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. 2019 Dec 9;12(24):4121.
doi: 10.3390/ma12244121.

The Effect of Stabilisation Agents on the Immunomodulatory Properties of Gold Nanoparticles Obtained by Ultrasonic Spray Pyrolysis

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Free PMC article

The Effect of Stabilisation Agents on the Immunomodulatory Properties of Gold Nanoparticles Obtained by Ultrasonic Spray Pyrolysis

Marina Bekić et al. Materials (Basel). .
Free PMC article

Abstract

Gold nanoparticles (GNPs) have been investigated extensively as drug carriers in tumour immunotherapy in combination with photothermal therapy. For this purpose, GNPs should be stabilised in biological fluids. The goal of this study was to examine how stabilisation agents influence cytotoxicity and immune response in vitro. Spherical GNPs, 20 nm in size, were prepared by ultrasonic spray pyrolysis (USP). Three types of stabilising agents were used: sodium citrate (SC), polyvinyl-pyrrolidone (PVP), and poly-ethylene glycol (PEG). Pristine, non-stabilised GNPs were used as a control. The culture models were mouse L929 cells, B16F10 melanoma cells and human peripheral blood mononuclear cells (PBMNCs), obtained from healthy donors. Control SC- and PEG-GNPs were non-cytotoxic at concentrations (range 1-100 µg/mL), in contrast to PVP-GNPs, which were cytotoxic at higher concentrations. Control GNPs inhibited the production of IFN-ϒ slightly, and augmented the production of IL-10 by PHA-stimulated PBMNC cultures. PEG-GNPs inhibited the production of pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α) and Th1-related cytokines (IFN-ϒ and IL-12p70), and increased the production of Th2 cytokines (IL-4 and IL-5). SC-PEG inhibited the production of IL-8 and IL-17A. In contrast, PVP-GNPs stimulated the production of pro-inflammatory cytokines, Th1 cytokines, and IL-17A, but also IL-10. When uptake of GNPs by monocytes/macrophages in PBMNC cultures was analysed, the ingestion of PEG- GNPs was significantly lower compared to SC- and PVP-GNPs. In conclusion, stabilisation agents modulate biocompatibility and immune response significantly, so their adequate choice for preparation of GNPs is an important factor when considering the use of GNPs for application in vivo.

Keywords: cytokines; cytotoxicity; gold nanoparticles; immune response; stabilisation agent.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterisation of ultrasonic spray pyrolysis (USP)-generated gold nanoparticles (GNPs). (a) TEM, and (b) SEM image of c-GNPs; (c) UV-vis spectra of c-GNPs or GNPs coated with sodium citrate (SC), polyvinyl-pyrrolidone (PVP), or poly-ethylen glycol (PEG), with indicated peak values for surface plasmon resonance (SPR); (d) Hydrodynamic size by dynamic light scattering (DLS) analysis of GNPs is shown as intensity %, number %, and volume %.
Figure 1
Figure 1
Characterisation of ultrasonic spray pyrolysis (USP)-generated gold nanoparticles (GNPs). (a) TEM, and (b) SEM image of c-GNPs; (c) UV-vis spectra of c-GNPs or GNPs coated with sodium citrate (SC), polyvinyl-pyrrolidone (PVP), or poly-ethylen glycol (PEG), with indicated peak values for surface plasmon resonance (SPR); (d) Hydrodynamic size by dynamic light scattering (DLS) analysis of GNPs is shown as intensity %, number %, and volume %.
Figure 2
Figure 2
The effect of GNPs stabilised differently on the metabolic activity of cells in vitro. Different concentrations (12.5 µg/mL–100 µg/mL) of non-stabilised GNPs (c-GNPs), or stabilised with SC, PVP and PEG, were incubated with (a) L929 cells; (b) B16F10 or (c) peripheral blood mononuclear cells (PBMNCs), for 24 h; (d) PBMNCs were also cultivated with the corresponding concentrations of PVP (0.1%–0.01%) as present in PVP-GNP, or PVP-GNP (12.5 µg/mL–100 µg/mL) that where washed in DI water twice and then used. After that, the relative metabolic activity of the cells was determined by 3-[4.5 dimethyl-thiazol-2lyl]-2.5 diphenyl tetrazolium bromide (MTT), taking that the metabolic activity of control non-treated cells was 100%, in each assay. The results are shown as mean ± SD of three independent experiments. *** p < 0.005, ** p < 0.01, compared to corresponding control cells.
Figure 3
Figure 3
Effects of GNPs on production of pro-inflammatory cytokines. Cytokines’ production was determined by measuring the levels of indicated cytokines in Peripheral Blood Mononuclear Cells (PBMNC) cultures treated with c-GNPs, SC-GNPs, PVP-GNPs and PEG-GNPs (all at the concentration of 50 μg/mL) for 72 h. Values of IL-8, TNF-α, IL-6 and IL-1β are shown as mean pg/mL ±SD of three independent experiments; * p < 0.05, ** p < 0.01 compared to control phytohemagglutinin (PHA)-stimulated PBMNC.
Figure 4
Figure 4
Effects of GNPs on the production of IFN-γ, IL-12 and IL-17A cytokines. Cytokines’ production was determined by measuring the cytokine levels in PBMNC cultures treated with non-toxic concentrations (50 μg/mL) of c-GNPs, SC-GNPs, PVP-GNPs and PEG-GNPs for 72 h. The values of IFN-γ, IL-12 and IL-17A are shown as mean pg/mL ± SD of three independent experiments; * p < 0.05, ** p < 0.01 compared to the corresponding control.
Figure 5
Figure 5
Effects of GNPs on the production of IL-4 and IL-10 cytokines. Cytokine production was determined by measuring cytokine levels in PBMNC cultures treated with a non-toxic concentration (50 μg/mL) of c-GNPs, SC-GNPs, PVP-GNPs and PEG-GNPs for 72 h. The values of IL-4 and IL-10 are shown as mean pg/mL ± SD of three independent experiments; * p < 0.05, ** p < 0.01 compared to the corresponding control.
Figure 6
Figure 6
Internalisation of GNPs by PBMNC. PBMNCs were co-cultivated with c-GNPs, SC-GNPs, PVP-GNPs and PEG-GNPs (25 μg/mL) for 24 h, after which the cells were washed to remove non-internalised GNPs, and the samples were prepared as cytospins. (a) May-Gunwald Giemsa (MGG) stained preparation of PBMNCs treated with PVP-GNPs is shown. The scores (0–4) shown beside the indicated cells represent the arbitrary score used for quantification of the GNPs’ internalisation by PBMNC; (b) and the results of internalisation scoring are shown from three independent experiments as mean ± SD; in each experiment at least 500 cells were analysed. ** p < 0.01 compared to bare c-GNPs.

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