Rheology of Graphene Nanomaterials Change in Biological Fluid

Graphene is a two-dimensional carbon sheet composed of sp2 hybridized carbon atoms arranged in a regular hexagonal structure on an atomic scale. The remarkable mechanical properties of this material have piqued people’s interest.

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Most of those fascinating properties of graphene are preserved in graphene-based materials like graphene oxide (GO) and graphene nanoplatelets (GNP). Quantum physics, catalysis, nanocomposites, nanoelectronics, and sensor technology are just a few of the applications for graphene nanomaterials.

Due to their large surface area and delocalized π electrons, they can solubilize and adsorb drug molecules, with many attempts to use them for drug delivery or imaging. However, before doing so, the behavior of these materials in biological media must be established. Proteins are physically adsorbed onto the exterior of graphene oxide through non-covalent interactions.

Because of its large percentage of sequence identities (76%), in comparison to human serum albumin (HSA), high structural stability, and low cost, bovine serum albumin (BSA) is frequently used as a model protein. The high levels of growth stimulatory factors and low levels of growth-inhibitory factors make fetal bovine serum (FBS), whose chief component is BSA, the most extensively used growth supplement for cell cultures.

Some studies have considered the rheological properties of protein solutions, but there are concerns about maintaining the physiological medium’s viscosity. When graphene-based nanoparticles are added, viscosity changes occur, which can affect the structure of protein solutions.

However, there are no studies in the literature on the rheological behavior of graphene-related substances in biological media. In light of the foregoing, a structural and rheological analysis of the interaction of three graphene-based nanomaterials, namely graphene oxide (GO), expanded graphene oxide (EGO), and graphene nanoplatelets (GNP), with two different biological media, FBS and BSA, was carried out in this study.

Methodology

Biological fluids, bovine fetal serum (FBS) and bovine serum albumin (BSA), as well as graphene nanoplatelets (GNP) and graphene oxide (GO), were obtained.

The three graphenoids were dispersed in the biological fluid to make suspensions with concentrations of 1; 1.5; 2; and 3 mg/mL (Figure 1).

Figure 1. Photographs describing dispersions preparation. (a) Nanomaterial powder and FBS; (b) homogenization of samples and (c) dispersions obtained. Image Credit: Cerpa-Naranjo et al., 2022

The nanomaterials and the nanomaterials dispersed in biological fluids’ structural characterization were carried out with microscopy techniques: transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic forces microscopy (AFM).

Microscopy techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy were used to characterize the structural properties of nanomaterials and nanomaterials dispersed in biological fluids (AFM).

At neutral pH values, zeta potential measurements were taken with a Zetasizer Nano ZS instrument. A Haake RheoStress 6000 rotational rheometer was used to make the rheological measurements.

Various concentrations of GO, GNP, and EGO suspensions were measured. The rheological measurements at high shear rates were conducted in a rheometer with micrometric capillaries on a chip Fluidicam^Rheo due to the constraints of rotational rheometry for low viscosities.

Results and Discussion

Microscopy techniques were used to characterize the structure. The images acquired by TEM are shown in Figure 2. The superposition of many layers can be seen in GO, the wrinkled surface with deformed edges owing to the formation of folds can be seen in GNP, and the superposition of several layers can be seen in EGO.

Figure 2. TEM images of the starting carbonaceous materials: (a) GO; (b) GNP; and (c) EGO powder. Image Credit: Cerpa-Naranjo et al., 2022

SEM was used to examine the morphology of the particles in greater detail. GO SEM images reveal a sheet-like structure with a smooth surface, illustrating that the layers of GO shrink when dispersed in FBS medium or water (Figure 3d, g). An equivalent morphology is noted in GNP and EGO powders, which are made up of random aggregates. It has been shown that when a nanomaterial comes into contact with a protein solution, its toxicity decreases.

Figure 3. SEM images of GO, GNP and EGO starting powders (a, b, c, respectively) and the same powders dispersed in water (d, e, f, respectively) and dispersed in FBS (g, h, i, respectively). Image Credit: Cerpa-Naranjo et al., 2022

Figure 4 shows the 2D and 3D AFM images of the three graphene-based nanomaterials and their morphologies after interaction with the FBS.

Figure 4. AFM 2D, 3D morphology and high profile of GO, GO + FBS, GNP, GNP + FBS, EGO and EGO + FBS. Image Credit: Cerpa-Naranjo et al., 2022

The stability of powders in suspension can be determined quantitatively using zeta potential measurements. The electrophoretic mobility values obtained after three distinct measurements for each sample are used to calculate zeta potentials.

Table 1 shows the average of these three measurements along with their standard deviations. The findings show that GO and EGO dispersions are more stable, whereas GNP dispersions have a higher tendency to aggregate.

