Polyacrylic-Coated Solid Nanoparticles Increase the Aquaporin Permeability to Hydrogen Peroxide

Abstract

Aquaporins (AQPs) allow the diffusion of hydrogen peroxide (H₂O₂) and act as ROS scavenging systems, which are important for controlling the redox state of cells. Recently, cerium oxide nanoparticles were found to increase the water and H₂O₂ permeability by modulating AQPs. To further analyze the action of nanoparticles (NPs) on AQP, we examined the effect of the NPs presenting different core compositions (CeO₂, Gd₂O₃, Fe₃O₄, and TiO₂), hydrodynamic sizes, and surface functionalization. The NPs produced an increase in H₂O and H₂O₂ permeability as a general trend. The hydrodynamic sizes of the NPs in the range of 22-100 nm did not produce any significant effect. The chemical nature of the NPs' core did not modify the effect and its intensity. On the other hand, the NPs' functionalized surface plays a major role in influencing both water and H₂O₂ permeability. The results suggest that NPs can play a significant role in controlling oxidative stress in cells and might represent an innovative approach in the treatment of a number of pathologies associated with an increased oxidative status.

Keywords: CeO2NP; Fe3O4NP; Gd2O3NP; HeLa; HyPer7 biosensor; TiO2NP; oxidative stress; peroxiporin; water channels.

Figure 1

(A) Hydrodynamic size graph (distribution by number) of both small and large FeNPs, GdNPs, TiNPs, and CeNPs and dextran-coated CeNPs (S-CeNPdexs) with the corresponding TEM images. Note that the aggregate aspect of the crystals visible in the TEM images is the result of the sample preparation and does not reflect the real aggregation state in the original suspension. Bars, 100 nm. (B) Summary of hydrodynamic size, zeta potential, and polydispersity index values for all nanoparticles.

Figure 2

Effect of S-FeNPs (A), L-FeNPs (B), S-GdNPs (C), L-GdNPs (D), S-CeNPs (E), L-CeNPs (F), S-TiNPs (G), and S-CeNPdexs (H) on the cell viability of HeLa. Cells were incubated for 2 h with nanoparticles diluted 1:10, 1:20, 1:50, 1:100, and 1:200 in PBS. Cell viability was calculated by measuring the blue (live cells; Ex 360nm–Em 460 nm) over the green (dead cells; Ex 420 nm–Em 535 nm) fluorescence signal ratio in treated cells versus untreated cells (Ctr). Ratio values expressed in percentage (normalized to the total protein content) are the mean ± S.E.M. of cells for each of the three different experiments. a, p < 0.05 versus Ctr; b, p < 0.001 versus Ctr (ANOVA, followed by Dunnett’s multiple comparison test). Concentration of the NPs’ stock solutions: S-FeNP and L-FeNP, 5.4 mg/mL; S-GdNP and L-GdNP, 6.4 mg/mL; S-CeNP (E) and L-CeNP, 6.6 mg/mL; S-TiNP, 6.0 mg/mL; S-CeNPdex, 6.5 mg/mL.

Figure 3

Hydrogen peroxide and water permeability in HeLa cells treated with S-FeNPs. H₂O₂ permeability in HeLa control cells (Ctr) and in HeLa cells treated for 2 h (S-FeNP) was evaluated after the addition of 50 μM H₂O₂. (A) Representative images extracted from videos showing the kinetics of H₂O₂ permeability in control and treated cells. The increased HyPer7-NES fluorescence is shown in pseudo color (upper panel; the scale used is indicated in the insert). (B) Representative time course of H₂O₂ fluorescence into control and treated cells. (C) Bars represent the maximal H₂O₂ fluorescence, which was obtained by computerized least squares regression, fitting the experimental points of the time courses of H₂O₂ transported curves with a one-phase exponential association equation (GraphPad Prism 4.00 2003). Maximal fluorescence values are means ± S.E.M. of 3 different experiments. a, p < 0.0005 (Student’s t-test). (D) Control cells (Ctr) and cells incubated with nanoparticles for 2 h (S-FeNP) were exposed to an osmotic gradient of 150 mOsm. Bars represent the osmotic water permeability of HeLa cells expressed as a percent of k relative. Values are means ± S.E.M. of 10–20 single shots for each of the 3 different experiments. b, p = 0.0176 (Student’s t-test).

Figure 4

Hydrogen peroxide and water permeability in HeLa cells treated with L-FeNP. H₂O₂ permeability in HeLa control cells (Ctr) and HeLa cells treated for 2 h (L-FeNP) was evaluated after the addition of 50 μM H₂O₂. (A,B) See the legend of Figure 3. (C) Bars represent the maximal H₂O₂ fluorescence, which was obtained by computerized least squares regression, fitting the experimental points of the time courses of H₂O₂ transported curves with a one-phase exponential association equation (GraphPad Prism 4.00 2003). Maximal fluorescence values are means ± S.E.M. of 3 different experiments. a, p < 0.0001 (Student’s t-test). (D) Control cells (Ctr) and cells incubated with nanoparticles for 2 h (L-FeNP) were exposed to an osmotic gradient of 150 mOsm. Bars represent the osmotic water permeability of HeLa cells expressed as a percent of k relative. Values are means ± S.E.M. of 10–20 single shots for each of the 3 different experiments (Student’s t-test).

