Surface Charge-Modulated Toxicity of Cysteine-Stabilized Silver Nanoparticles

Abstract

The toxicity of silver nanoparticles (AgNPs) depends on their physicochemical properties. The ongoing research aims to develop effective methods for modifying AgNPs using molecules that enable control over the processes induced by nanoparticles in both normal and cancerous cells. Application of amino acid-stabilized nanoparticles appears promising, exhibiting tunable electrokinetic properties. Therefore, this study focused on determining the influence of the surface charge of cysteine (CYS)-stabilized AgNPs on their toxicity towards human normal B (COLO-720L) and T (HUT-78) lymphocyte cell lines. CYS-AgNPs were synthesized via the chemical reduction. Transmission electron microcopy (TEM) imaging revealed that they exhibited a quasi-spherical shape with an average size of 18 ± 3 nm. CYS-AgNPs remained stable under mild acidic (pH 4.0) and alkaline (7.4 and 9.0) conditions, with an isoelectric point observed at pH 5.1. Following a 24 h treatment of lymphocytes with CYS-AgNPs, concentration-dependent alterations in cell morphology were observed. Positively charged CYS-AgNPs notably decreased lymphocyte viability. Furthermore, they exhibited grater genotoxicity and more pronounced disruption of biological membranes compared to negatively charged CYZ-AgNPs. Despite both types of AgNPs interacting similarly with fetal bovine serum (FBS) and showing comparable profiles of silver ion release, the biological assays consistently revealed that the positively charged CYS-AgNPs exerted stronger effects at all investigated cellular levels. Although both types of CYS-AgNPs have the same chemical structure in their stabilizing layers, the pH-induced alterations in their surface charge significantly affect their biological activity.

Keywords: amino acids; charge inversion; cysteine; cytotoxicity; genotoxicity; impact of surface charge; lymphocytes; protein adsorption; silver nanoparticles; streaming potential; zeta potential.

Figure 1

TEM micrographs of CYS-AgNPs dispersed in aqueous suspensions of (a) pH 4.0, (b) pH 9.0, and (c) in full RPMI medium supplemented by 10% FBS of pH 7.4; (d) size distribution of CYS-AgNPs after 24 h of incubation in pH 4.0 (red bars), pH 9.0 (blue bars) and 10% FBS RPMI 1640 medium (green bars). The histogram was constructed by analyzing the surface areas and diameters of 1000 CYS-AgNPs from the TEM micrographs.

Figure 2

(a) Effect of Bt-BSA deposition on CYS-AgNPs determined as the dependence of absorption (at λ = 450 nm) of Bt-BSA on CYS-AgNPs(+) or CYS-AgNP(−). (b) Effect of FBS deposition on CYS-AgNP monolayers determined as the dependence of zeta potential of monolayers on ionic strength at selected pH based on the streaming potential measurements. The solid lines are a fit of the experimental data.

Figure 3

The impact of CYS-AgNPs on the viability of (a) COLO-720L and (b) HUT-78 cells was assessed. The viability of the lymphocytes was evaluated after 24 h of CYS-AgNP treatment using the MTT assay. Data obtained for control samples (without CYS-AgNPs) are marked in gray. Data points are means ± SD (five replicate trials). Different letters indicate significant (p < 0.05) differences between treatments.

Figure 4

Impact of CYS-AgNPs on the LDH release and membrane integrity of (a) COLO-720L and (b) HUT-78 cells. The LDH release from the lymphocyte cells was evaluated after 24 h of CYS-AgNP treatment using LDH assay. Data points are means± SD (five replicate trials). Different letters indicate significant (p < 0.05) differences between treatments.

Figure 5

TEM micrographs of COLO-720L cells: (a) before and after 24 h of exposure to (b) CYS-AgNPs(+) and (c) CYS-AgNPs(−) at concentrations of 5 mg L⁻¹. The scale bar represents 3000 nm.

Figure 6

TEM micrographs of HUT-78 cells: (a) before and after 24 h of exposure to (b) CYS-AgNPs(+) and (c) CYS-AgNPs(−) at a concentration of 10 mg L⁻¹. The scale bar represents 2000 nm for panel (a) and 1000 nm for panels (b) and (c).