3.2.1. The Effect of Polystyrene Polymers Concentration

The ELS technique was used to provide insight into the possible changes in zeta potential values, due to treating erythrocytes and thrombocytes' cell membranes with PS-100, PS-200, PS-NH2-100, or PS-NH2-200. In order to obtain the pH-dependent *ζ* value, the systems, suspended in a 0.155 M NaCl solution, were titrated to the appropriate pH with concentrated NaOH or HCl.

Figure 3 shows representative plots of the electrokinetic potential vs. pH obtained for cell membranes of red blood cells modified with submicron polystyrene particles differing in size and/or the presence of amino groups on their surface. As it can be observed, an increase in the positive value of the zeta potential was observed alongside a decrease in the pH value. Conversely, as the pH increased, the negative values of *ζ* increased until they reached a plateau.

Upon analyzing data depicted in Figure 3, it can be noted that mixing red blood cells with three types of polystyrene particles (PS-100, PS-200, and PS-NH2-100, Figure 3a–c) did not cause statistically significant changes in *ζ* values compared to the control sample, which was pure erythrocytes suspended in 0.155 M sodium chloride. These changes were not noticeable in almost the entire range of polymer concentrations, with exceptions at extreme pH values where destruction of the membrane structure occurred. This was seen for membranes measured in the electrolyte solution containing 0.01 mg/mL PS-200 at pH~3 or 0.1 mg/mL PS-100 at pH~12. The lack of zeta potential changes allowed us to conclude that PS-100, PS-200, and PS-NH2-100 were internalized by the cell. The entry mechanisms for nanoparticles into cells are still not yet understood. None of the endocytic pathways, all of which involve vesicle formation in an actin-mediated process, are likely to account for nanoparticle translocation. The translocation of particles may also occur via non-specific pathways, including diffusion, trans-membrane channels, electrostatic, hydration, van der Waals forces, or steric interactions [40]. Moreover, PS particles within cells are not membrane-bound and hence have direct access to intracellular proteins, organelles, and DNA, which may greatly enhance their toxic potential [41].

In contrast, statistically significant changes in the *ζ* values of the erythrocyte cell membranes were observed after their treatment with PS-NH2-200 in the entire pH range studied, as shown in Figure 3d and Table 2.



**Table 2.** The zeta potential of the erythrocytes' cell membranes after exposure to PS-NH<sup>2</sup>


<sup>a</sup> Statistically significant differences vs. control group, *p* < 0.05. <sup>b</sup> Statistically significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.002 mg/mL), *p* < 0.05. <sup>c</sup> Statistically significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.01 mg/mL), *p* < 0.05. <sup>d</sup> Statistically significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.1 mg/mL), *p* < 0.05.

**Figure 3.** The zeta potential of the erythrocytes' cell membranes as a function of the pH of the electrolyte solution. Membranes untreated (●) or treated with 0.002 (♦), 0.01 (■), 0.1 (▲), and 0.5 (×) mg/mL of various types of polystyrene polymer: (**a**) PS–100, (**b**) PS–200, (**c**) PS–NH2–100, and (**d**) PS–NH2–200. **Figure 3.** The zeta potential of the erythrocytes' cell membranes as a function of the pH of the electrolyte solution. Membranes untreated (•) or treated with 0.002 (), 0.01 (), 0.1 (N), and 0.5 (×) mg/mL of various types of polystyrene polymer: (**a**) PS–100, (**b**) PS–200, (**c**) PS–NH2–100, and (**d**) PS–NH2–200.

Erythrocytes, the same as most biological surfaces, can be characterized by the negative charge occurring on their surface. This attribute is due to the presence of sialic acid located on the glycoproteins on the exposed parts of the cell [42]. As it is rather troublesome to measure the true surface charge atop cells, zeta potential is used interchangeably, having been deduced from electrophoretic mobility measurements. This parameter is a crucial characteristic of biological membranes, as it is bound to stabilize red blood cells dispersed in an electrolyte solution as it repels erythrocytes from other types of cells and, especially, themselves. This way, adhesion between RBCs and interactions with the endothelium is regulated [43]. Durocher et al. [44] proved mature cells have less sialic acid and less surface charge than those still maturing. These changes were theorized to occur during the senescence.

Due to electrostatic interactions between negatively charged erythrocytes and ions of positive charge present in the structure of polystyrene, a disruption of the natural process occurs. Furthermore, while treating red blood cells with PS-NH2-200, the zeta potential values change as a result of generating additional surface charge atop the cells. Based on the data presented in Figure 3d, it can be presumed that PS-NH2-200 is attached to the RBC's membrane. As a consequence of this phenomenon, the zeta potential values of erythrocytes treated with this type of plastic are increased in the range of pH 2–7. However, *ζ* tends to decrease in a solution with a pH above 7 as OH− ions start to react with the amine groups of the PS-NH2-200 particles.

The process of adhesion to the membranes and internalization into the cells of many particles is influenced by their physical properties, including size, shape, solubility, surface composition, and surface charge [45,46]. This leads to polymers of identical structures but distinct diameters causing different, sometimes contrary, effects on the natural surfaces, as size is a determinant of the mechanism of interaction between plastic and biological membranes [47].

