, 1, 5, and 10 mM was prepared and dispersed in a 1:1 ratio of salt solution to a 1:1 solution of water to egg albumen. The resulting solutions were then mixed for 2 min before a solution of NaOH was added in the amount 1 mL per 10 mL of the total solution. The vials of mixed solution were then wrapped in tin foil and incubated at room temperature for 24 h. The FeNC synthesis followed a similar protocol. Then, 5 mL of a 1:1 dilution of EW and H2O was made and mixed with 5 mL of FeCl3. For this experiment, five different concentrations of the salt solution, namely, 10, 5, 1, 0.5, and 0.1 mM were used. Upon combining the aqueous egg white with the metal salt, the resulting solutions turned turbid. The solution was then manually mixed for 2 min and followed immediately by the addition of 2 mL of 1.0 M NaOH. Upon the addition of NaOH, the solution became transparent. The samples were then left to incubate for 24–48 h at room temperature in the dark.

Exposures

Following 24 h of incubation, the growth medium was replaced with a fresh medium containing either gold or iron salt or pre-synthesized gold or iron nanoclusters. Gold and iron salt exposures were performed at concentrations of 1, 10, 100, and 1000 μM (higher concentrations were utilized to ensure cellular response for comparison as gold salt appears to possess low cytotoxicity), while presynthesized gold and iron nanoclusters were administered at 10 and 100 nM, and 1, 10, and 100 μM concentrations.

Viability, Apoptosis, and Necrosis

Cell viability, apoptosis, and necrosis were determined using the Viacount assay (Millipore). The culture supernatant was collected, and cells were detached from the culture surface using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Following cell detachment, trypsin was inactivated via addition of the collected growth medium. Detached cells were centrifuged at 100 ×g for 7 min to pellet cells, and the supernatant was discarded. Cells were resuspended in 100 μL of DMEM, subsequently diluted 1:1 with Viacount reagent (120 μL of total volume) in 96-well plates, and incubated in the dark at room temperature for 5 min. Following incubation, the 96-well plate was loaded into the Guava EasyCyte HT and analyzed for viability, apoptosis, and necrosis using built-in Viacount assay.

Reactive Oxygen Species

The production of ROS was measured in a 48-well microplate using the fluorescent 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) ROS probe (Fisher Scientific). Plated cells were incubated in phosphate-buffered saline (PBS) containing 100 μM H2DCFDA for 30 min at 37 °C. Following incubation, PBS was removed, and cells were treated with gold nanoclusters (GNCs) accordingly. At designated time points (0, 4, 6, 8, 12, 24, 48, and 72 h), the microplate was placed in a Flexstation3 (Molecular Probes), and the probe was excited at 488 nm, and emission was read at 538 nm wavelengths.

Nanocluster Uptake

Scanning transmission electron microscopy (STEM) was utilized to determine uptake of metallic compounds into cells. Samples were fixed in 4% paraformaldehyde for 2 h, washed with 1X PBS to remove any remaining aldehydes, stained with 4% osmium tetroxide for 2 h at room temperature, washed with ultrapure water, then dehydrated via serial incubation with 35, 50, 70, 90, and 100% ethanol (EtOH). Following dehydration, samples were suspended in EtOH/LR white resin (1:1, vol/vol) overnight at room temperature, then 100% LR white resin for 2 h. Samples were next transferred to BEEM capsules in 100% LR white resin for 1 h then cured at 60 °C under negative pressure (−20 psi) for 24 h. Nanocluster confirmation was obtained on a JEOL JEM-2100 LaB6 TEM. Cells were lysed with 1X RIPA buffer for 10 min followed by centrifugation at 15,000 ×g for 10 min. Samples were then immediately frozen at −80 °C until imaging. The sample for TEM imaging were prepared by dropping 10 μL of the solution onto a suspended 400 mesh ultrathin lacy carbon grid using a micropipette. Suspension is used to prevent clusters from contaminating both sides of the grid. After being left to dry for an hour, images were taken on the TEM at 200 kV.

Fluorescence Microscopy

Cellular fluorescence was determined using an Olympus Fluoview 1000 fluorescent microscope. Cells were stained for nuclear DNA with 4,6-Diamidino-2-phenylindole (DAPI). Cells were first fixed with paraformaldehyde (4%) for 15 min followed by washing with PBS. Cell membranes were then permeabilized using Triton-X 100 (0.1%) for 15 min, washed with PBS, and blocked using BSA (1%) for 30 min. Cells were then incubated in DAPI stain for 7 min. Cells were imaged for nuclear DNA using 405 nm laser and nanocluster fluorescence via 488 nm laser.

