Morphology, Aggregation Properties, Cytocompatibility, and Anti-Inflammatory Potential of Citrate-Stabilized AuNPs Prepared by Modular Ultrasonic Spray Pyrolysis

1 Institute of Materials Technology, University of Maribor, SI-2000 Maribor, Slovenia Zlatarna Celje d.d., SI-3000 Celje, Slovenia Institute for the Application of Nuclear Energy, University of Belgrade, 11000 Belgrade, Serbia Indian Institute of Technology (ISM), Dhanbad, Jharkhand 826 004, India Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia National Institute of Chemistry, SI-1000 Ljubljana, Slovenia IME Institute, RWTH Aachen University, Aachen, Germany Institute for Medical Research, Medical Faculty of the Military Medical Academy, University of Defense, 11000 Belgrade, Serbia


Introduction
Gold nanoparticles (AuNPs) possess excellent Surface Plasmon (SP) properties, enabling tunable size-dependent optical properties of AuNPs. When the SP of AuNPs, that is, the oscillation of conduction electrons on the surface of the nanoparticles, is in resonance with the incident light, a strong nonfading emission can be obtained [1]. Shi et al. [2] and Jain et al. [3] reported that the magnitude of light scattering by However, it is not known whether the citrate stabilization of such AuNPs could affect their cytocompatibility. Although citrate is considered as a good and nontoxic AuNP stabilizer [22,23], it has been reported that the citrate-stabilized AuNPs applied in high concentrations could affect the actin cytoskeleton and the proliferation of fibroblasts [24] and HEPG2 cells [25]. Additionally, we showed previously that nontoxic AuNPs produced by USP [16], similar to those obtained commercially [26], can induce immunomodulatory effects by modulating the functions of Antigen Presenting Cells (APCs). Monocytes represent a major population of p h a g o c y t i cA P C si nb l o o d ,a n dt h e yg i v er i s et ot h et i s s u e macrophages and inflammatory DCs [27], both of which are responsible for immune and inflammatory responses to nanoparticles in tissues [28]. However, the data on the effects of citrate-capped AuNPs on human monocytes are quite scarce. Therefore, besides the cytotoxicity evaluation, we investigated the response of primary human monocytes to citrate-capped AuNPs produced by modular USP.

Experimental Details
2.1. Synthesis of Gold Nanoparticles. The synthesis of AuNPs was carried out on the modular USP device at the IME Institute of Process Metallurgy and Metal Recycling, RWTH Aachen University, Germany ( Figure 1). An aqueous solution of Hydrogen Tetrachloroaurate (either 2.5 g/L or 0.5 g/L, Sigma-Aldrich) in deionized water (Millipore) was used as the precursor. The prepared precursor solutions were fed into the Ultrasonic Aerosol Generator (Gapusol, RBI, France, piezoelectric transducer membrane frequency: 2.5 MHz) to form aerosol droplets with diameters ranging from 1 to 15 m [29]. A nitrogen gas flow range of 1.0 to 4.5 L/min was used as the carrier gas to transport these formed aerosol droplets to the two heating zones through a quartz glass tube of 2 cm diameter. A hydrogen gas flow, set to 1.0 to 2.0 L/min, was added as a reducing agent for the gold chloride solution to pure gold metal nanoparticles. The first heating zone was set at a dispersed temperature range from 50 to 100 ∘ Cf o r droplets' evaporation and particle drying and also allowed Journal of Nanomaterials an optimal material diffusion inside the droplet ([AuCl4]− and H+). The second reactor furnace was set at a temperature range from 260 to 500 ∘ C and was responsible for the chemical reactions required for obtaining pure AuNPs. The ambient temperature was 21 ∘ C. The two sets of parameters ( Table 1) enabled generation of two types of AuNPs (AuC18 and AuC20: "Au" is for gold nanoparticles, "C" is the series separator, and "18" and "20" are the experiments in the series). The parameters presented in this article have been chosen based on the results of a series of experiments, while these have yielded the best results for our target nanoparticles. The nanoparticles were collected in 0.1% solution of Sodium Citrate (Alfa Aesar) in Millipore water (pH 3.5 ± 1). The lack of pH change after the collection of AuNPs in citrate solution indicated that Au was reduced completely by the hydrogen gas flow rather than by the citrate in the collection bottle. All synthesized AuNPs were stored in Miron glass at 8 ∘ Ca w a y from light for at least 3 months. In the conditioning experiments, dispersed AuNPs (50 g/mL) were incubated in a complete RPMI medium (basic RPMI 1640 (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 2-mercaptoethanol (Sigma), penicillin, streptomycin, and gentamicin (1% each, ICN Galenika)) for 24 h and then analyzed by Zeta Nano ZS, as described.