Functionalized polystyrene particles become adhered to erythrocytes due to distinct mechanisms, hydrogen bonding, hydrophobic interactions, and specific van der Waals forces, to name a few [48,49]. As has already been described, PS with carboxyl groups present on their surface ranging in size from 100 nm to 1.1 µm and non- and aminefunctionalized PS 200 nm in diameter become easily attached to erythrocyte surfaces [48,49]. It was also proven that increasing the number of particles present per cell led to more particles attaching to the red blood cells' membranes. Erythrocytes' morphology did not change due to the adhesion, and the investigated process was observed to be non-specific to any area of the cell surfaces [48,49]. However, polystyrene submicron particles of no charge, as well as of positive and negative charges, were found inside the red blood cells (while they were smaller than 0.1 µm in diameter) in addition to being present on the cell surface (while ranging from 0.2 to 1 µm in size) [40]. What is more, PS 78 nm and 200 nm in diameter were found within erythrocytes without inhabiting RBC's membranes [41].

Platelets, similarly to erythrocytes, possess membrane glycoproteins that play an important role during two crucial processes: Adhesion to the subendothelial matrix and platelet–platelet cohesion, or aggregation [50]. These glycoproteins in their structure contain carboxyl groups that provide a negative charge to the surface of thrombocytes. The results for experimental studies of platelets' cell membranes, treated and untreated with polystyrene polymers, are shown in Figure 4. Slight *ζ* changes were observed for membranes in contact with PS-100, PS-200, and PS-NH2-100 (Figure 4a–c), and the most visible were noted at extreme pH values, e.g., at pH~12 when 0.1 or 0.5 mg/mL PS-200 was used (Figure 4b). It was theorized that the changes result from the destruction of the cell membrane structure in an excessively alkaline environment.

*Membranes* **2022**, *12*, x 10 of 17

cell membrane structure in an excessively alkaline environment.

polystyrene (0.002 and 0.01 mg/mL) were insignificant.

membranes [41].

to be non-specific to any area of the cell surfaces [48,49]. However, polystyrene submicron particles of no charge, as well as of positive and negative charges, were found inside the red blood cells (while they were smaller than 0.1 μm in diameter) in addition to being present on the cell surface (while ranging from 0.2 to 1 μm in size) [40]. What is more, PS 78 nm and 200 nm in diameter were found within erythrocytes without inhabiting RBC's

Platelets, similarly to erythrocytes, possess membrane glycoproteins that play an important role during two crucial processes: Adhesion to the subendothelial matrix and platelet–platelet cohesion, or aggregation [50]. These glycoproteins in their structure contain carboxyl groups that provide a negative charge to the surface of thrombocytes. The results for experimental studies of platelets' cell membranes, treated and untreated with polystyrene polymers, are shown in Figure 4. Slight *ζ* changes were observed for membranes in contact with PS-100, PS-200, and PS-NH2-100 (Figure 4a–c), and the most visible were noted at extreme pH values, e.g., at pH~12 when 0.1 or 0.5 mg/mL PS-200 was used (Figure 4b). It was theorized that the changes result from the destruction of the

Statistically significant changes, as in the case of erythrocytes, were observed for the thrombocytes' cell membranes treated with PS-NH2-200 (Figure 4d), and the most visible in the entire pH range were noted for polymer concentrations of 0.1 and 0.5 mg/mL. At an acidic pH (pH~3 and pH~4), the *ζ* reached higher positive values, while in the remaining pH range (pH 5 to 12), the zeta potential increased towards negative values. The changes that occurred in the parameter with the participation of lower concentrations of

**Figure 4.** The zeta potential of the platelets' cell membranes as a function of the pH of the electrolyte solution. Membranes untreated (●) or treated with 0.002 (♦), 0.01 (■), 0.1 (▲), and 0.5 (×) mg/mL of various types of polystyrene polymer: (**a**) PS–100, (**b**) PS–200, (**c**) PS–NH2–100, and (**d**) PS–NH2–200. **Figure 4.** The zeta potential of the platelets' cell membranes as a function of the pH of the electrolyte solution. Membranes untreated (•) or treated with 0.002 (), 0.01 (), 0.1 (N), and 0.5 (×) mg/mL of various types of polystyrene polymer: (**a**) PS–100, (**b**) PS–200, (**c**) PS–NH2–100, and (**d**) PS–NH2–200.

