Gold nanoclusters elicit homeostatic perturbations in glioblastoma cells and adaptive changes of lysosomes

Abstract

Unique physicochemical features place gold nanoclusters at the forefront of nanotechnology for biological and biomedical applications. To date, information on the interactions of gold nanoclusters with biological macromolecules is limited and restricts their use in living cells. Methods: Our multidisciplinary study begins to fill the current knowledge gap by focusing on lysosomes and associated biological pathways in U251N human glioblastoma cells. We concentrated on lysosomes, because they are the intracellular destination for many nanoparticles, regulate cellular homeostasis and control cell survival. Results: Quantitative data presented here show that gold nanoclusters (with 15 and 25 gold atoms), surface-modified with glutathione or PEG, did not diminish cell viability at concentrations ≤1 µM. However, even at sublethal concentrations, gold nanoclusters modulated the abundance, positioning, pH and enzymatic activities of lysosomes. Gold nanoclusters also affected other aspects of cellular homeostasis. Specifically, they stimulated the transient nuclear accumulation of TFEB and Nrf2, transcription factors that promote lysosome biogenesis and stress responses. Moreover, gold nanoclusters also altered the formation of protein aggregates in the cytoplasm. The cellular responses elicited by gold nanoclusters were largely reversible within a 24-hour period. Conclusions: Taken together, this study explores the subcellular and molecular effects induced by gold nanoclusters and shows their effectiveness to regulate lysosome biology. Our results indicate that gold nanoclusters cause homeostatic perturbations without marked cell loss. Notably, cells adapt to the challenge inflicted by gold nanoclusters. These new insights provide a framework for the further development of gold nanocluster-based applications in biological sciences.

Keywords: cell organelle; cellular stress response; lysosome positioning; nanomaterials; organellar pH; proteostasis.

Figure 1

Synthesis and UV-vis absorption spectra of AuNCs. (A) Schematic representation of AuNC synthesis. AuNCs surfaces were modified with glutathione (SG) and PEG. Following the controlled reduction of gold with L-glutathione, AuNCs were PEGylated using the peptide coupling method described in the Methods section. (B) Absorbance spectra of Au₁₅ and Au₂₅ nanoclusters. Absorbance spectra for the different AuNCs indicated were obtained at a final concentration of 100 µM in water.

Figure 2

Impact of AuNCs on cell numbers. (A) U251N cells were treated with Au₁₅SG₁₃ for 72 hours with the concentrations indicated. Cells were identified with Hoechst 33342 staining of the nuclei as described in the Methods section. (B) Upon incubation with different AuNCs, cell numbers were quantified and results were normalized to vehicle controls. Graphs depict the average ± SEM for three independent experiments. Significant differences between the vehicle control and AuNC-treated cells are marked. *, p<0.05; ***, p<0.001.

Figure 3

Evaluation of lysosomes in U251N cells. All graphs depict results normalized to the vehicle control; data are shown as average ± SEM; *p<0.05, **p<0.01, ***p<0.001; AU, arbitrary units. (A) Detection of LAMP-2. Cells were treated with 1 μM Au₁₅SG₁₃ or 1 μM Au₁₅PEG for 4 or 24 hours. LAMP-2 was located by immunocytochemistry (red); nuclei were demarcated with Hoechst 33342 (blue). Fluorescence intensities/area were quantified for at least 97 cells per condition. (B) Lysosome staining with Lysotracker Red. Cells were incubated with vehicle, 1 μM Au₁₅SG₁₃ or 1 μM Au₁₅PEG for 4 or 24 hours. The bars depict the average fluorescence intensity/area ± SEM; 52 to 134 cells were assessed per condition. (C) The distribution of Lysotracker Red signals was determined for the 4- and 24-hour treatment shown in part B. The fluorescence intensities were quantified for a 5-μm area adjacent to the nuclear margin (perinuclear) and for peripheral cell regions. The ratio of perinuclear/peripheral signals was calculated for 44 to 58 cells for each condition. Results are depicted as average ± SEM. (D) Staining of U251N cells with Lysosensor Green. U251N cells treated with 1 μM Au₁₅SG₁₃ or 1 μM Au₁₅PEG for 4 or 24 hours were incubated with Lysosensor Green and imaged as described in the Methods section. Graphs depict average fluorescence intensities per area ± SEM; measurements were performed for a minimum of 81 cells per condition and at least two independent experiments.

