Gold Nanoparticles and Graphene Oxide Flakes Synergistic Partaking in Cytosolic Bactericidal Augmentation: Role of ROS and NOX2 Activity

Gold nanoparticles (GNPs) and graphene oxide flakes (GOFs) exerted significantly (p < 0.0001) supportive roles on the phagocytosis bioactivity of the immune cells of phagocytic nature against the Gram-positive and Gram-negative human pathogenic bacteria Staphylococcus aureus and Escherichia coli. Under experimental conditions, upon bacterial exposure, the combined GNPs and GOFs induced significant clearance of bacteria through phagosome maturation (p < 0.0001) from time-points of 6 to 30 min and production of reactive oxygen species (ROS, p < 0.0001) through the NADPH oxidase 2 (NOX2, p < 0.0001)-based feedback mechanism. The effects of the combined presence of GNPs and GOFs on phagocytosis (p < 0.0001) suggested a synergistic action underway, also achieved through elevated signal transduction activity in the bone-marrow-derived macrophages (BMDM, p < 0.0001). The current study demonstrated that GNPs’ and GOFs’ bactericidal assisting potentials could be considered an effective and alternative strategy for treating infections from both positive and negative bacterial strains.


Introduction
Various nanoparticles (NPs) are employed extensively in nanobiotechnology and biomedicine fields due to their unique physicochemical features of size, shape, and other physicochemical characteristics, including their reactivity toward biocomponents. NPs function as antigen carriers [1], imaging reagents, molecular tags [2], therapeutic, diagnostic, and theranostic entities, and also as part of simultaneous delivery modules [3][4][5]. The multifunctional core-shell nanoplatforms made from gold nanoparticles (GNPs) and graphene oxide flakes (GOFs) for miRNA delivery and their study on the delivery and release profile were reported [6,7]. Notably, as NPs are foreign to the body, macrophages, characterized by plasticity and heterogeneity [8], have to play crucial roles in in vivo situations to recognize, process, and clean-off NPs [9]. When activated, macrophages exert M1 or pro-inflammatory and M2 as anti-inflammatory phenotypes [10], with M1 exerting crucial effector cells during the resistance responses against intracellular pathogens and tumour growths [11,12].

Materials
The gold nanoparticle (GNPs, Cat.NO.741965-25ML) and graphene oxide flakes (GOFs, Cat. NO. 763713-250MG) were purchased from Sigma (Milwaukee, WI, USA). The carbon contents in GOFs were between 42% and 52% for the product according to the specification supplied by Sigma. The GOFs were used after sonication in deionized water to exfoliate into monolayers as an aqueous dispersion. Deionized water, a strong repulsive media for the GOFs, ensured that aggregation did not persist, and the GOFs were available as single-layer flakes. GNPs and GOFs were mixed at a concentration of 10 µg/mL each. Moreover, the samples were freshly prepared and utilized immediately. GNPs and GOFs were characterized by UV-visible spectroscopy in a quartz cuvette and Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), respectively. The GNPs were of 21.33 ± 4.51 nm in diameter size.

Bone Marrow-Derived Macrophages (BMDMs)
Male C57/BL6 mice (7-8 weeks old) were used as the source for isolation of primary BMDMs, following the previously reported method [47]. All experiments using mice were performed in accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised in 1996) and were approved by the Animal Care and Ethics Committee at Biotechnology Division, Applied Sciences Department, University of Technology, Baghdad, Iraq.

Blood Sampling and Preparation 2.3.1. Blood Samples
Fresh samples of blood were taken from 10 healthy donors and collected in heparincoated tubes as described [48] based on Helsinki's declaration and regulation of 1975 as a statement of ethical principles. All the methods were carried out in accordance with all the relevant guidelines and regulations for working with human origin samples. Permission was obtained from the hospitals of the medical city, Baghdad, Iraq. The institutional ethics committee approved all experimental protocols of the University of Technology, Baghdad, Iraq (Ref. No. AS 20-8-01-2020). The participants were informed about the study before the collection of any data or samples. Informed consent was obtained from the study participants.