The data of the zeta potential of thrombocytes during PS-NH2-200 treatment as a function of pH is compiled in Table 3. **Table 3.** The zeta potential of the platelets' cell membranes after exposure to PS-NH2-200 (C = 0.002, 0.01, 0.1, and 0.5 mg/mL). Statistically significant changes, as in the case of erythrocytes, were observed for the thrombocytes' cell membranes treated with PS-NH2-200 (Figure 4d), and the most visible in the entire pH range were noted for polymer concentrations of 0.1 and 0.5 mg/mL. At an acidic pH (pH~3 and pH~4), the *ζ* reached higher positive values, while in the remaining

15.28 ± 0.89 a,b,c

Statistically

−10.78 ± 0.76 a,b,c −21.14 ± 0.95 a,b,c,d

−13.10 ± 1.21 a,b,c −22.48 ± 1.61 a,b,c,d

−15.10 ± 0.72 a,b,c −22.40 ± 0.90 a,b,c,d

−17.30 ± 1.65 a,b,c −22.98 ± 0.78 a,b,c,d

Statistically significant differences

vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.002 mg/mL), *p* < 0.05. <sup>c</sup>

6 −8.48 ± 1.72 −7.03 ± 0.45 a −8.53 ± 0.91 <sup>b</sup>

7 −8.35 ± 1.24 −7.12 ± 0.60 −9.31 ± 0.30 <sup>b</sup>

8 −9.42 ± 0.74 −8.15 ± 0.75 −10.24 ± 0.94 <sup>b</sup>

10 −10.48 ± 1.49 −9.41 ± 0.52 −11.88 ± 0.66 <sup>b</sup>

Statistically significant differences vs. control group, *p* < 0.05. <sup>b</sup>

a

= 0.1 mg/mL), *p* < 0.05.

4 2.49 ± 0.77 −0.02 ± 1.65 6.85 ± 0.67 a,b 9.25 ± 1.62 a,b 7.17 ± 0.93 a,b 5 −4.19 ± 0.85 −2.37 ± 0.52 0.00 ± 0.83 a,b −8.24 ± 1.00 a,b,c −22.18 ± 1.82 a,b,c,d

9 −11.20 ± 0.97 −8.43 ± 1.16 a −10.43 ± 0.64 −14.68 ± 1.16 a,b,c −21.98 ± 1.54 a,b,c,d

11 −10.76 ± 0.38 −12.84 ± 0.80 a −13.76 ± 1.41 a −17.94 ± 0.61 a,b,c −23.30 ± 1.03 a,b,c,d 12 −13.44 ± 1.08 −14.74 ± 0.82 −17.26 ± 0.70 a,b −19.34 ± 0.15 a,b,c −24.92 ± 1.16 a,b,c,d

significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.01 mg/mL), *p* < 0.05. d Statistically significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C

pH range (pH 5 to 12), the zeta potential increased towards negative values. The changes that occurred in the parameter with the participation of lower concentrations of polystyrene (0.002 and 0.01 mg/mL) were insignificant.

The data of the zeta potential of thrombocytes during PS-NH2-200 treatment as a function of pH is compiled in Table 3.

**Table 3.** The zeta potential of the platelets' cell membranes after exposure to PS-NH<sup>2</sup> -200 (C = 0.002, 0.01, 0.1, and 0.5 mg/mL).


<sup>a</sup> Statistically significant differences vs. control group, *p* < 0.05. <sup>b</sup> Statistically significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.002 mg/mL), *p* < 0.05. <sup>c</sup> Statistically significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.01 mg/mL), *p* < 0.05. <sup>d</sup> Statistically significant differences vs. modified erythrocyte membranes with PS-NH2-200 (C = 0.1 mg/mL), *p* < 0.05.

There is much evidence in the literature that suggests the possibility of interactions between submicron polymer particles and elements/components of the circulatory system—blood and blood vessels. The existence of such interactions would explain the proatherothrombic effect observed in a number of models. Smyth et al. [51] conducted research investigating the ability of non-functionalized and functionalized polystyrene particles of different sizes (50 and 100 nm) to cause thrombocytes' aggregation in vitro and in vivo. Their results confirmed that the process of aggregation was influenced by the physical properties of PS. Tested particles caused GPIIb/IIIa-mediated aggregation of thrombocytes to a certain degree, varying due to the size and presence of surface groups. PS-NH<sup>2</sup> of 50 nm in diameter acted in an enhanced agonist-induced aggregation by linking adjacent platelets, many of which were not even activated. Nemmar et al. [52] incubated thrombocytes with 60 nm polystyrene particles with different surface chemistries and found that the potency of causing aggregation was highest when using aminated particles, followed by carboxylated ones. Unmodified polystyrene was observed to induce aggregation to the lowest degree. Although these findings were to be generally confirmed by McGuinnes et al. [53], a difference in causing aggregation between PS particles of amino and carboxyl groups present on their surfaces was not to be found.

Given the data acquired using the ELS technique presented in Figures 3 and 4 and Tables 2 and 3, it can be recognized that significant changes of zeta potential were observed when treating erythrocytes and thrombocytes' membranes using PS-NH2-200. The exposition of cells' surfaces to 200 nm amine-functionalized polystyrene caused the electrokinetic potential to shift towards more negative values. As it was reported in the scientific literature, the larger the PS-NH<sup>2</sup> was, the higher *ζ* tended to increase [54], which was confirmed in Section 3.2.1. We hypothesized that the interaction between erythrocyte and thrombocyte membranes and the PS-NH2-200 underlie the increased membrane perturbation. McGuinnes et al. [53] demonstrated that amino-functionalized PS appear to act via an unexplained mechanism that results in the display of anionic phospholipids on the outer surface of platelets and erythrocyte membranes and suggested that unusual protein adsorption patterns might lead to membrane perturbation-mediated aggregation.