Figure 4

(A) Ratiometric measurement of cytoplasmic pH. U251N cells were treated for 4 hours with 1 µM Au₁₅SG₁₃ or 1 µM Au₁₅PEG in serum-free medium. Following treatment, cells were incubated with 5 μM SNARF®-1 in serum-free DMEM for 45 min at 37^oC. The pH values were determined by ratiometric fluorescence measurements and extrapolation from the calibration curve. The graph shows averages ± SEM for one experiment with triplicate samples. (B) Subcellular distribution of STAT3 and LAMP-1. U251N cells were incubated with vehicle, 1 µM Au₁₅SG_13, 1 µM Au₁₅PEG or 20 µM chloroquine (CQ) in serum-free medium for 4 hours or 24 hours as indicated. Cells were fixed and processed for immunocytochemistry as described in the Methods section. Scale bar is 20 µm. Selected regions of the overlay image were magnified 5-fold. Yellow color in the overlay images indicates co-localization of STAT3 and LAMP-1.

Figure 5

Au₁₅SG₁₃ increases the concentration of filamentous actin. U251N cells were incubated with 10 µM Au₁₅SG₁₃ for 24 hours, fixed and processed for immunohistochemistry to detect α-tubulin. F-actin was stained with Alexa Fluor® 488 phalloidin (Phalloidin); nuclei were demarcated with DAPI. Scale bar is 20 µm. Fluorescence intensities for phalloidin and α-tubulin were quantified for vehicle (105 cells) and Au₁₅SG₁₃ treated samples (126 cells). Graphs depict average + SEM for one representative experiment. Size was measured for 210 (vehicle) and 231 (Au₁₅SG₁₃) cells. Nuclear size was determined for 371 (vehicle) and 385 (Au₁₅SG₁₃) cells. AU, arbitrary units. Statistical evaluation was performed with Student's t-test; **, p<0.01; ***, p<0.001.

Figure 6

Effects of AuNCs on TFEB nucleocytoplasmic distribution and lysosomal activity. (A) U251N cells were treated with vehicle, 1 μM Au₁₅SG₁₃ or 1 μM Au₁₅PEG in DMEM for the times indicated. Panels depict the results for cells incubated for 30 min with vehicle or AuNCs. TFEB was detected by immunocytochemistry, phalloidin stained F-actin, and Hoechst 33342 demarcated nuclei. Whole cell and nuclear fluorescence signals were quantified, and the nuclear/cytoplasmic ratio was calculated. The percentage of cells with elevated nuclear TFEB signals was determined for two independent experiments. For each condition and time point 80 to 101 cells were scored; bars show averages ± SEM. (B) AuNCs increase cathepsin B activity. Cathepsin B activity was measured in U251N cells treated with vehicle, 1 μM Au₁₅SG₁₃ or 1 μM Au₁₅PEG. Cells were incubated with Magic Red as described in the Methods section. Hoechst 33342 identified nuclei. For quantification, Magic Red fluorescence intensity was normalized to the vehicle control. Bars depict average ± SEM for at least two independent experiments. Between 34 and 92 cells were analyzed per condition and time point; **, p<0.01; ***, p<0.001.

Figure 7

AuNCs promote the nuclear accumulation of Nrf2. U251N cells were treated for the times indicated with vehicle, 300 μM of H₂O₂, 10 μM of Au₁₅SG₁₃ or 10 μM Au₁₅PEG for the times indicated. Cells were processed for immunocytochemistry with antibodies against Nrf2. Nuclei were detected with Hoechst 33342 and F-actin stained with phalloidin. After incubation for 10, 30 or 60 minutes, Nrf2 nuclear translocation was quantified by assessing Nrf2 fluorescence in the nucleus and cytosol. Three independent experiments were conducted and a total of 120 cells were analyzed. The graph shows average ± SEM; *, p<0.05; **, p< 0.01; ***, p<0.001.

Figure 8

Effect of AuNCs on protein aggregation. (A) U251N cells treated with vehicle, 1 μM Au₁₅SG₁₃ or Au₁₅PEG in DMEM for 24 hours were fixed and protein aggregation was evaluated with Proteostat. Some of the aggregates are marked with arrowheads. Fluorescence intensities/area were quantified for 97 to 105 cells for each condition. Results normalized to the vehicle control are shown as average ± SEM; ***, p<0.001. (B) U251N cells were incubated as described in part A. Chloroquine (CQ) was present during treatment as indicated. Proteostat signals were quantified for 88 to 105 cells per condition. Results were normalized to the vehicle control/minus chloroquine. Bars show average ± SEM; *p<0.05, **p<0.01, ***p<0.001.

Figure 9

Simplified model depicting the cellular components that are affected, at least transiently, by AuNCs. AuNCs in this study were surface-modified with GSH (Au-SG) or PEGylated (Au-PEG). Lysosomes with more acidic pH (light green) are perinuclear, whereas peripheral lysosomes are less acidic (dark green). AuNCs stimulate the transient nuclear accumulation of transcription factors TFEB and Nrf2. PEGylated AuNCs may increase protein aggregation. See text for details.