Stabilization of Graphene Oxide Dispersion in Plasma-like Isotonic Solution Containing Aggregating Concentrations of Bivalent Cations

Abstract

Graphene oxide's (GO) intravascular applications and biocompatibility are not fully explored yet, although it has been proposed as an anticancer drug transporter, antibacterial factor or component of wearable devices. Bivalent cations and the number of particles' atom layers, as well as their structural oxygen content and pH of the dispersion, all affect the GO size, shape, dispersibility and biological effects. Bovine serum albumin (BSA), an important blood plasma protein, is expected to improve GO dispersion stability in physiological concentrations of the precipitating calcium and magnesium cations to enable effective and safe tissue perfusion.

Methods: Four types of GO commercially available aqueous dispersions (with different particle structures) were diluted, sonicated and studied in the presence of BSA and physiological cation concentrations. Nanoparticle populations sizes, electrical conductivity, zeta potential (Zetasizer NanoZS), structure (TEM and CryoTEM), functional groups content (micro titration) and dispersion pH were analyzed in consecutive preparation stages.

Results: BSA effectively prevented the aggregation of GO in precipitating concentrations of physiological bivalent cations. The final polydispersity indexes were reduced from 0.66-0.91 to 0.36-0.43. The GO-containing isotonic dispersions were stable with the following Z-ave results: GO1 421.1 nm, GO2 382.6 nm, GO3 440.2 nm and GO4 490.1 nm. The GO behavior was structure-dependent.

Conclusion: BSA effectively stabilized four types of GO dispersions in an isotonic dispersion containing aggregating bivalent physiological cations.

Keywords: aqueous dispersion; bovine serum albumin; graphene oxide; ion-induced aggregation; nanoparticle size; stability of dispersion.

Figure 1

Typical course of the conductometric titration curve of GO dispersions with two distinct subcourses corresponding to the two relevant functional groups significantly differing in acidity, i.e., sulfonic (-SO3H) and carboxylic (-COOH) groups.

Figure 2

Size distribution by intensity, charts for purchased dispersions of GO samples: GO1pu (A), GO2pu (B), GO3pu (C), GO4pu (D). Data shown as averaged results of 30 measurements in 10 samples. Intensity expressed in percentages represents the percentage of given particle population in the entire particle population of the analyzed sample; size represents hydrodynamic particle diameter expressed in nm.

Figure 3

Stock dispersions of GO. (A) The mean particle size of the most frequent fraction (Int1) presented together with the percentage of the fraction in peak means analysis (Int1%) (results expressed as mean values of the single measurements). (B) Zeta potential of the original dispersions expressed in mV. (C) The conductivity of the original dispersions of GO in mS/cm. (D) pH measured in the original dispersions of GO (* p < 0.05; ** p < 0.01).

Figure 4

Size distribution by the intensity of dispersions of GO (red) and diluted dispersions in ultrapure water in a concentration of 140 µg/mL (green). GO1 (A), GO2 (B), GO3 (C), GO4 (D). Charts represent the averaged (pooled) results of 30 measurements in 10 samples.

Figure 5

Diluted GO dispersions (140 µg/mL). (A) Mean particle sizes of the most frequent fractions are presented together with a percentage of the fraction in peak means analysis of single measurements. The GO4 bar represents only 15 measurements out of the 42 measurements performed. (B) Zeta potential of diluted GO dispersions (140 µg/mL) in mV. (C) Conductivity of the diluted GO dispersions (140 µg/mL) expressed in mS/cm. (D) pH measured in diluted dispersions of graphene oxides (140 µg/mL). (p < 0.05 *; p < 0.01 **).

Figure 6

Size distribution by the intensity of four types of GO dispersions before (red) and after six sonication steps (50 W) (green). GO1 (A), GO2 (B), GO3 (C), GO4 (D). Charts represent the averaged results of 30 measurements in 10 samples.

Figure 7

Sonicated dispersions of GO (140 µg/mL). (A) Mean particle sizes of the most frequent particle size fractions are presented together with a percentage of each fraction in peak means analysis. (B) Zeta potential of sonicated water GO dispersions (140 µg/mL) in mV. (C) The conductivity of the sonicated GO dispersions (140 µg/mL) expressed in mS/cm. (D) pH measured in sonicated GO dispersions (140 µg/mL (* p < 0.05; ** p < 0.01)).

Figure 8

Size distribution in four types of GO dispersions after six sonication steps (red) and after adding BSA (green). GO1 (A), GO2 (B), GO3 (C), GO4 (D). Charts represent mean values for 10 samples measured 3 times each.