Figure 5

Hydrogen peroxide and water permeability in HeLa cells treated with S-GdNP. H₂O₂ permeability in HeLa control cells (Ctr) and HeLa cells treated for 2 h (S-GdNP) was evaluated after the addition of 50 μM H₂O₂. (A,B) See the legend of Figure 3. (C) Bars represent the maximal H₂O₂ fluorescence, which was obtained by computerized least squares regression, fitting the experimental points of the time courses of H₂O₂ transported curves with a one-phase exponential association equation (GraphPad Prism 4.00 2003). Maximal fluorescence values are means ± S.E.M. of the 3 different experiments. a, p < 0.0239 (Student’s t-test). (D) Control cells (Ctr) and cells incubated with nanoparticles for 2 h (S-GdNP) were exposed to an osmotic gradient of 150 mOsm. Bars represent the osmotic water permeability of HeLa cells expressed as a percent of k relative. Values are means ± SEM of 10–20 single shots for each of the 3 different experiments. a, p = 0.0317 (Student’s t-test).

Figure 6

Hydrogen peroxide and water permeability in HeLa cells treated with L-GdNP. H₂O₂ permeability in HeLa control cells (Ctr) and HeLa cells treated for 2 h (L-GdNP) was evaluated after the addition of 50 μM H₂O₂. (A,B) See the legend of Figure 3. (C) Bars represent the maximal H₂O₂ fluorescence, which was obtained by computerized least squares regression, fitting the experimental points of the time courses of H₂O₂ transported curves with a one-phase exponential association equation (GraphPad Prism 4.00 2003). Maximal fluorescence values are means ± S.E.M. of the 3 different experiments. a, p = 0.045 (Student’s t-test). (D) Control cells (Ctr) and cells incubated with nanoparticles for 2 h (L-GdNP) were exposed to an osmotic gradient of 150 mOsm. Bars represent the osmotic water permeability of HeLa cells expressed as a percent of k relative. Values are means ± S.E.M. of 10–20 single shots for each of the 3 different experiments. a, p < 0.01 (Student’s t-test).

Figure 7

Hydrogen peroxide and water permeability in HeLa cells treated with L-CeNP. H₂O₂ permeability in HeLa control cells (Ctr) and HeLa cells treated for 2 h (L-CeNP) was evaluated after the addition of 50 μM H₂O₂. (A,B) See the legend of Figure 3. (C) Bars represent the maximal H₂O₂ fluorescence, which was obtained by computerized least squares regression, fitting the experimental points of the time courses of H₂O₂ transported curves with a one-phase exponential association equation (GraphPad Prism 4.00 2003). Maximal fluorescence values are means ± S.E.M. of the 3 different experiments (Student’s t-test). (D) Control cells (Ctr) and cells incubated with nanoparticles for 2 h (L-CeNP) were exposed to an osmotic gradient of 150 mOsm. Bars represent the osmotic water permeability of HeLa cells expressed as a percent of k relative. Values are means ± S.E.M. of 10–20 single shots for each of the 3 different experiments. a, p = 0.0147 (Student’s t-test).

Figure 8

Hydrogen peroxide and water permeability in HeLa cells treated with TiNP. H₂O₂ permeability in HeLa control cells (Ctr) and HeLa cells treated for 2 h (TiNP) was evaluated after the addition of 50 μM H₂O₂. (A,B) See the legend of Figure 3. (C) Bars represent the maximal H₂O₂ fluorescence, which was obtained by computerized least squares regression, fitting the experimental points of the time courses of H₂O₂ transported curves with a one-phase exponential association equation (GraphPad Prism 4.00 2003). Maximal fluorescence values are means ± S.E.M. of the 3 different experiments (Student’s t-test). (D) Control cells (Ctr) and cells incubated with nanoparticles for 2 h (TiNP) were exposed to an osmotic gradient of 150 mOsm. Bars represent the osmotic water permeability of HeLa cells expressed as a percent of k relative. Values are means ± S.E.M. of 10–20 single shots for each of the 3 different experiments. a, p < 0.0001 (Student’s t-test).

Figure 9

Hydrogen peroxide and water permeability in HeLa cells treated with S-CeNPdexs. H₂O₂ permeability in HeLa control cells (Ctr) and HeLa cells treated for 2 h (S-CeNPdex) was evaluated after the addition of 50 μM H₂O₂. (A,B) See the legend of Figure 3. (C) Bars represent the maximal H₂O₂ fluorescence, which was obtained by computerized least squares regression, fitting the experimental points of the time courses of H₂O₂ transported curves with a one-phase exponential association equation (GraphPad Prism 4.00 2003). Maximal fluorescence values are means ± S.E.M. of the 3 different experiments (Student’s t-test). (D) Control cells (Ctr) and cells incubated with nanoparticles for 2 h (S-CeNPdex) were exposed to an osmotic gradient of 150 mOsm. Bars represent the osmotic water permeability of HeLa cells expressed as a percent of k relative. Values are means ± S.E.M. of 10–20 single shots for each of the 3 different experiments (Student’s t-test).

Figure 10

Summary of the NPs’ characteristics and permeability changes. Gray circles indicate small and large NPs. Green circles indicate the negative, while the gray indicates the neutral functionalization. Red arrows indicate H₂O₂ permeability, while blue arrows indicate H₂O permeability. Arrows pointing up or down indicate increased or decreased permeability. PAA, poly(acrylic) acid; dex, dextran.