Influence of size and surface capping on photoluminescence and cytotoxicity of gold nanoparticles

Abstract

Hydrophilic and homogeneous sub-10 nm blue light-emitting gold nanoparticles (NPs) functionalized with different capping agents have been prepared by simple chemical routes. Structure, average, size, and surface characteristics of these NPs have been widely studied, and the stability of colloidal NP solutions at different pH values has been evaluated. Au NPs show blue PL emission, particularly in the GSH capped NPs, in which the thiol-metal core transference transitions considerably enhance the fluorescent emission. The influence of capping agent and NP size on cytotoxicity and on the fluorescent emission are analyzed and discussed in order to obtain Au NPs with suitable features for biomedical applications. Cytotoxicity of different types of gold NPs has been determined using NPs at high concentrations in both tumor cell lines and primary cells. All NPs used show high biocompatibility with low cytotoxicity even at high concentration, while Au-GSH NPs decrease viability and proliferation of both a tumor cell line and primary lymphocytes.

Keywords: Agent; Capping; Cell viability; Cytotoxicity; GSH; Gold nanoparticles; Photoluminescence; Proliferation; Semiconfocal microscopy.

Fig. 1

Schematic representation of different Au NP capping agents. a NP obtained by synthesis A or B (named Au-C and Au-TC, respectively): spherical gold core surrounded by citrate ligands. b NP obtained by synthesis C (named Au-CYS): the gold core can link to the thiol groups on the CYS molecule. c NP obtained by synthesis D (named Au-GSH): the gold core is bound to GSH thiol groups

Fig. 2

TEM micrographs obtained from different Au NP capping agents: a Au-C, b Au-TC after 5 min of synthesis, c Au-TC after 45 min of synthesis, d Au-CYS, e Au-GSH. Corresponding histograms displaying particle size distribution are shown in the right panels: f and g are high-resolution images for Au-TC and Au-CYS, respectively

Fig. 3

a FTIR spectra for the different Au NPs. b Amplification of the region between wavenumber 1500 and 900 cm⁻¹

Fig. 4

UV-visible spectra for the four different Au NP solutions (a) and for Au-TC NPs during the growing process (b)

Fig. 5

Absorption spectra at different pH values for Au-C (a), Au-TC (b), Au-CYS (c), and Au-GSH (d) colloidal NP solutions

Fig. 6

PL emission spectra for Au NP solutions excited at 317 nm (a). PL emission spectra for Au-CYS NP colloidal solutions at different pH values (b)

Fig. 7

Fluorescence microscopy images of cells that were incubated with different NPs (Au-C, Au-Cys, Au-GSH, or Au-TC) or cultured in the absence of NPs as a control. The corresponding transmitted light microscopy images are shown in the lower panel. Images from three different experiments were analyzed and both total and positive cells were counted with the aid of FIJI® software. The percentage of fluorescent cells from three different experiments was analyzed for statistical significance by ANOVA (only NP-TC shows a statistically significant increase in fluorescence above control cells [p < 0.01])

Fig. 8

MTT cell viability assay of primary lymphocytes (upper panels) or Jurkat tumor cells (lower panels), incubated with the indicated NPs at 15 μg/mL (left panels) or 1.5 μg/mL (right panels). Results are represented as the mean ± standard deviation of at least three independent experiments performed in triplicate. Statistical analysis was performed using the GraphPad Prism 5.00 software. Significance was determined using a t test. Statistical significance is indicated by an asterisk (*p < 0.05)

Fig. 9

Flow cytometry dot plot showing the cytotoxicity profile of primary lymphocytes—PBMC—(upper panels) or Jurkat tumor cells (lower panels), cultured with the indicated NPs. Cells (at least 10,000) were electronically gated according to their size/granularity distribution. Histograms corresponding to three Live/dead™ cell staining (dead cells) experiments are shown for each Np. Within each histogram, different experiments for each NP are shown in different colors, with the control sample with no NP shown in black. According to a one-way ANOVA analysis (XLSTAT® Excell®), only PBMC cultured in the presence of Au-GSH showed a significant increase in cell dead when compared to cells cultured in the absence of NPs (p < 0.002)

Fig. 10

Detection of proliferating cells by EdU incorporation and labeling. Jurkat tumor cells were cultured with 10 μM EdU, in the presence or absence of the indicated nanoparticles at 15 μg/mL. Proliferation was determined as a function of Edu incorporation into cells’ DNA, detected by means of a Pacific blue fluorescent dye. EdU+ proliferating (gated in P) and non-proliferating cells (NP) were clearly and distinctly separated by FACS. Jurkat cells in the absence of NP were used as a positive control (+ control) for proliferation, as they are a tumor cell line that spontaneously proliferate in culture. Cells that have not incorporated Edu were used as negative control (− control). a Overlay histogram showing the proliferation profile of all samples. Samples had been electronically gated according to their size/granularity distribution. Individual samples as well as the gating strategy are shown in supplementary Fig. S.3. An overlay histogram of the ungated populations as well as results corresponding to individual ungated samples is shown in Fig. S.4.b) Average and standard errors for the percentage of non-proliferating cells under each condition (**p < 0.001)