Characterization
The UV-vis spectra of AuNPs dispersed in 0.03% Nacitrate solution and in complete RPMI medium (1 : 2 AuNP in 0.1% Na-citrate : medium vol. ratio) for 2 h or 24 h were analyzed using Ultrospec 2000 (Pharmacia Biotech) within range of 200-900 nm. The blank samples, 0.03% citrate solution and corresponding concentrations of citrate in complete RPMI medium, were used for background subtraction. The SPR curves detected between 400 and 700 nm were averaged from 10 measurements and normalized to peak value 1 in each sample to enable comparison between samples.

L929 Cells.
The following cells were used in the cytocompatibility assays: L929 cells and primary human monocytes. L929, a mouse fibroblast cell line, was obtained from ATCC (Washington DC, USA). Prior to the experiments, L929 cells were thawed from liquid nitrogen and cultivated in complete RPMI medium at 37 ∘ C, 5% CO 2 ,u n t i lt h e yr e a c h e d9 0 % confluence. After that, the cells were trypsinized with 0.25% trypsin solution (Sigma) in 0.02% NaEDTA/RPMI. After two passages, L929 cells were seeded in 96-well plate (1 × 10 4 /well) f o r2 4h ,w a s h e dw i t hc o m p l e t eR P M Im e d i u m ,a n dt h e n treated with AuC18 or AuC20 NPs (12.5-200 g/mL).
The viability of L929 cells was determined after 24 h by harvesting all cells and staining them with 1% Trypan blue. The labeled cells, identified by light microscopy, were considered as dead, predominantly necrotic cells. The percentages of dead cells were determined on the basis of at least 500 total cells from one well. The percentage of viable cells was calculated as 100% of total cells − %ofdeadcells.
Additionally, the percentage of apoptotic/necrotic L929 cells after the cultures was determined by Annexin-V-FITC/Propidium Iodide (PI) labelling kit (R&D Systems) and analyzing them on a flow cytometer (Partec Cube 6).
An MTT assay was performed to determine the metabolic activity of L929 cells cultivated with AuNPs. L929 cells (2 × 10 4 /well of 96-well plate) were cultivated overnight to reach subconfluence after which they were washed with complete RPMI medium twice and then treated with AuC18 or AuC20 NPs (12.5-200 g/mL in medium) or medium alone (nontreated control), in 6-plicates for the next 24 h. The corresponding cell-free cultures were prepared as blank controls. After the cultivation, all cultures were washed in phenol-red free RPMI medium twice to remove free AuNPs, and a tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (1 mg/mL in phenol-red free RPMI) was added for the next 4 h. The formazan crystals generated by NADH-oxidoreductase enzymes were dissolved by using 10% Sodium Dodecyl Sulphate (SDS) in 0.01 N HCl overnight, and the absorbance was read at 570 nm (Behring ELISA Processor II, Heidelberg, Germany). The absorbance measured in cell-free blank controls was subtracted from the absorbance of corresponding experimental cultures. The absorbance (metabolic activity) detected in the treated cult u r e sw a se x p r e s s e da st h ep e r c e n t a g eo fa b s o r b a n c ei nt h e nontreated control cultures (100%).
The proliferation of L929 cells (seeded initially at 0.5 × 10 4 /well) which were cultivated with AuNPs (12.5-200 g/mL) was determined after 3 days of cultivation. Duringthelast18hours,thecellswerepulsedwith1 Ci/well [3H] thymidine (6.7 Ci/mmol, Amersham, Bucks, UK) to estimate the level of DNA synthesis in the log-phase of cellular proliferation. The cells were harvested onto glass fiber filters and the incorporation of the radionuclide into DNA was measured by -scintillation counting (LKB-1219 Rackbeta, Finland).