Table 1. Zeta potential, mobility, and pH of GO; GNP; EGO suspended in FBS at 1 mg/mL and temperature 25 °C. Source: Cerpa-Naranjo et al., 2022

Figure 5 depicts the effect of various graphene-based nanomaterials on viscosity in the presence of biologic fluid and fetal bovine serum. The viscosity values for FBS alone and combined with the three nanomaterials are exceptionally low in all cases, indicating that the flow should be Newtonian. The samples were characterized using a rotational rheometer and a microfluidic chip rheometer to acquire a dependable rheological characterization.

Figure 5. Viscosity (η) vs. shear rate (γ˙) for 1 mg/mL dispersions of FBS alone and different graphene-based nanomaterials with FBS biological medium. Temperature: 37 °C. Image Credit: Cerpa-Naranjo et al., 2022

The effect of nanomaterial concentration on viscosity was investigated by maintaining a constant temperature of 37 °C and a shear rate of 200 s⁻¹. Figure 6 shows the viscosity curves collected for four different concentration dispersions. The EGO + FBS dispersion at 1 mg/mL has the lowest viscosity value of 0.6 mPa·s, while the GO + FBS dispersion at 3 mg/mL has the highest viscosity value of 1.3 mPa·s.

Figure 6. Viscosity of GO, GNP and EGO dispersed in FBS at different nanomaterial concentrations, temperature 37 °C and γ˙: 200 s⁻¹. Image Credit: Cerpa-Naranjo et al., 2022

The study was supplemented with several measurements of viscosity in other fluids, like water and BSA, to better understand the behavior of nanomaterial dispersions in biological fluids other than FBS. The rotational rheometer was used to measure the viscosity of GNP, GO, and EGO dispersed in the three fluids studied (bovine serum albumin, fetal bovine serum, and water) at 1 mg/mL concentration.

Figure 7 depicts the viscosity values for the three fluids for the analyzed samples at 37 °C. The viscosity of the nanomaterials GO and GNP is higher in the presence of FBS; however, the viscosity of EGO is nearly identical in the presence of all three fluids.

Figure 7. Viscosity values for dispersions of different nanostructures in several fluids. Concentration: 1 mg/mL; temperature 37 °C, γ˙: 200 s⁻¹. Image Credit: Cerpa-Naranjo et al., 2022

Figure 8 and Table 2 show the effect of temperature on the viscosity of GO, EGO, and GNP, dispersed in FBS at a concentration of 1 mg/mL. All measurements were made with the two rheometers (rotational and microchip fluid) to find out the differences in viscosity of the three samples analyzed. For low viscous Newtonian fluids, the microfluidic chip rheometer proved to be more accurate.

Figure 8. Viscosity (η) vs. shear rate (γ˙) at different temperatures of (a) GO + FBS, (b) GNP + FBS and (c) EGO + FBS at 1 mg/mL. Rotational rheometer (left) and microfluidic chip rheometer (right). Image Credit: Cerpa-Naranjo et al., 2022

Table 2. Viscosity values (mPa·s) of GO; EGO, GNP suspended in FBS at 1 mg/mL in rotational rheometer and Fluidicam equipments. Source: Image Credit: Cerpa-Naranjo et al., 2022

At low shear rates, the viscosity values measured in the rotational rheometer are a little higher than that observed at higher shear rates using a microfluidic chip. The viscosity values for the GNP + FBS, GO + FBS, and EGO + FBS dispersions in the temperature range of 25–40 °C are between 1.1 and 0.6 mPa·s.

When fetal bovine serum is being used as the dispersion medium, it can be seen that the type of graphene-based structure has no effect on the viscosity. Figure 9 depicts the effect of temperature on viscosity measurements in both rheometers. It has been demonstrated that as temperature rises, viscosity decreases.

Figure 9. Effect of the temperature on the viscosity of GO + FBS, GNP + FBS and EGO + FBS at 1 mg/mL comparing rotational rheometer and microfluidic rheometer (Fluidicam). Image Credit: Cerpa-Naranjo et al., 2022

Conclusion

The current research analyzed the rheological behavior of dispersions of three graphenoid nanomaterials in biological fluids. Variables like the kind of nanomaterial, type of fluid, concentration, and temperature were examined.

Journal Reference:

Cerpa-Naranjo, A., Pérez-Piñeiro, J., Navajas-Chocarro, P., Arce, M. P., Lado-Touriño, I., Barrios-Bermúdez, N., Moreno, R., Rojas-Cervantes, M. L. (2022) Rheological Properties of Different Graphene Nanomaterials in Biological Media. Materials, 15(10), p. 3593. Available Online: https://www.mdpi.com/1996-1944/15/10/3593/htm.

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