Human Neutrophils Isolation
Neutrophil isolation medium (5.0 mL) was taken into a centrifuge tube; the blood sample (5.0 mL) was carefully added, and centrifuged (2000 RPM, 10 min, 4 • C). The detailed procedure was followed according to the reported protocol [49]. The cells were counted and adjusted to 1 × 10 6 /mL, and pretreated with GNPs and GOFs (1 h, 10 µg/mL each), followed by infection with S. aureus (multiplicity of infection (MOI)-1:100) and incubation (37 • C, 30 min). The phagocytic index was calculated using the following equation: where a refers to nonphagocytic cells and b refers to phagocytic cells.

Intracellular Bacterial Killing Assay
S. aureus and E. coli were cultured (37 • C) in Lysogeny Broth (LB) broth until the mid-log phase (optical density (OD), 0.4-0.6). Following centrifugation (3500× g, 15 min, 4 • C), sterile Phosphate Buffered Saline (PBS) was used to wash the pellets 3 times. The concentration of the re-suspended bacteria was determined by measuring OD at 550 nm. The BMDMs were used alone or pretreated with GNPs, GOFs, and combined GNPs and GOFs mixture at 10 µg/mL each. The cells were then infected with S. aureus or E. coli at M.O.I. 1:50 and incubated at 37 • C for 60 min. The BMDMs were treated with lysis buffer.
To determine total and extracellular bacterial killings, serial dilutions (10×) of the lysates were incubated with Luria-Bertani (LB) agar plates (37 • C, 24 h). Values of the total and extracellular bacterial killings were used to derive the intracellular bacterial killing values.

Determination of pH of Phagosomes
The luminal pH of the phagosomes was measured according to the previous method [50]. Briefly, double-labelling of the heat-killed isolates of S. aureus and E. coli were performed using 5 mg/mL carboxy-fluorescein-succinimidyl ester (SE), a fluorescent probe with sensitivity to pH; Molecular Probes, Eugene, OR, USA, and 10 mg/mL carboxy-tetraethylrhodamine-SE (a fluorescent probe with no sensitivity to pH; Molecular Probes, Eugene, OR, USA). Next, a pulse process for the isolated BMDMs was conducted with the labelled bacteria (MOI = 1:50; 30 min) and pursued at 37 • C for each of the mentioned periods, the presence and absence of GNPs, and GOFs. Phagosome pH was extracted using the ratio of fluorescein/rhodamine fluorescence using a standard curve.

Assay of Phagosome/Lysosome Fusion
The BMDMs were isolated and plated into 4-well chamber-slides at a concentration of 1 × 10 5 cells/mL in RPMI-1640 media. After 10 h, the BMDMs were pretreated with GNPs and GOFs at a concentration of 10 µg/mL each for 1 h. The Lysotracker Red was used to load the cells (25 nM, 37 • C, 60 min), then incubated with Fluorescein isothiocyanate (FITC)conjugated S. aureus (MOI-1:50, 2 h). The Lysotracker Red was added during infection time. Sterile, cold PBS was used to wash the cells five times, followed by fixation with 4% paraformaldehyde and nuclei staining using 4 ,6-diamidino-2-phenylindole (DAPI). Samples were mounted and examined with a fluorescent microscope (Olympus, Tokyo, Japan). Unfused phagosomes containing FITC-bacteria appeared with a green stain, whereas the lysosomes labelled with Lysotracker appeared red. The fusion of these phagosomes and lysosomes seemed to be yellow as a result of the fusion of the two labelled fluorochromes.

Phagocytosis of pHrodo E. coli BioParticles by BMDMs
The BMDM cells were plated in a 4-well plate. The cells were pretreated with GNPs and GOFs at a concentration of 10 µg/mL each. pHrodo-particles were mixed in 1 × PBS (pH 7.4), vortexed, and 20 µL of the solution was added to the cells. Phagocytosis was allowed for 2 h, and the cells were fixed and stained with DAPI; images were captured using a Zeiss confocal microscope at 400×. The blue colour represented the cells' nucleus, and the red dots showed the E. coli-pHrodo constituent in phagocytes.