Primary Human
Monocytes. Primary human monocytes were isolated from Peripheral Blood Mononuclear Cells (PBMCs) from healthy volunteers who provided consent forms. All studies on human blood cells were approved by the EthicalCommitteeoftheMilitaryMedicalAcademy.PBMCs (10 6 cells/cm 2 ) were allowed to adhere for 1.5 hours, after which nonadherent cells were washed out thoroughly with prewarmedRPMImedium.Afterthat,AuC18orAuC20NPs were added to the cell cultures. Additionally, some cultures were treated simultaneously with LPS (500 ng/mL) to induce the activation of monocytes. After 24 h of cultivation, the internalization of AuNPs by monocytes was studied, as well as the apoptosis/necrosis of monocytes and their phenotypic properties.
For the internalization studies, cultivated monocytes were harvested, washed in PBS solution, and added to a microscopic slide prior to their analysis by phase contrast microscopy. Alternatively, the cells were analyzed by flow cytometry (Partec Cube 6) to assess the granularity of the cells by monitoring the Side Scatter (SS) parameter.
2.5. Statistics. Data are presented as a representative experi m e n to ra sam e a n± Standard Deviation (SD) of at least 3 independent experiments. The differences between control experimental samples were analyzed using the Friedman test with Dunn's posttest, and the values with < 0.05 or less were considered to be statistically significant.

Properties of USP-Generated Citrate-Stabilized AuNPs.
USP has a good potential for removing technological issues such as small production rates with major variations in shape and sizes of the nanoparticles, but additional improvements o ft h ep r o c e s sa r er e q u i r e dt ol i m i tt h es i z ev a r i a b i l i t yo f AuNPs and to improve their stability. In this paper, we applied an innovation by separating the heating chambers of the UPS into the evaporation/drying chamber for drying of the droplets generated by the ultrasound (2.5 MHz) and the thermal decomposition chamber (Figure 1), leading to better control of AuNP size [21]. Considering that the dynamics of droplets formation depend strongly on the precursor concentrations and the gas flow rate [18,19], we applied two sets of parameters (Table 1) to generate two types of AuNPs (AuC18 and AuC20), followed by their stabilization in Na-citrate solution. ICP-OES measurements of Au 3+ in the collection citrate solution suggested that the concentrations of AuC18 and AuC20 NPs were 14 g/L and 4.1 g/L, respectively.
To analyze the size and morphology of AuC18 and AuC20, first we conducted TEM analysis on freshly synthesized s a m p l e sa n do nt h o s es t o r e di nt h ec i t r a t es o l u t i o nf o r3 months ( Figure 2). It was observed that AuNPs possess predominantly circular shape with no visible defects (cracks, pores, etc.). The circularity of AuC18 and AuC20 samples was 0.86 ± 0.03 and 0.88 ± 0.02,r e s p e c t i v e l y( 0s i g n i fi e s the irregular shape and 1 signifies the perfect circle), and the density of AuNPs was uniform within each sample. The average core size of AuC18 was 14.7±13.9 nm ( = 200)with median (range) at 9.3 (3.7-76.6) nm. The average core size of AuC20 was 36.8 ± 10.4 nm ( = 212)withmedian(range) at 35.1 (15.2-88.2) nm (Figure 2(a)). Similar results were obtained on the samples stored in citrate buffer for 3 months, suggesting that AuNPs had a stable size and morphology during the storage (Figure 2(b)). Based on the ICP-OES detected concentration of Au and the average size of AuC18 and AuC20 NPs observed by TEM, the theoretical number of NPs/mL was 4.25 × 10 11 and 2.71 × 10 10 ,r e s p e c t i v e l y (Appendix). Energy Dispersive Spectroscopy (EDS) analysis confirmed a high purity content of 99.9 wt.% Au (Figure 3(a)). Besides, the electron diffraction analysis of AuC18 and AuC20 samples showed a space group Fm-3m in both types of AuNPs, which corresponds to the face-centred cubic lattice structure present in Au (Figure 3(b)). The surface energies for grain growth of the Au face-centred cubic lattice were in accordance with those previously described [30] No apparent grain orientation was present. There was a high concentration of grain boundaries and twins present in the AuNPs, while there were no visible vacancies or porosities. This suggested that the growth of the AuNPs during USP was heterogeneous.
