Direct Conjugation of Resveratrol on Hydrophilic Gold Nanoparticles: Structural and Cytotoxic Studies for Biomedical Applications

Abstract

Strongly hydrophilic gold nanoparticles (AuNPs), functionalized with citrate and L-cysteine, were synthetized and used as Resveratrol (RSV) vehicle to improve its bioavailability. Two different conjugation procedures were investigated: the first by adding RSV during AuNPs synthesis (1) and the second by adding RSV after AuNPs synthesis (2). The two different conjugated systems, namely AuNPs@RSV1 and AuNPs@RSV2 respectively, showed good loading efficiency (η%): η₁ = 80 ± 5% for AuNPs@RSV1 and η₂ = 20 ± 3% for AuNPs@RSV2. Both conjugated systems were investigated by means of Dynamic Light Scattering (DLS), confirming hydrophilic behavior and nanodimension (<2R_H> ₁ = 45 ± 12 nm and <2R_H> ₂ = 170 ± 30 nm). Fourier Transform Infrared Spectroscopy (FT-IR), Synchrotron Radiation induced X-Ray Photoelectron Spectroscopy (SR-XPS) and Near Edge X-ray Absorption Fine Structure (NEXAFS) techniques were applied to deeply understand the hooking mode of RSV on AuNPs surface in the two differently conjugated systems. Moreover, the biocompatibility of AuNPs and AuNPs@RSV1 was evaluated in the concentration range 1.0-45.5 µg/mL by assessing their effect on breast cancer cell vitality. The obtained data confirmed that, at the concentration used, AuNPs do not induce cell death, whereas AuNPs@RSV1 maintains the same anticancer effects as the unconjugated RSV.

Keywords: bioconjugates; conjugation; drug delivery systems; gold nanoparticles; immobilization; resveratrol.

Figure 1

(a) Scheme of gold nanoparticles (AuNPs); (b) AuNPs Uv-vis spectrum in water with Table 550 nm.

Figure 2

(a) Loading efficiency η (%) for AuNPs@RSV1 (η (%) = 80 ± 5%) and AuNPsRSV2 (η (%) = 20 ± 3%); (b) <2R_H> in water of AuNPs in red (530 ± 60 nm nm), AuNPs@RSV1 in blue (45 ± 12 nm) and AuNPs@RSV2 in black (170 ± 30 nm).

Figure 3

C1s spectra of (a) AuNPs, (b) Resveratrol (RSV), (c) AuNPs@RSV1 and (d) AuNPs@RSV2.

Figure 4

Fourier Transform Infrared Spectroscopy (FT-IR) spectra in the 2000–600 cm⁻¹ range of samples AuNPs, AuNPs@RSV1 and AuNPs@RSV2. Markers represent peaks related to resveratrol.

Figure 5

Near Edge X-ray Absorption Fine Structure (NEXAFS) C K edge NEXAFS spectra of samples AuNPs, AuNPs@RSV1 and AuNPs@RSV2 recorded at grazing (Th20) and normal (Th90) incidence.

Figure 6

Analyses of cellular DNA content obtained from propidium iodine assay (PI). (a) MCF-7 cells were treated for 48 h with either vehicle (ethanol/DMEM, 1/10) or E2 (10 nM) or staurosporine (100 nM; STS) or with different AuNPs concentrations. Data are means ± SD of six different experiments. (*) p < 0.001 was calculated with Student’s t test versus vehicle. (b) Western blot (left) and densitometric analyses (right) of PARP-1 cleavage in MCF-7 cells. Cells were treated with staurosporine (10 or 100 nM, STS) or with different AuNPs concentrations. The amount of cleaved protein was normalized by comparison with α-tubulin levels. Data are the mean ± SD of five different experiments. (*) p < 0.001 was determined with Student t-test with respect to the vehicle.

Figure 7

Analyses of cellular DNA content obtained from propidium iodine assay (PI). (a) MCF-7 cells were treated for 48 h with either vehicle (ethanol/DMEM, 1/10) or E2 (10 nM) or staurosporine (100 nM; STS) or with different RSV or concentrations. Data are means ± SD of six different experiments. p < 0.001 was calculated with ANOVA followed by Bonferroni test versus vehicle (a), versus RSV 0.1 and 1 µM (b), versus the same concentrations of AuNPS@RSV1 (c), versus AuNPS@RSV1 0.1 µM (d), and versus the same concentrations of RSV (e). (b) Western blot (left) and densitometric analyses (right) of PARP-1 cleavage in MCF-7 cells. Cells were treated with E2 (10 nM) or staurosporine (10 nM, STS) or with different AuNPs@RSV1 concentrations. The amount of cleaved protein was normalized by comparison with α-tubulin levels. Data are the mean ± SD of three different experiments. (*) p < 0.001 was determined with Student t-test with respect to the vehicle.