NADPH Oxidase Assay
Lucigenin (bis-N-methyl acridinium nitrate; Sigma-Aldrich, USA) was used to determine the NADPH oxidase (NOX2) activity of BMDMs. Briefly, an incubation step (37 • C for different periods) was performed for BMDMs with heat-killed and opsonized isolates of S. aureus and E. coli (each separately with phagocytes/bacteria ratio = 1:50) in the presence and absence of GNPs and GOFs at the concentration of 10 µg/mL each.

Statistical Analysis
The data ere expressed as the mean ± SE. All experiments were carried out in triplicate. Differences between groups were analyzed using one-way ANOVA for the phagocytic index, intracellular killing, and phagocytosis of pHrodo E. coli BioParticles, while two-way ANOVA was used for phagosomal maturation, ROS, and NOX2 followed by a posthoc test using Tukey's multigroup comparisons on GraphPad Prism 8.0.2, San Diego, FL, USA. The data were considered significant if p < 0.05 [51].

Characterization of the GNPs and GOFs
The GNPs and GOFs were analyzed by UV-visible (UV-VIS) spectroscopic method and were found to exhibit the λmax absorption values at 528 nm and 230 nm for the GNPs and GOFs, respectively ( Figure 1A). The morphology of the gold nanoparticles and graphene oxide was studied using SEM ( Figure 1B) and was found to be spherical for the GNPs with a size of 21.33 ± 4.51 nm and flakes for the GOFs. TEM results are shown in Figure 1C. The GNPs were dispersed on the surface of GOFs. Most of the GNPs were located on the surface of the GOFs and did not protrude from the surface, indicating very strong interactions between GOFs and GNPs. GOFs in the aqueous media ruled out any aggregations of GOFs, which are lipophilic. The Raman spectra confirm the difference between the single-and multilayer GO sheets by exhibiting single peaks at~2679 cm −1 [52][53][54].

GNPs and GOFs Increase the Phagocytic Index of Human Neutrophils
GNPs and GOFs were tested for their capability to induce human neutrophils ( Figure  2A-E). The results showed an increase in phagocytic human neutrophil activity, followed by infection with S. aureus after adding the GNPs and GOFs at a concentration of 10 µg/mL each ( Figure 2F). The nanoparticles increased the phagocytic cells' activity known to contribute to IL-8 production [55]. An increase indicated the nanoparticles' capacity as im-

GNPs and GOFs Increase the Phagocytic Index of Human Neutrophils
GNPs and GOFs were tested for their capability to induce human neutrophils (Figure 2A-E). The results showed an increase in phagocytic human neutrophil activity, followed by infection with S. aureus after adding the GNPs and GOFs at a concentration of 10 µg/mL each ( Figure 2F). The nanoparticles increased the phagocytic cells' activity known to contribute to IL-8 production [55]. An increase indicated the nanoparticles' capacity as immune-modulators by increasing the phagocytic cells' activity to engulf the bacteria. Phagocytic cells, such as human neutrophils, were previously shown to possess the ability to induce cytokines, such as IL-8, after treatment with silver nanoparticles (AgNPs) [56].
The entry of the GNPs and GOFs demonstrated significantly (p < 0.0001) increased phagocytosis activity at the cellular scale experiment compared to the control ( Figure 2F). The biological activity details on the antibacterial action of the GOFs, as shown in Figure 2D, did not exhibit any significant morphological changes for the GOFs. According to reference [57], bacteria and GOF interactions are expected to reduce GOFs, resulting in a lesser oxygen component ratio in the GOFs. However, the quantitation of the carbon could not be estimated at the cellular level. The interaction of the bacteria and GOFs affects the morphology and chemical contents of the GOFs. The GOFs are biocompatible for the bacteria, and the reduced GOFs were expected to increase the antibacterial action slightly. The sheets also work as adsorption sites for bacteria, thereby inhibiting the proliferation of bacteria in cellular compartments.