Considering that hydrodynamic size and surface charge are the most relevant for biological systems, we analyzed -potential and hydrodynamic sizes of the two types of AuNPs in citrate buffer by using DLS analysis ( Figure 4). DLS measurements of AuNPs in citrate buffer suggested that AuC18NPshadtwosizepeaks(bimodalsizedistribution)of 9.8 ± 3.4 nm (95.3% of AuNPs based on volume distribution analysis) and 58 ± 33.6 nm (4.7% of AuNPs). In contrast, AuC20 had unimodal size distribution of 32.0 ± 19.5 nm (99.7% of AuNPs) in the same buffer. The zeta-potential of AuC18 and AuC20 AuNPs in citrate buffer was negatively charged at −23.2±1.3 mV and −28.3±1.7 mV, resp ec tively. The negative zeta-potential of AuNPs most probably helped in the stability and high dispersity of these AuNPs due to repulsive forces, since the hydrodynamic size of AuNPs also did not change significantly more than three months after their initial preparation (data not shown).
Although DLS measurements were in general agreement withthesizemeasuredbyTEM,somediscrepanciesbetween the expected and the obtained values were probably a consequence of large variability in AuNPs' size combined with the 6 Journal of Nanomaterials On the other hand, DLS sizing of NPs also has certain limitations, especially when the solution contains larger NPs due to their intense light scattering properties [31]. However, both sizing methods indicated that although AuNPs generated b ym o d u l a rU S Ph a dal o w e rs i z ev a r i a b i l i t yc o m p a r e dt o standard USP [21], the process needs additional improvements for better size control. The bimodal size distribution of AuC18 was probably a consequence of two mechanisms of their generation during USP: the Gas-to-Particle (GTP) and Droplet-to-Particle (DTP) mechanisms. We showed previously that the GTP mechanism results in the synthesis of much smaller nanoparticles and DTP results in larger nanoparticles [21]. According to the results obtained in this study, the GTP mechanism was most probably predominant, as compared to the DTP for the sample AuC18, which resulted in the synthesis of a larger number of small sized AuNPs.
On the other hand, the formation of the AuNPs in sample AuC20 was most probably due to GTP only, which yielded unimodal size distribution in the sample. The reason behind this mechanism is most probably the higher gas flow rates of nitrogen and hydrogen. In high gas flows rates, more turbulence in the system is expected, causing more collisions of aerosol droplets, precursor vapors, and nanoparticles. An increased number of eddies in the gas flow probably caused the increase of nanoparticle sizes with unimodal size distribution. Besides the better size control, the modular USP will require an increase of AuNPs' yield. Namely, taking into account the initial precursor concentration, the amount of precursor solution consumed, and the concentration of AuNPs collected in the final volume, the yields for AuC18 and AuC20 samples were 4.67% and 6.83%, respectively. The low yield of AuNPs by this USP equipment most probably occurred due to the losses of deposition of AuNPs in the transport tubes, impaction, gravitational sedimentation in the transport tubes and diffusion onto the transport tube walls, turbulence, and thermophoresis.

Properties of USP-Generated AuNPs in Cell Culture
Medium. Besides AuNPs size and surface charge, the optical properties of AuNPs are the most relevant for their biomedical application [1]. In contrast to the acidic environment of  AuNPs storage (Na-citrate, pH 3.5 ± 0.1), biological fluids are pH neutral and contain different proteins and ions. Barreto et al. [32] showed recently by using UV-vis spectrometry that citrate-capped AuNPs of different sizes prepared by chemical synthesis were unstable in different ionic strength solutions, including cell culture media, displaying a significant increase in size after conditioning due to agglomeration/aggregation. To examine whether USP-generated AuNPs behave similarly, the UV-vis spectra of AuC18 and AuC20 in citrate solution and after their incubation in the complete RPMI medium for 2 h or 24 h were analyzed (Figure 4(a)). AuC18 NPs had t h eS P Rp e a ka t5 2 8n mi nc i t r a t es o l u t i o n .Ar e ds h i fto f 2 nm was observed after their conditioning in the complete RPMI medium for 2 h. A further increase in SPR peak was observed after 24 h of conditioning by a total of 6 nm. AuC20 NPs had the SPR peak at 532 nm in the citrate solution due to their larger average size compared to AuC18 NPs. A similar red shift of 2 nm was observed after the incubation of AuC20 in the complete RPMI medium for 2 h. However, after 24 h of conditioning, a loss of SPR band and a strong red shift absorbance were observed. The detected SPR peaks in citrate solution were probably a result of optical activity of all NPs present in the solution, which could explain their higher SPR values as compared with NPs with a small size distribution.The2nmredshiftsintheSPRpeaksaftershortterm conditioning could be explained by the change of local refraction index at the surface of AuNPs due to adsorption of medium components [3][4][5]. In contrast, larger red shifts and 8 Journal of Nanomaterials a loss of the SPR band could indicate their increase in size due to agglomeration [32].
