Ligand impact on reactive oxygen species generation of Au10 and Au25 nanoclusters upon one- and two-photon excitation

Abstract

In photodynamic therapy (PDT), light-sensitive photosensitizers produce reactive oxygen species (ROS) after irradiation in the presence of oxygen. Atomically-precise thiolate-protected gold nanoclusters are molecule-like nanostructures with discrete energy levels presenting long lifetimes, surface biofunctionality, and strong near-infrared excitation ideal for ROS generation in PDT. We directly compare thiolate-gold macromolecular complexes (Au₁₀) and atomically-precise gold nanoclusters (Au₂₅), and investigate the influence of ligands on their photoexcitation. With the ability of atomically-precise nanochemistry, we produce Au₁₀SG₁₀, Au₁₀AcCys₁₀, Au₂₅SG₁₈, and Au₂₅AcCys₁₈ (SG: glutathione; AcCys: N-acetyl-cysteine) fully characterized by high-resolution mass spectrometry. Our theoretical investigation reveals key factors (energetics of excited states and structural influence of surface ligands) and their relative importance in singlet oxygen formation upon one- and two-photon excitation. Finally, we explore ROS generation by gold nanoclusters in living cells with one- and two-photon excitation. Our study presents in-depth analyses of events within gold nanoclusters when photo-excited both in the linear and nonlinear optical regimes, and possible biological consequences in cells.

Fig. 1. Type I and type II mechanism of ROS generation using photoexcited gold nanoclusters (upon one- and two-photon excitation).

CT charge transfer, ISC intersystem crossing, ${{\mathrm{O}}_2}^{\cdot -}$, superoxide anion, •OH hydroxyl radical, H₂O₂ hydrogen peroxide, ³O₂ triplet state oxygen (molecular oxygen), ¹O₂ singlet oxygen.

Fig. 2. Comparison of Au₁₀AcCys₁₀ and Au₁₀SG₁₀ illustrating ligand effects by hydrogen bonding network.

Structures are presented for a Au₁₀AcCys₁₀ and b Au₁₀SG₁₀. Gold atoms in structures are labeled yellow. Ligands are shown in windows. For details on structures optimization see Computational Approach. SG glutathione, AcCys N-acetyl-cysteine.

Fig. 3. Comparison of Au₂₅AcCys₁₈ and Au₂₅SG₁₈ illustrating ligand effects by hydrogen bonding network.

Structures are presented for a Au₂₅AcCys₁₈ and b Au₂₅SG₁₈. Gold atoms in structures are labeled yellow. Ligands are shown in windows. For details on structures optimization see Computational Approach. SG glutathione, AcCys N-acetyl-cysteine.

Fig. 4. Time-dependent density functional theory (TDDFT) energies for singlets (S_n, n = 1–5) and triplets (T_n, n = 1–5) states.

TDDFT energies are shown for a Au₁₀AcCys₁₀ and b Au₁₀SG₁₀. Singlet-triplet (E_S1-E_T1) energy gaps are: ΔE_S-T (Au₁₀AcCys₁₀) = 0.68 eV and ΔE_S-T (Au₁₀SG₁₀) = 0.67 eV. For details on structures optimization see Computational Approach. Gold atoms in structures are labeled yellow. Ligands are detailed in windows of Fig. 2.

Fig. 5. Time-dependent density functional theory (TDDFT) energies for singlets (S_n, n = 1–5) and triplets (T_n, n = 1–5) states.

TDDFT energies are shown for a Au₂₅AcCys₁₈ and b Au₂₅SG₁₈. Singlet-triplet (E_S1-E_T1) energy gaps are: ΔE_S-T (Au₂₅AcCys₁₈) = 0.15 eV and ΔE_S-T (Au₂₅SG₁₈) = 0.13 eV. For details on structures optimization see Computational Approach. Gold atoms in structures are labeled yellow. Ligands are detailed in windows of Fig. 3.

Fig. 6. Oxidative stress in human microglia in response to single- and two-photon-stimulated gold nanoclusters.

a Representative fluorescence micrographs of ROS level in human microglia loaded with CellROX (red) and treated with Au₁₀AcCys₁₀, Au₁₀SG₁₀, Au₂₅AcCys₁₈ or Au₂₅SG₁₈ at 100 μM in serum-deprived conditions before stimulation with a one-photon laser (473 nm) for 3 min. Nuclei (blue) were labeled with Hoechst 33342. b Shown are the average level of ROS per individual cell (white dot) and the average ROS level per condition (black bar ±SD) in microglia treated as in a), normalized to the fluorescence intensity of the untreated control (set to 1). f.i. a.u. fluorescence intensity arbitrary units. At least 540 cells were analyzed from three independent experiments. c Representative fluorescence confocal micrographs of singlet oxygen levels (red, white arrows) in human microglia treated with gold nanoclusters Au₁₀AcCys₁₀, Au₁₀SG₁₀, Au₂₅AcCys₁₈, or Au₂₅SG₁₈ at 100 μM before exposure to two-photon laser (720 nm) for 3 min to induce singlet oxygen production. Singlet oxygen level was detected using the fluorescent probe Si-DMA. Nuclei (blue) are labeled with Hoechst 33342. d Shown are the average level of singlet oxygen in individual microglia cells (white dot) treated as in (a)), and the average level per condition (black bar), normalized to the fluorescence intensity prior to laser exposure (0 min, set to 1) from at least 60 cells per condition and at least two independent experiments. ***p < 0.001.