GNPs and GOFs Increase Bacterial Intracellular Killings
The numbers of internalized S. aureus and E. coli in BMDMs were tested following infection in the presence or absence of GNPs and GOFs to gauge the activity of the phagocytosis process, intracellular killings, and comparison between the two conditions of the presence and absence of the nanoparticles. Both the GNP-and GOF-pretreated phagocytes showed a significant increase in their intracellular killings of the ingested, live S. aureus, and E. coli, as exhibited in Figure 3A,B, while combined GNP-and GOF-treated cells demonstrated maximum significant increment (p < 0.0001). Thus, the GNP and GOF-pre-treated phagocytic cells demonstrated an increased bactericidal response to Gram-positive and Gram-negative strains.

The GNPs and GOFs in Phagosome Maturation
Following the ingestion of bacteria, acidification by phagosomes and the fusion of phagosomes and lysosomes are two main characteristics of phagocyte maturation [58]. Our results showed a significant increase in phagosomal acidification of the GNP-and GOF-pretreated BMDMs following the ingestion of S. aureus and E. coli, as compared with the control BMDMs ( Figure 4). Further loading of BMDMs with Lysotracker red, which serves in labelling the late endosomes/lysosomes selectively, allowed monitoring of the maturation events of the S. aureus-FITC ingesting phagosomes through testing their overtime capability of co-localization with Lysotracker red. The results demonstrated the occurrence of this co-localization at 30 min in BMDMs pretreated with GNPs and GOFs. In parallel, most S. aureus-FITC entities showed less co-localization with the Lysotracker in control BMDMs ( Figure 5).
Moreover, following the ingestions of S. aureus, an increase in phagosomal acidification in GNP-and GOF-pretreated BMDMs was observed. Again, the nanoparticle-pretreated macrophages demonstrated significantly increased phago-lysosome fusions in response to S. aureus and other bacterial challenges, such as E. coli. These results, therefore, demonstrated that BMDMs, which were pretreated with GNPs and GOFs, possessed improved phagosome maturation following the ingestion of S. aureus and E. coli. The entry of the GNPs and GOFs demonstrated significantly (p < 0.0001) increased phagocytosis activity at the cellular scale experiment compared to the control ( Figure 2F). ocytosis process, intracellular killings, and comparison between the two conditions of the presence and absence of the nanoparticles. Both the GNP-and GOF-pretreated phagocytes showed a significant increase in their intracellular killings of the ingested, live S. aureus, and E. coli, as exhibited in Figure 3A,B, while combined GNP-and GOF-treated cells demonstrated maximum significant increment (p < 0.0001). Thus, the GNP and GOFpre-treated phagocytic cells demonstrated an increased bactericidal response to Grampositive and Gram-negative strains.

The GNPs and GOFs in Phagosome Maturation
Following the ingestion of bacteria, acidification by phagosomes and the fusion of phagosomes and lysosomes are two main characteristics of phagocyte maturation [58]. Our results showed a significant increase in phagosomal acidification of the GNP-and GOF-pretreated BMDMs following the ingestion of S. aureus and E. coli, as compared with the control BMDMs ( Figure 4). Further loading of BMDMs with Lysotracker red, which serves in labelling the late endosomes/lysosomes selectively, allowed monitoring of the maturation events of the S. aureus-FITC ingesting phagosomes through testing their overtime capability of co-localization with Lysotracker red. The results demonstrated the occurrence of this co-localization at 30 min in BMDMs pretreated with GNPs and GOFs. In parallel, most S. aureus-FITC entities showed less co-localization with the Lysotracker in control BMDMs ( Figure 5).   Moreover, following the ingestions of S. aureus, an increase in phagosomal acidification in GNP-and GOF-pretreated BMDMs was observed. Again, the nanoparticle-pretreated macrophages demonstrated significantly increased phago-lysosome fusions in response to S. aureus and other bacterial challenges, such as E. coli. These results, therefore, demonstrated that BMDMs, which were pretreated with GNPs and GOFs, possessed improved phagosome maturation following the ingestion of S. aureus and E. coli.