To determine how the results from UV-vis analysis relate to the change in AuNPs' hydrodynamic size after conditioning, DLS measurements of AuNPs conditioned for 2 4hi nc o m p l e t em e d i u mw e r ec a r r i e do u ti nc o m p a r i s o n to DLS size in citrate buffer. The size distribution of sample AuC18 after the conditioning increased 1.5-2 times (size after conditioning was 15.7 ± 4.9 nm (95% of AuNP) and 99.9 ± 62.5 nm (5% of AuNPs)), as compared to AuC18 size in Na-citrate solution, which could explain the 6 nm shift in the SPR peak. In the case of the AuC20 samples, the size distribution values increased up to 3 times (size after conditioning was 102.3 ± 44.4 nm) (Figure 4(b)). This c o r r e s po n d st ot h ec h a n g e so b se rv e di nt h eS P Ro fA u C 2 0 , as NPs larger than 100 nm do not have a clearly defined S P Rb a n dw i t h i n5 0 0 -6 0 0n md u et ot h ed o m i n a n tc o ntributions from higher-order electron oscillations [33]. The aggregation of AuNPs in the complete RPMI medium was not immediate, most probably due to adsorption of serum proteins on their surface. In line with this, it was shown that aggregation/agglomeration of chemically synthesized citrate-capped AuNPs occurs immediately upon placing them in artificial saliva or serum-free cell culture media (RPMI or DMEM), whereas the agglomeration in the serumcontaining media was much slower [32,34,35]. These studies suggested that agglomeration/aggregation of citrate-capped AuNPs results from changes on the extension of capping by the citrate anions at the surface [36]. Although the increase in hydrodynamic size of USP-generated AuNPs in biological media was much smaller compared to those described for the chemically synthesized citrate-AuNPs, the expected size-dependent changes in the optical properties of USPgenerated AuC20 NPs due to agglomeration/aggregation do not allow their easy application in diagnostics. However, t h es i z eo fb o t hA u N P si nm e d i u mw a ss t i l ls u i t a b l ef o r potential development of drug-delivery systems. Therefore, we next investigated the cytocompatibility of AuNPs on L929 cells, which are recommended by the ISO 10993-5 Standard, and primary human monocytes to examine their potential immunomodulatory properties.

Cytocompatibility of AuNPs.
Our previous studies suggested that pure AuNPs produced by USP are not cytotoxic for L929 cells, rat thymocytes, and splenocytes up to 100 g/mL [16,17]. Furthermore, the improvement of USP by incorporation of the preheating chamber does not compromise the good biocompatibility of these AuNPs [21]. However, the biocompatibility of citrate-capped AuNPs is still controversial [23][24][25]37]. Therefore, we evaluated whether citrate-stabilized AuNPs produced by modular USP induce thecytotoxicityofL929cellsafter24handwhethertheyaffect the proliferation of these cells after 3 days of cultivation.
Although the DLS spectra showed a large variation in the size of AuNPs and their increase upon interaction with medium, L929 clearly internalized AuNPs after 24 h of incubation ( Figure 5(a)). Considering that AuNPs did not agglomerate immediately upon their interaction with medium and that the internalization of AuNPs is a relatively quick process [38], it is possible that the majority of AuNPs were internalized by L929 cells in their native size rather than as agglomerates. Besides, it is not known whether L929 cells internalize monodispersed or agglomerated AuNPs at a higher rate as is known for other cell types [39].
