**3. Results and Discussion**

An investigation into the characteristics of the polymers used in the research was carried out to analyze the occurrence of possible flaws in the commercial product that could affect the conducted analysis. The hydrodynamic size and stability of the submicron polystyrene particles were measured using the DLS method. FTIR spectroscopy was also utilized during the research for identity analysis.

To investigate the interactions between the cell membranes of morphotic blood components (erythrocytes and thrombocytes) and various polystyrene particles (PS-100, PS-200, PS-NH2-100, PS-NH2-200), several experimental studies were carried out using the ELS method. These interactions could potentially change the values of the electrokinetic potential of the systems studied. Both the influence of the polymer concentration and the exposure time of the cell membrane to the polymer were taken into the account during the experiment. Each of the measurements was performed as a function of the H<sup>+</sup> ion concentration.

#### *3.1. Characteristics of Polymers Used in the Study 3.1. Characteristics of Polymers Used in the Study*

ion concentration.

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also utilized during the research for identity analysis.

Structural studies of PS particles differing in size and/or the presence of amine groups on their surface were carried out using FTIR spectroscopy. Firstly, each of the analyzed polymers was suspended in water, and then the obtained sample was frozen and freezedried. The FTIR analysis of PS-100, PS-200, PS-NH2-100, and PS-NH2-200 is shown in Figure 1. Structural studies of PS particles differing in size and/or the presence of amine groups on their surface were carried out using FTIR spectroscopy. Firstly, each of the analyzed polymers was suspended in water, and then the obtained sample was frozen and freeze-dried. The FTIR analysis of PS-100, PS-200, PS-NH2-100, and PS-NH2-200 is shown in Figure 1.

cron polystyrene particles were measured using the DLS method. FTIR spectroscopy was

To investigate the interactions between the cell membranes of morphotic blood components (erythrocytes and thrombocytes) and various polystyrene particles (PS-100, PS-200, PS-NH2-100, PS-NH2-200), several experimental studies were carried out using the ELS method. These interactions could potentially change the values of the electrokinetic potential of the systems studied. Both the influence of the polymer concentration and the exposure time of the cell membrane to the polymer were taken into the account during the experiment. Each of the measurements was performed as a function of the H<sup>+</sup>

**Figure 1.** FTIR spectra of (**a**) PS–100, (**b**) PS–200, (**c**) PS–NH2–100, and (**d**) PS–NH2–200. **Figure 1.** FTIR spectra of (**a**) PS–100, (**b**) PS–200, (**c**) PS–NH2–100, and (**d**) PS–NH2–200.

As expected, the recorded spectra of all samples showed bands confirming the structure of polystyrene [36–38]. The bands at approximately 3025–3033 cm−1 were attributed to the C–H stretching vibration of the benzene ring, while the signals at approximately 2917–2935 and 2836–2853 were assigned to the aliphatic –CH2– stretching vibration. Further proof of the presence of the benzene ring in the structure of the analyzed particles is the bands at 1605, 1498, and 1451 cm−1 (e.g., values for PS-100, Figure 1a), corresponding to the stretching vibrations of the aromatic C=C bonds. The bands at approximately 756 and 696 cm−1 (e.g., values for PS-100, Figure 1a) were characteristic of the aromatic substitution pattern [36,37] and were assigned to the C–H out-of-plane bending vibration. A broad peak at approximately 3442–3454 cm−1, corresponding to the stretching vibrations of O–H, indicated the existence of hydroxyl groups likely coming from the water. All obtained spectra (Figure 1a–d) looked much the same, differing slightly in the position of the bands that are characteristic of polystyrene. Additionally, the size of the analyzed particles did not result in any significant structural differences (PS-100, PS-200, Figure 1a,b). However, in the case of the amino-functionalized PS parti-As expected, the recorded spectra of all samples showed bands confirming the structure of polystyrene [36–38]. The bands at approximately 3025–3033 cm−<sup>1</sup> were attributed to the C–H stretching vibration of the benzene ring, while the signals at approximately 2917–2935 and 2836–2853 were assigned to the aliphatic –CH2– stretching vibration. Further proof of the presence of the benzene ring in the structure of the analyzed particles is the bands at 1605, 1498, and 1451 cm−<sup>1</sup> (e.g., values for PS-100, Figure 1a), corresponding to the stretching vibrations of the aromatic C=C bonds. The bands at approximately 756 and 696 cm−<sup>1</sup> (e.g., values for PS-100, Figure 1a) were characteristic of the aromatic substitution pattern [36,37] and were assigned to the C–H out-of-plane bending vibration. A broad peak at approximately 3442–3454 cm−<sup>1</sup> , corresponding to the stretching vibrations of O–H, indicated the existence of hydroxyl groups likely coming from the water. All obtained spectra (Figure 1a–d) looked much the same, differing slightly in the position of the bands that are characteristic of polystyrene. Additionally, the size of the analyzed particles did not result in any significant structural differences (PS-100, PS-200, Figure 1a,b). However, in the case of the amino-functionalized PS particles (PS-NH2-100, PS-NH2-200, Figure 1c,d) additional bands were observed, thus confirming their surface modification. The signal observed in the PS-NH2-200 spectrum (Figure 1d) at 1672 cm−<sup>1</sup> and the bands appearing at 1187 and 1032 cm−<sup>1</sup> were assigned to the N-H bending vibrations characteristic of primary amines and the stretching vibrations of C-N bonds, respectively. The analogous peaks in the FTIR spectrum recorded for PS-NH2-100 (Figure 1c) exhibited a low intensity and are not clearly visible. The reason for this is likely the smaller size of the tested submicron particles and the proportionally smaller number of –NH<sup>2</sup> groups on its surface.