GNPs and GOFs Increase Phagocytosis of pHrodo E. coli Bioparticles by BMDMs
The phagocytic activity of BMDMs was determined by monitoring the uptake of tagged E. coli that showed a fluorescent signal only upon their trafficking into an acidic compartment of the lysosome (Figure 6A-E). A comparison of phagocytosis of pHrodo E. coli BioParticles followed by pretreatment of BMDMs with GNPs and GOFs was carried out. The results showed that the control BMDMs cells had less phagocytosis potential than the BMDMs cells, which were pretreated with GNPs and GOFs, Figure 6F.

GNPs and GOFs Increase Phagocytosis of pHrodo E. coli Bioparticles by BMDMs
The phagocytic activity of BMDMs was determined by monitoring the uptake of tagged E. coli that showed a fluorescent signal only upon their trafficking into an acidic compartment of the lysosome (Figure 6A-E). A comparison of phagocytosis of pHrodo E. coli BioParticles followed by pretreatment of BMDMs with GNPs and GOFs was carried out. The results showed that the control BMDMs cells had less phagocytosis potential than the BMDMs cells, which were pretreated with GNPs and GOFs, Figure 6F.  Cells were quantified in 10 fields using Image J software version 1.43 (U.S. National Institutes of Health, Bethesda, MD, USA). Data are represented as mean ± SE. Statistical significance by one-way ANOVA (p < 0.0001) followed by posthoc testing using Tukey's multi group comparison with control cells: ** p < 0.01, **** p < 0.0001 indicate statistically different from control BMDMs.

GNPs and GOFs Induce NOX2 Function in BMDMs
The phagocytes were able to kill the ingested bacteria through the crucial mechanism of the activation of the NOX2 enzyme complex, leading to ROS production (reactive oxygen species), i.e., superoxide anions. In the membranes of the phagosomes, these bacterial anions are produced by the activity of the NADPH oxidase complex NOX2, thereby leading the phagocytes to undergo more active killing of the ingested bacteria [59,60]. To test the NOX2 and ROS activities in the phagocytes, superoxide anion production by murine BMDMs was assayed. Compared with control BMDMs, the GNP-and GOFpretreated BMDMs showed increased ROS and NOX2 activities, supposedly with the substantial release of superoxide anions in response to either S. aureus or E. coli presence (Figures 7 and 8). Thus, the pretreatment of BMDMs with GNPs and GOFs was associated with increased NOX2 activity in response to improved ROS production, which is possibly one of the principal mechanisms leading to enhanced killing clearance of bacteria [61]. However, other mechanisms cannot be ruled out, since cell wall damage [62] and DNA/RNA destruction [63] was not monitored. In most probability, bacteria trappings in the aggregated GOFs and cell wall damage by the sharp edges of the GOFs pieces cannot be ruled out [64,65]. The DNA/RNA damage needs further work to estimate the damage to the nucleic acid component.