Statistical Analyses

All analyses were performed in GraphPad Prism 7 using 2-way ANOVA with Bonferroni’s multiple hypothesis adjustment where applicable data are expressed as average ± standard error of the mean (S.E.M.). Statistical significance was considered met if p-values were less than 0.05. ANOVA F-values and associated degrees of freedom can be found in Table S2.

Acknowledgments

S.K. and R.G. would like to thank Mr. Michael J. Leggieri, Jr. at DoD Blast Injury Research Coordination Office for his encouragement and interest in this work.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02455.

  • (Table S1) Cellular fluorescence difference between 48 h treatments, (Table S2) ANOVA summary, (Figure S1) transmission electron microscopy of the cell following exposure to iron salt and presynthesized gold and iron nanoclusters, (Figure S2) scanning transmission electron microscopy of the cell following FeCl3 exposure, (Figure S3) scanning transmission electron microscopy of the cell following presynthesized AuNC exposure, (Figure S4) scanning transmission electron microscopy of the cell following presynthesized FeNC exposure, (Figure S5) cellular fluorescence following 48 h exposures to presynthesized nanoclusters, (Figure S6) cellular fluorescence following 48 h exposures to iron(III) chloride, (Figure S7) apoptotic subpopulations following exposures, (Figure S8) dead subpopulations following exposures, and (Figure S9) reactive oxygen species production following exposure to (a) iron(III) chloride or presynthesized (b) gold and (c) iron nanoclusters (PDF)

Financial support for this work at ARL by DoD Blast Injury Research Program, Medical Research and Materiel Command through agreement #12480607 between ARL and BLAST (06/12/2018) is gratefully acknowledged.

The authors declare no competing financial interest.

Supplementary Material

ao0c02455_si_001.pdf (1.4MB, pdf)