Parameters (size and zeta potential) characterizing the polymers were measured in 0.155 M sodium chloride at pH = 7.4 with the Zetasizer Nano ZS apparatus. The results obtained using the DLS technique are shown in Figure 2.

**Figure 2.** Polystyrene size distribution by (**a**) number, (**b**) intensity. **Figure 2.** Polystyrene size distribution by (**a**) number, (**b**) intensity.

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smaller number of –NH2 groups on its surface.

obtained using the DLS technique are shown in Figure 2.

cles (PS-NH2-100, PS-NH2-200, Figure 1c,d) additional bands were observed, thus confirming their surface modification. The signal observed in the PS-NH2-200 spectrum (Figure 1d) at 1672 cm−1 and the bands appearing at 1187 and 1032 cm−1 were assigned to the N-H bending vibrations characteristic of primary amines and the stretching vibrations of C-N bonds, respectively. The analogous peaks in the FTIR spectrum recorded for PS-NH2-100 (Figure 1c) exhibited a low intensity and are not clearly visible. The reason for this is likely the smaller size of the tested submicron particles and the proportionally

Parameters (size and zeta potential) characterizing the polymers were measured in 0.155 M sodium chloride at pH = 7.4 with the Zetasizer Nano ZS apparatus. The results

The plots of the polymer size distribution by number and intensity indicated the formation of monomodal fractions. The data obtained from dependences of the particles' diameter by number range from 110 to 207 nm, and regarding intensity, range from 160 nm to 249 nm. The values registered for PS-100 and PS-NH2-100 differ slightly from the ones confirmed by the companies, while for PS-200 and PS-NH2-200, the measurements are consistent with the information provided by the producer. The plots of the polymer size distribution by number and intensity indicated the formation of monomodal fractions. The data obtained from dependences of the particles' diameter by number range from 110 to 207 nm, and regarding intensity, range from 160 nm to 249 nm. The values registered for PS-100 and PS-NH2-100 differ slightly from the ones confirmed by the companies, while for PS-200 and PS-NH2-200, the measurements are consistent with the information provided by the producer.

The presence of other fractions, above 3000 nm in diameter, should not be ruled out. All size data obtained, including the standard deviation (SD) and polydispersity index (PDI) values, are collated in Table 1. The presence of other fractions, above 3000 nm in diameter, should not be ruled out. All size data obtained, including the standard deviation (SD) and polydispersity index (PDI) values, are collated in Table 1.


**Table 1.** The parameters characterizing the polymers (0.155 M NaCl, pH = 7.4). **Table 1.** The parameters characterizing the polymers (0.155 M NaCl, pH = 7.4).

Summaries for the values of zeta potential are also compiled in Table 1, measured for each polymer using the ELS technique. The lowest zeta potential values were recorded for plain polystyrene particles with a size of 200 nm (−46.10 ± 2.11 mV). The remaining polymers (PS-100, PS-NH2-100, and PS-NH2-200) show *ζ* values ranging from −24.90 ± 1.20 mV to −31.00 ± 0.99 mV.

Generally, a large positive or negative value of the zeta potential (lower than −30 mV and higher than +30 mV) indicates physical stability due to the electrostatic repulsion of individual particles. On the other hand, a small value of the electrokinetic potential may cause the aggregation or flocculation of particles due to the van der Waals forces [39]. Based on data collected in Table 1, it can be concluded that all polymers are characterized by good stability.
