3.5.1. In Vitro Antibacterial Activity

The in vitro antibacterial activity of the prepared nanoparticles was compared with that of the starting chitosan, iron(III) chloride hexahydrate and deferoxamine. For the in vitro evaluation of the antibacterial effect, we used the conventional agar well diffusion method. This method allows one to directly estimate the diameter of the microbial colonies' growth inhibition zone. The compound that provokes the largest zone of inhibition of bacterial growth is considered the most active antibacterial agent. The results of the experiments are presented in Table 2.


**Table 2.** Antibacterial effects of the elaborated nanoparticles.

\* Mean value ± SD, n = 3.

Both the starting chitosan and iron(III) hexahydrate are characterized by moderate antibacterial activity, while their composite POX-1 is ca. 1.5 times more effective toward the tested bacteria. This can be explained by the formation of highly positively charged nanoparticles of POX-1. Our previous studies showed that, in many instances, chitosanbased nanoparticles exhibit much stronger antibacterial effects than the starting chitosan in its native form of a molecular coil [39].

Deferoxamine practically does not have an antibacterial effect. In contrast, the blending of deferoxamine with POX-1 results in POX-2 with outstanding antibacterial activity, which is comparable to that of the reference antibiotics ampicillin and gentamicin. We speculate that the high antibacterial activity of POX-2 is associated with the symbatic action of POX-1/deferoxamine in their complex with POX-2, but a fuller understanding of their mechanism requires additional biological studies.

The antibacterial effect of POX-3 essentially does not differ from that of POX-2.

The most effective antibacterial nanoparticles proved to be POX-2 and POX-3. The minimum inhibitory concentration (MIC) values were 0.14 μg/mL (*S. aureus*) and 0.19 μg/mL (*E. coli*) (compare with MIC values of ampicillin 0.18 μg/mL (*S. aureus*), gentamicin 0.23 μg/mL (*E. coli*)). Thus, the most active elaborated antibacterial nanoparticles, POX-2 and POX-3, are not inferior in their in vitro effect to the conventional antibiotics ampicillin and gentamicin.

#### 3.5.2. Effect of the Integrity of the Bacterial Membrane

The main recognized model of the mechanism of the antibacterial effect of chitosan is associated with its polycationic nature [40]. Due to the protonation of primary amino functionalities, the neutral chitosan (pKa = 6.5) is converted into its polycation. The polycation interacts with the anionic moieties of the microbial cell, and this results in ionic pumps' dysfunction, osmotic imbalance, and overall membrane dysfunction, followed by cell membrane rupture and bacterial death [41].

To study the effects of the elaborated systems on the integrity of the microbial membrane, we used spectrophotometry of a suspension of bacterial cells in a 0.5% aqueous solution of sodium chloride in the UV region [42]. This approach is based on the fact that intracellular components are characterized by strong absorption at 260 nm [43]. In preliminary experiments, we found that both deferoxamine and iron(III) chloride did not damage the membranes of the tested bacteria. Thus, we compared the effect of starting chitosan and POX-1, POX-2 and POX-3 on the integrity of the cell membranes of *S. aureus* and *E. coli*. The results of the study are summarized in Figures 6 and 7.

In general, POX-1, POX-2 and POX-3 disrupt the integrity of the bacterial cell to a greater extent than the initial chitosan, and the effect of POX-2 and POX-3 is more pronounced than that of the starting chitosan. In addition, both in the case of *Staphylococcus aureus* and in the case of *E. coli*, POX-2 and POX-3 require less time to reach a plateau, i.e., their effect develops faster than that of chitosan of POX-1. Thus, the main mode of the

antibacterial effect of POX-2 and POX-3 is the destruction of the integrity of the bacterial cell membrane.

**Figure 6.** The effects of chitosan (CH), POX-1 (A), POX-2 (B), POX-3 (C) on the integrity of the cell membranes of *E. coli*.

**Figure 7.** The effects of chitosan (CH), POX-1 (A), POX-2 (B), POX-3 (C) on the integrity of the cell membranes of *S. aureus*.

#### 3.5.3. In Vitro Toxicity

The in vitro toxicity of the leading POX-2 and POX-3 was evaluated using the classic MTT test and compared with that of the starting chitosan, POX-1, deferoxamine and iron(III) chloride. As a quantitative measure of toxicity, we used the cell viability (CV, %) of the HEK-293 line after the cells were treated with a solution of the test substance (300 μg/mL). The highest toxicity was found for iron(III) chloride (CV = 72%). The incorporation of iron(III) chloride into the chitosan polymeric matrix dramatically reduces its toxicity (CV = 94%). As a result, the formed nanoparticles POX-1 are characterized by the same toxicity as the starting chitosan (CV = 96%), which is considered a non-toxic polymer. The introduction of deferoxamine into POX-1 to give POX-2 or POX-3 did not lead to any noticeable changes in toxicity (for POX-1, CV = 93%; for POX-2, CV = 96%). However, it should be noted that the described nanoparticles are characterized by high positive values of the zeta potential; therefore, their intravenous administration into the general systemic circulation is undesirable, since it can cause the aggregation of negatively charged platelets. In this regard, in further experiments in vivo, we decided to use the intracavitary method of administration for these nanoparticles.