Figure 9

Sonicated GO dispersions with BSA. (A) Mean particle sizes of the most frequent fractions are presented together with a percentage of the fraction in peak means analysis and statistical analysis performed on single measurements. GO in concentration of 50 µg/mL and BSA in concentration of 400 mg/L. (B) Zeta potential of sonicated water GO dispersions with BSA in mV. (C) The conductivity of the sonicated GO dispersions with BSA expressed in mS/cm. (* p < 0.05; ** p < 0.01).

Figure 10

Size distribution by the intensity of sonicated dispersions with BSA (red) and dispersion of GO with BSA and Krebs–Henseleit (KH) buffer (green) for each analyzed GO. GO1 (A), GO2 (B), GO3 (C), GO4 (D). Charts represent mean values for 10 samples measured 3 times each.

Figure 11

Sonicated GO dispersions with BSA in KH buffer. (A) Mean particle sizes of the most frequent fractions are presented together with the percentage of the fraction in peak means analysis. (B) Zeta potential of sonicated water GO dispersions with BSA in KH buffer in mV. (C) Electrical conductivity of the sonicated GO dispersions with BSA in KH buffer expressed in mS/cm. (D) pH values of the sonicated water GO dispersions with BSA in KH buffer (* p < 0.05; ** p < 0.01).

Figure 12

The GO1 at three chosen stages of preparation visualized with TEM and cryo-TEM. Stage and calibration description under the pictures. (A): GO1 stock dispersion in TEM. The calibration bars in the bottom of the pictures represent, from left to right, 2 µm; 100 nm; 200 nm. (B): GO1 dispersion with BSA in TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 100 nm; 1 µm. (C): The GO1-KH-BSA dispersion visualized with CRYO-TEM. The calibration bars in the bottom of the pictures represent, from left to right, 200 nm; 200 nm; 200 nm.

Figure 13

The GO2 at three chosen stages of preparation seen in TEM and with cryo-TEM. Stage description under the pictures. (A): GO2 stock dispersion The calibration bars in the bottom of the pictures represent, from left to right, 200 nm; 100 nm; 50 nm. (B): The GO2 dispersion with BSA. The calibration bars in the bottom of the pictures represent, from left to right, 20 nm; 50 nm; 500 nm. (C): The GO2-KH-BSA dispersion visualized with CRYO-TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 50 nm; 100 nm.

Figure 14

The GO3 at three chosen stages of preparation seen in TEM and with CRYO-TEM. Stage description under the pictures. (A): The GO3 stock dispersion visualized with TEM. The calibration bars in the bottom of the pictures represent, from left to right, 50 nm; 5 µm; 100 nm. (B): The GO3 dispersion with BSA visualized with TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 50 nm; 100 nm. (C): The GO3-KH-BSA dispersion visualized with CRYO-TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 100 nm; 100 nm.

Figure 15

GO4 at three chosen stages of preparation visualized with TEM and cryo-TEM. Stage description under the pictures. (A): The GO4 stock dispersion visualized with TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 50 nm; 200 nm. (B): The GO4-BSA dispersion visualized with TEM. The calibration bars in the bottom of the pictures represent, from left to right, 500 nm; 1 µm; 200 nm. (C): The GO4-KH-BSA dispersion visualized with CRYO-TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 100 nm; 200 nm.

Figure 15

GO4 at three chosen stages of preparation visualized with TEM and cryo-TEM. Stage description under the pictures. (A): The GO4 stock dispersion visualized with TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 50 nm; 200 nm. (B): The GO4-BSA dispersion visualized with TEM. The calibration bars in the bottom of the pictures represent, from left to right, 500 nm; 1 µm; 200 nm. (C): The GO4-KH-BSA dispersion visualized with CRYO-TEM. The calibration bars in the bottom of the pictures represent, from left to right, 100 nm; 100 nm; 200 nm.

Figure 16

The atomic force microscope (AFM) of four types of GO with BSA: (A): GO1, (B): GO2, (C): GO3, (D): GO4. The granules of the BSA are easily visible in GO2 and GO4, but not in the case of GO1 and GO3. The red triangles depict the points of the measurements both for vertical and horizontal directions. The vertical distance depicted in the measurement result area refers to the thickness of the GO particle.

Figure 17

Illustration of hydrolysis of anhydride (and lactone) groups accompanied by desulfonation of graphene-oxide-based materials obtained by wet chemistry exfoliation. Dashed ovals surround the areas of the particle modifications.