GNPs and GOFs Induce NOX2 Function in BMDMs
The phagocytes were able to kill the ingested bacteria through the crucial mechanism of the activation of the NOX2 enzyme complex, leading to ROS production (reactive oxygen species), i.e., superoxide anions. In the membranes of the phagosomes, these bacterial anions are produced by the activity of the NADPH oxidase complex NOX2, thereby leading the phagocytes to undergo more active killing of the ingested bacteria [59,60]. To test the NOX2 and ROS activities in the phagocytes, superoxide anion production by murine BMDMs was assayed. Compared with control BMDMs, the GNP-and GOF-pretreated BMDMs showed increased ROS and NOX2 activities, supposedly with the substantial release of superoxide anions in response to either S. aureus or E. coli presence (Figures 7 and  8). Thus, the pretreatment of BMDMs with GNPs and GOFs was associated with increased NOX2 activity in response to improved ROS production, which is possibly one of the principal mechanisms leading to enhanced killing clearance of bacteria [61]. However, other mechanisms cannot be ruled out, since cell wall damage [62] and DNA/RNA destruction [63] was not monitored. In most probability, bacteria trappings in the aggregated GOFs and cell wall damage by the sharp edges of the GOFs pieces cannot be ruled out [64,65]. The DNA/RNA damage needs further work to estimate the damage to the nucleic acid component.  BMDMs were pre-treated with GNPs, and GOFs were then infected with Staphylococcus aureus or Escherichia coli. ROS was measured using Ampliflu. Kinetic readings of representative data are shown as mean ± SE at the indicated time. Statistical significance by two-way ANOVA (p < 0.0001) followed by posthoc testing using Tukey's multi group comparison with control cells: *** p = 0.001, **** p < 0.0001 indicate statistically different for (A) S. aureus while ** p = 0.002, *** p = 0.0003, **** p < 0.0001 indicate statistically different for (B) E. coli. data are shown as mean ± SE at the indicated time. Statistical significance by two-way ANOVA (p < 0.0001) followed by posthoc testing using Tukey's multi group comparison with control cells: *** p = 0.001, **** p < 0.0001 indicate statistically different for (A) S. aureus while ** p = 0.002, *** p = 0.0003, **** p < 0.0001 indicate statistically different for (B) E. coli.

Conclusions
A significant increase in phagocytic cell activity was observed after introducing GNPs and GOFs in the experimental set-up studying cellular behaviour. The incremental phagocytosis activity was linked to the ROS and NOX2 pathway feedback. Presumably, the presence of immune-modulator entities to the involvement of increased levels of phagocytic cells, together with the presence of GNPs and GOFs leading to maturation of the phagocytic cells and involvement of the NOX2 pathway is a plausible explanation of the increased immune response and phagocytosis activity, which has the potential to be utilized for better antibacterial designs and preparation. The study also indicated that GNPs and GOFs could contain chemical components that act as immune-enhancers to increase phagocytic cell activity to engulf bacteria and other xenobiotics. However, the nanoparticles' exact chemical roles need to be ascertained in further detail, and the chemical aspect of the interaction needs exploration. Taken together, the data demonstrated that GNPs Figure 8. GNPs and GOFs improved NADPH oxidase 2 (NOX2) activity in BMDMs infected with Staphylococcus aureus or Escherichia coli in the presence and absence of GNPs and GOFs. NOX2 activity in BMDMs was assessed kinetically with lucigenin. Data are shown as mean ± SE at the indicated time. Statistical significance by two-way ANOVA (p < 0.0001) followed by posthoc testing using Tukey's multi group comparison with control cells: *** p = 0.0001, **** p < 0.0001 indicate statistically different for (A) S. aureus while ** p = 0.002, *** p = 0.0004, **** p < 0.0001 indicate statistically different for (B) E. coli.

Conclusions
A significant increase in phagocytic cell activity was observed after introducing GNPs and GOFs in the experimental set-up studying cellular behaviour. The incremental phagocytosis activity was linked to the ROS and NOX2 pathway feedback. Presumably, the presence of immune-modulator entities to the involvement of increased levels of phagocytic cells, together with the presence of GNPs and GOFs leading to maturation of the phagocytic cells and involvement of the NOX2 pathway is a plausible explanation of the increased immune response and phagocytosis activity, which has the potential to be utilized for better antibacterial designs and preparation. The study also indicated that GNPs and GOFs could contain chemical components that act as immune-enhancers to increase phagocytic cell activity to engulf bacteria and other xenobiotics. However, the nanoparticles' exact chemical roles need to be ascertained in further detail, and the chemical aspect of the interaction needs exploration. Taken together, the data demonstrated that GNPs and GOFs contribute to the two main bactericidal processes represented by the maturation of phagosomes and the production of ROS derived from NOX2 activation in this scenario. It can be concluded that the GNPs and GOFs are important contributors to the amplification of antibacterial responses initiated by the host's innate immune system.