To analyze the cytocompatibility of AuNPs in L929 cell culture, viability, apoptosis, metabolic activity, and proliferation dynamics were analyzed. The viability assay, based on Trypan blue staining, showed no signs of cytotoxicity of AuC18 and AuC20 in L929 cell culture up to 200 g/mL ( Figure 5(b)). Similar results were obtained by Annexin-V/PI staining of cells after the culture, as only a mild increase in early and late apoptotic cells (Annexin-V+/PI− and Annexin-V+/PI+) was observed in the cultures with 200 g/mL of AuNPs, whereas lower concentrations had no cytotoxic effects ( Figure 5(c)). Additionally, the MTT assay carried out to determine the total metabolic activity of L929 cell cultures inthepresenceofAuNPsalsosuggestedthatAuNPsprepared by USP do not affect the metabolic activity in the culture at the concentrations lower than 200 g/mL ( Figure 5(d)). The dose-dependent effect of AuC18 and AuC20 NPs on proliferation of L929 cells was carried out using the 3H-thymidine incorporation assay as the most sensitive method for the cell proliferation studies (Figure 5(b)). The results showed that both types of nanoparticles inhibited the proliferation of L929 cells at a concentration of 100 g/mL and higher. Such a ne ff e c tw a ss i m i l a rt ot h eo n ew eo b s e r v e dp r e v i o u s l y ,i n which nonstabilized AuNPs prepared by modular USP did not inhibit the proliferation of L929 cells at the concentrations lower than 100 g/mL [21]. Considering that 100 g/mL of USP-generated AuNPs was not toxic for L929, the observed antiproliferative effect could be explained by the fact that AuNPs can affect the cytoskeleton upon their internalization and impair the cellular processes necessary for proliferation [24,25]. However, this hypothesis needs to be tested independently for the USP-generated AuNPs.
3.4. Anti-Inflammatory Properties of AuNPs. Previously, we showed that AuNPs prepared by USP [16] and those synthesized chemically [26] can induce direct immunomodulatory effects when applied at nontoxic concentration (50 g/mL) predominantly by modulating the functions of APCs. Considering these results, as well as those obtained in this study, we chose the concentration of 50 g/mL to investigate the immunomodulatory/anti-inflammatory properties of AuC18 and AuC20 nanoparticles, using primary human monocytes as a model system. Monocytes represent the major population of phagocytic APCs [27], but the data on the effects of citratecapped AuNPs on human monocytes are quite scarce.
Therefore, the monocytes were cultivated with AuC18 or AuC20 for 24 h, in either the presence or absence of LPS, followed by assessment of the monocytes' viability and phenotypic and functional properties. After 24 h cultures, monocytes internalized AuNPs, as judged by phase contrast microscopy ( Figure 6(a)) and flow cytometry (Figure 6(b)). These results suggested that AuNPs, irrespective of their large size variability, were internalized easily by monocytes, most probably as provisionally stabilized by serum proteins. Upon the internalization, AuC18 and AuC20 did not induce

Control
AuC18 AuC20         significant apoptosis of monocytes compared to the control, as judged by Annexin-V and PI staining (Figures 6(c) and 6(d)). The apoptosis of monocytes in the presence of LPS was significantly lower, as expected from previous findings [40], and AuNPs did not modify significantly the prosurvival effect ofLPSonmonocytes.Theseresultsareinlinewiththerecent finding of Chhour et al. [41], showing that AuNPs stabilized with different capping agents do not cause the apoptosis of the mouse monocyte cell line RAW 264.7 after internalization, even when applied in much higher concentrations. The low toxicity of citrate-capped AuNPs was also shown in experiments with different cell lines [42]. To our knowledge, this is the first finding indicating that good cytocompatibility of citrate-capped AuNPs could be expected for primary human monocytes as well.
Peripheral blood monocytes constitute two functionally distinct subpopulations, classical CD14 + (which could be subdivided further into CD14 + CD16 − and CD14 + CD16 + ) and CD14 low CD16 + monocytes, and the changes in these subpopulation ratios were shown to correlate with the inflammatory response of the host [43,44]. However, the analysis of monocytes subpopulations upon their interaction with AuNPs has not been carried out so far. To observe whether AuNPs affect the distribution of these subpopulations, the monocytes cultivated with AuC18 and AuC20 nanoparticles, in either the presence or absence of LPS, were analyzed by flow cytometry. The results suggested that the population of analyzed monocytes contained more than 83% of CD14 + cells, and neither AuNPs nor LPS affected the expression of this molecule (Figure 7). However, both types of AuNPs