3.5.4. In Vivo Antibacterial Activity

At the next stage of the current work, we evaluated the in vivo antibacterial activity and toxicity of the leading systems, i.e., POX-2 and POX-3, in rats and compared their effects with those of antibiotics ampicillin and gentamicin.

The rats were subjected to the so-called model peritonitis. To imitate peritonitis, we infected the rats with a microbial mixture containing hospital strains of *S. aureus* and *E. coli*. Six hours after the introduction of microorganisms, all rats showed the conventional symptoms of peritonitis: lethargy, food refusal, rapid breathing and abdominal distention. In the control groups, exudate (200 μL) was collected with a sterile syringe 24 h after infection. A day later, all other rats were injected with a solution of the tested POX-2 or POX-3, or ampicillin or gentamicin. Then, 200 μL of exudate was taken after 7 h. To 200 μL of exudate, 1000 μL of 0.9% NaCl aqueous solution was added. Next, 100 μL of the resulting solution was evenly applied to a Petri dish with meat peptone agar. Colonies were counted 24 h after incubation at 37 ◦C. Subsequently, colony-forming units (CFU) were recalculated per 1 mL of exudate (Table 3).


**Table 3.** In vivo antibacterial effect.

All tested samples demonstrated high in vivo antibacterial effects. However, POX-2 and POX-3 showed extremely high antimicrobial effects: no growth of colonies was found after collection of the exudate. The lower efficacy of the antibiotics can be explained by their rapid elimination after intracavitary administration. The elimination of polymerbased nanoparticles is much slower, and this leads to an increase in antibacterial activity compared low-molecular compounds, i.e., antibiotics, ampicillin and gentamicin. It should be noted that the results of this in vivo study in rats may not be directly applicable to human patients, and further studies, including clinical trials, are necessary to confirm the efficacy and safety of these nanoparticles in humans.

#### **4. Conclusions**

The results of this work can be considered from the following main perspectives.

Firstly, we elaborated the following types of chitosan-based nanoparticles: chitosan/ Fe(III) (POX-1) and chitosan/Fe(III)/deferoxamine (POX-2 and POX-3, which differs from POX-2 in that it has been stirred in water for 24 h rather than 1 h). The resulting nanoparticles are characterized by close values of the hydrodynamic diameter and zeta potential. Characterization of the obtained POX-2 and POX-3 by physicochemical methods of analysis

clearly demonstrated that POX-2 and POX-3 are practically identical nanoparticles. The equal biological effects of POX-2 and POX-3 support this conclusion. Therefore, POX-2 is stable in water for at least 24 h, retaining all the characteristic features of its structure and biological effects.

Secondly, we evaluated the in vitro and in vivo antibacterial activity of the prepared POX-1, POX-2 and POX-3. The most effective antibacterial species are POX-2 and POX-3 and their antimicrobial efficiency is equal, which is not surprising since POX-2 and POX-3 are the same system. POX-2 and POX-3 are characterized by outstanding in vivo antibacterial effects, and their activity exceeds even that of commercial antibiotics ampicillin and gentamicin. Moreover, POX-2 and POX-3 are non-toxic.

Thirdly, we concluded that the introduction of iron ions into the chitosan matrix increases the ability of the resulting nanoparticles to disrupt the integrity of the membranes of microorganisms in comparison with pure chitosan. The introduction of deferoxamine into the obtained nanoparticles sharply expands their effect of destruction of the bacterial membrane.

Finally, we provide a fast, simple and convenient route to obtain highly effective, non-toxic antibacterial systems. This route is based on non-covalent chemistry and does not require laborious and sophisticated organic synthesis methods. The obtained antibacterial nanoparticles are promising for further preclinical studies, and this project is underway in our group.

**Author Contributions:** Conceptualization, O.M.K. and A.S.K.; methodology, O.M.K. and A.R.E.; software, A.E.B.; validation, A.A.K., A.G.T. and A.S.K.; formal analysis, N.N.L.; investigation, O.M.K. and V.E.E.; resources, R.A.G.; data curation, V.E.E.; writing—original draft preparation, A.R.E. and O.M.K.; writing—review and editing, A.G.T.; visualization, M.V.T.; supervision, A.S.K.; project administration, A.S.K.; funding acquisition, A.R.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Russian Science Foundation, grant № 23-23-00021.

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee and followed the recommendations of European Directive 2010/63/EU of 22 September 2010.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