References

  1. Dykman L.; Khl ebtsov N. Gold nanoparticles in biomedical applications: recent advances and perspectives. Chem. Soc. Rev. 2012, 41, 2256–2282. 10.1039/C1CS15166E. [DOI] [PubMed] [Google Scholar]
  2. Matulionyte M.; Dapkute D.; Budenaite L.; Jarockyte G.; Rotomskis R. Photoluminescent Gold Nanoclusters in Cancer Cells: Cellular Uptake, Toxicity, and Generation of Reactive Oxygen Species. Int. J. Mol. Sci. 2017, 18, 378. 10.3390/ijms18020378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Qu X.; Li Y.; Li L.; Wang Y.; Liang J.; Liang J. Fluorescent Gold Nanoclusters: Synthesis and Recent Biological Application. J. Nanomater. 2015, 2015, 784097. 10.1155/2015/784097. [DOI] [Google Scholar]
  4. West A. L.; Schaeublin N. M.; Griep M. H.; Maurer-Gardner E. I.; Cole D. P.; Fakner A. M.; Hussain S. M.; Karna S. P. In situ Synthesis of Fluorescent Gold Nanoclusters by Nontumorigenic Microglial Cells. ACS Appl. Mater. Interfaces 2016, 8, 21221–21227. 10.1021/acsami.6b06624. [DOI] [PubMed] [Google Scholar]
  5. Zheng Y.; Lai L.; Liu W.; Jiang H.; Wang X. Recent advances in biomedical applications of fluorescent gold nanoclusters. Adv. Colloid Interface Sci. 2017, 242, 1–16. 10.1016/j.cis.2017.02.005. [DOI] [PubMed] [Google Scholar]
  6. Huang X.; Zhang F.; Wang Y.; Sun X.; Choi K. Y.; Liu D.; Choi J. S.; Shin T. H.; Cheon J.; Niu G.; Chen X. Design considerations of iron-based nanoclusters for noninvasive tracking of mesenchymal stem cell homing. ACS Nano 2014, 8, 4403–4414. 10.1021/nn4062726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cui M.; Zhao Y.; Song Q. Synthesis, optical properties and applications of ultra-small luminescent gold nanoclusters. TrAC, Trends Anal. Chem. 2014, 57, 73–82. 10.1016/j.trac.2014.02.005. [DOI] [Google Scholar]
  8. Duan H.; Nie S. Etching Colloidal Gold Nanocrystals with Hyperbranched and Multivalent Polymers: A New Route to Fluorescent and Water-Soluble Atomic Clusters. J. Am. Chem. Soc. 2007, 129, 2412–2413. 10.1021/ja067727t. [DOI] [PubMed] [Google Scholar]
  9. Muhammed M. A. H.; Verma P. K.; Pal S. K.; Arun Kumar R. C.; Paul S.; Omkumar R. V.; Pradeep T. Bright, NIR-emitting Au23 from Au25: characterization and applications including biolabeling. Chemistry 2009, 15, 10110–10120. 10.1002/chem.200901425. [DOI] [PubMed] [Google Scholar]
  10. Muhammed M. A. H.; Verma P. K.; Pal S. K.; Retnakumari A.; Koyakutty M.; Nair S.; Pradeep T. Luminescent quantum clusters of gold in bulk by albumin-induced core etching of nanoparticles: metal ion sensing, metal-enhanced luminescence, and biolabeling. Chemistry 2010, 16, 10103–10112. 10.1002/chem.201000841. [DOI] [PubMed] [Google Scholar]
  11. Chen D.; Zhao C.; Ye J.; Li Q.; Liu X.; Su M.; Jiang H.; Amatore C.; Selke M.; Wang X. In Situ Biosynthesis of Fluorescent Platinum Nanoclusters: Toward Self-Bioimaging-Guided Cancer Theranostics. ACS Appl. Mater. Interfaces 2015, 7, 18163–18169. 10.1021/acsami.5b05805. [DOI] [PubMed] [Google Scholar]
  12. Zhu M.; Lanni E.; Garg N.; Bier M. E.; Jin R. Kinetically controlled, high-yield synthesis of Au25 clusters. J. Am. Chem. Soc. 2008, 130, 1138–1139. 10.1021/ja0782448. [DOI] [PubMed] [Google Scholar]
  13. Yuan X.; Luo Z.; Zhang Q.; Zhang X.; Zheng Y.; Lee J. Y.; Xie J. Synthesis of highly fluorescent metal (Ag, Au, Pt, and Cu) nanoclusters by electrostatically induced reversible phase transfer. ACS Nano 2011, 5, 8800–8808. 10.1021/nn202860s. [DOI] [PubMed] [Google Scholar]
  14. Li H.; Zhu W.; Wan A.; Liu L. The mechanism and application of the protein-stabilized gold nanocluster sensing system. Analyst 2017, 142, 567–581. 10.1039/C6AN02112C. [DOI] [PubMed] [Google Scholar]
  15. Udayabhaskararao T.; Pradeep T. New Protocols for the Synthesis of Stable Ag and Au Nanocluster Molecules. J. Phys. Chem. Lett. 2013, 4, 1553–1564. 10.1021/jz400332g. [DOI] [PubMed] [Google Scholar]
  16. Patchin E. S.; Anderson D. S.; Silva R. M.; Uyeminami D. L.; Scott G. M.; Guo T.; Van Winkle L. S.; Pinkerton K. E. Size-Dependent Deposition, Translocation, and Microglial Activation of Inhaled Silver Nanoparticles in the Rodent Nose and Brain. Environ. Health Perspect. 2016, 124, 1870–1875. 10.1289/EHP234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pan Y.; Neuss S.; Leifert A.; Fischler M.; Wen F.; Simon U.; Schmid G.; Brandau W.; Jahnen-Dechent W. Size-dependent cytotoxicity of gold nanoparticles. Small 2007, 3, 1941–1949. 10.1002/smll.200700378. [DOI] [PubMed] [Google Scholar]
  18. Venkatesh V.; Shukla A.; Sivakumar S.; Verma S. Purine-stabilized green fluorescent gold nanoclusters for cell nuclei imaging applications. ACS Appl. Mater. Interfaces 2014, 6, 2185–2191. 10.1021/am405345h. [DOI] [PubMed] [Google Scholar]
  19. Wang J.; Zhang G.; Li Q.; Jiang H.; Liu C.; Amatore C.; Wang X. In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters. Sci. Rep. 2013, 3, 1157. 10.1038/srep01157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gao S.; Chen D.; Li Q.; Ye J.; Jiang H.; Amatore C.; Wang X. Near-infrared fluorescence imaging of cancer cells and tumors through specific biosynthesis of silver nanoclusters. Sci. Rep. 2015, 4, 4384. 10.1038/srep04384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Su M.-N.; Ye J.; Li Q.-W.; Li S.-H.; Ge W.; Chen X.; Jiang H.; Wang X.-M. Novel in situ biosynthesized fluorescent zinc nanoclusters for specific cellular bio-imaging. Chin. Chem. Lett. 2015, 26, 1400–1402. 10.1016/j.cclet.2015.07.021. [DOI] [Google Scholar]
  22. Fu X.; Wang H. Spatial Arrangement of Polycaprolactone/Collagen Nanofiber Scaffolds Regulates the Wound Healing Related Behaviors of Human Adipose Stromal Cells. Tissue Eng., Part A 2012, 18, 631. 10.1089/ten.tea.2011.0069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang M.; Gu X.; Zhang G.; Zhang D.; Zhu D. Continuous colorimetric assay for acetylcholinesterase and inhibitor screening with gold nanoparticles. Langmuir 2009, 25, 2504–2507. 10.1021/la803870v. [DOI] [PubMed] [Google Scholar]
  24. Greenhalgh A. D.; David S.; Bennett F. C. Immune cell regulation of glia during CNS injury and disease. Nat Rev Neurosci. 2020, 21, 139–152. 10.1038/s41583-020-0263-9. [DOI] [PubMed] [Google Scholar]
  25. Dong L.; Li M.; Zhang S.; Li J.; Shen G.; Tu Y.; Zhu J.; Tao J. Cytotoxicity of BSA-Stabilized Gold Nanoclusters: In Vitro and In Vivo Study. Small 2015, 11, 2571–2581. 10.1002/smll.201403481. [DOI] [PubMed] [Google Scholar]
  26. Soenen S. J.; Manshian B.; Montenegro J. M.; Amin F.; Meermann B.; Thiron T.; Cornelissen M.; Vanhaecke F.; Doak S.; Parak W. J.; De Smedt S.; Braeckmans K. Cytotoxic effects of gold nanoparticles: a multiparametric study. ACS Nano 2012, 6, 5767–5783. 10.1021/nn301714n. [DOI] [PubMed] [Google Scholar]
  27. Wang J. Y.; Chen J.; Yang J.; Wang H.; Shen X.; Sun Y. M.; Guo M.; Zhang X. D. Effects of surface charges of gold nanoclusters on long-term in vivo biodistribution, toxicity, and cancer radiation therapy. Int. J. Nanomed. 2016, 11, 3475–3485. 10.2147/IJN.S106073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Fernández T. D.; Pearson J. R.; Leal M. P.; Torres M. J.; Blanca M.; Mayorga C.; Le Guével X. Intracellular accumulation and immunological properties of fluorescent gold nanoclusters in human dendritic cells. Biomaterials 2015, 43, 1–12. 10.1016/j.biomaterials.2014.11.045. [DOI] [PubMed] [Google Scholar]
  29. Domercq M.; Vázquez-Villoldo N.; Matute C. Neurotransmitter signaling in the pathophysiology of microglia. Front. Cell. Neurosci. 2013, 7, 49. 10.3389/fncel.2013.00049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thomas A. G.; O’Driscoll C. M.; Bressler J.; Kaufmann W.; Rojas C. J.; Slusher B. S. Small molecule glutaminase inhibitors block glutamate release from stimulated microglia. Biochem. Biophys. Res. Commun. 2014, 443, 32–36. 10.1016/j.bbrc.2013.11.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Barger S. W.; Goodwin M. E.; Porter M. M.; Beggs M. L. Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation. J. Neurochem. 2007, 101, 1205–1213. 10.1111/j.1471-4159.2007.04487.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zhang X. D.; Luo Z.; Chen J.; Song S.; Yuan X.; Shen X.; Wang H.; Sun Y.; Gao K.; Zhang L.; Fan S.; Leong D. T.; Guo M.; Xie J. Ultrasmall glutathione-protected gold nanoclusters as next generation radiotherapy sensitizers with high tumor uptake and high renal clearance. Sci. Rep. 2015, 5, 8669. 10.1038/srep08669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Li M.; Yang D. P.; Wang X.; Lu J.; Cui D. Mixed protein-templated luminescent metal clusters (Au and Pt) for H2O2 sensing. Nanoscale Res. Lett. 2013, 8, 182. 10.1186/1556-276X-8-182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Bunce N. J.; Jimenez R. E.; Petsinger V. I.; Gupta R. K.; Karna S. P.. Mixed protein templated fluorescent gold-nanoclusters allow folic acid sensing. In 2017 IEEE 17th International Conference on Nanotechnology (IEEE-NANO); 25–28 July 2017, 2017; pp 74–77.
  35. Jimenez R. E.; Bunce N. J.; Peng Z.; Wang Y.; Gupta R. K.; Karna S. P.. Mixed protein templated iron-nanoclusters exhibit photoluminescence in the visible and near-infrared region. In 2017 IEEE 17th International Conference on Nanotechnology (IEEE-NANO); 25–28 July 2017, 2017; pp 78–82.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao0c02455_si_001.pdf (1.4MB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES