**4. Discussion**

The assessment of the interaction of the biomaterial structure and modulation of the host immune response represents one of most important strategies for effective in vivo integration of biomaterial. Moreover, immunomodulation strategies based on biomaterials can significantly improve the outcomes of medical implants and tissue engineering therapies. In general, the immunomodulation of immune responses based on the physicochemical modification of a biomaterial is important for effective biomaterial implementation [32]. The current medical research focuses on immunotherapy [32], development of safe vaccines [41] and combinations of therapies [42,43] to achieve the most effective treatment.

Our previous study revealed the immunomodulative properties of PIPOx based on follow-up of the immunocompetent cell proliferation following specific sensitization of splenic cell fractions [18]. The stimulation of splenocytes with PIPOx induced a significant increase of the proliferation rate. Co-cultivation of non-adherent spleen cells with PIPOxstimulated adherent spleen cells after the first adherence period did not cause an increase in cell proliferation compared with unstimulated control cells, but after coculturing of non-adherent fraction of splenocytes with PIPOx-stimulated adherent spleen cells from a second adherence period, a significant increase of the proliferation rate was observed [18]. These results indicated that PIPOx exerted different immunomodulative effectivity with an emphasis on the phenotype of stimulation-affected immunocompetent cells. Now, regarding these results of previous observations, we perform analysis of the splenic cellsecreted cytokine profile following stimulation, to ascertain character of specific induced immune response and polarization.

In this study, the myeloid phagocytes represented by adherent CD11c+ and CD14+ spleen cells significantly induced production of IFN-γ and increased production of IL-17 after PIPOx stimulation. Thus, polarization of immune response towards Th1 and Th17 over Th2 and Treg immune responses is indicated. The adherent spleen cells more enriched in CD11c<sup>+</sup> APCs produced significantly higher amount of IL-10 indicating Treg polarization of immune response. Evidently, in PIPOx-exposed adherent CD11c+ and CD14<sup>+</sup> spleen cells, pro-inflammatory Th1 and Th17 response is more profound, while in the adherent spleen cells with higher expression, CD11c<sup>+</sup> anti-inflammatory Treg polarized response is applied.

Generally, the Th1 response correlates with protective immunity, contrary to the Th2 responses, whose signature cytokine is IL-4, down-regulating Th1 immunity. Th17 cells were previously believed to be a subset of Th1 cells. They presumably have a regulatory role supporting the Th1-response and down-regulating the Th2-response. Th17 cells producing IL-17 have a crucial role in inflammatory processes. On the contrary, Treg subsets producing signature cytokine IL-10 exerted anti-inflammatory capacity to limit the pro-inflammatory T-cell responses [44].

DCs as APCs respond to molecular patterns by inducing differentiation of naive T-cells into effector T-helper subpopulations and activation of adaptive immunity and initiated both pro-inflammatory and anti-inflammatory immune responses. Moreover, DCs, by taking up, processing, and presenting foreign antigens to Th cells, have a critical role in connecting innate and adaptive immunities. Inflammatory DCs initiate Th17 and Th2 cell responses, while tolerogenic DCs activate Th1 and Tregs. Evidently, the engagement of CD11c+ CD14+-enriched spleen cells, CD11c+-enriched spleen cells, and CD3+, CD4+, CD8<sup>+</sup> cells as a result of PIPOx cell-processing, cell presentation, and immune response polarization (Figures 4 and 5) has been observed.

The results revealed that the phagocytic capacity of RAW 264.7 macrophages remained unchanged upon treatment with PIPOx, without down-regulation of cell engulfment and internalization. Previously published immunobiological effectivity of poly(2 ethyl-2-oxazoline) (PETOx) and selected poly [2-(4-aminophenyl)-2-oxazoline-co-2-ethyl-2-oxazoline] (AEOx) copolymer in lymphoid mouse macrophage P388.D1 (Clone 3124) revealed a similar trend of phagocytosis, without any destructive interference with cell viability and phagocyting capability [1].

The uptake of polymers by cells depends on molecular structure, molar mass and the type of cell [45]. High molar mass of polymers can be a limiting factor in cell internalization and therefore some polymer domains that help polymer cell internalization were

already identified [46]. In our previous work devoted to cytotoxicity study of PIPOx, we have shown that PIPOx is internalized to fibroblast cells by endocytic pathway [18]. In macrophages, which are professional phagocytes, the phagocytic/endocytic activity is expected. Here, we show that internalization of PIPOx into RAW 264.7 macrophages is concentration-dependent and fast, as after 1h only slow increase of cell-internalized PIPOx is shown during 24 h incubation.

The fate of cell-internalized PIPOx was studied in colocalization studies with organelle markers. As shown by CLSM, PIPOx-FITC is not localized in mitochondria and is localized in vesicular structures that only partially co-localize with lysosomes (Figure 8). This is the difference between PIPOx colocalization in macrophages compared to mouse 3T3 fibroblasts where after 24 h PIPOx was mostly localized in lysosomes [18]. These other vesicular structures in macrophages could resemble other organelles of phagocytic or endocytic pathways, such as phagosomes or endosomes, which supports the hypothesis that PIPOx can be taken up and processed by APCs.

Biomaterials can thus act as synthetic adjuvants or DCs activators. Following internalization of the biomaterial vehicles by DCs, the antigens could be released intracellularly and activate MHC class I and II pathways and induce CD4<sup>+</sup> and CD8+ T-cell immunity [41]. It was shown that polymers can fulfill not only an indirect role as drug carriers [43], agents solubilizing hydrophobic drugs, materials capable of reducing drug cytotoxicity, etc., but they can also function as therapeutic molecules.

It was already mentioned previously that poly(2-oxazoline)s are biocompatible polymers with increasing interest as biomaterials for drug, gene, protein, and radionuclide delivery [2]. For example, novel delivery platforms based on poly(2-oxazolines) with different molar masses have been reported as promising conjugates with interleukins and growth factors [47,48].

They are, however, relatively new in comparison to other classes of water-soluble polymers already established for such use. Besides intensive study of poly(2-oxazoline)s biological properties, only a limited number of them comprises immunomodulatory activity (mainly our previous studies) [1,49,50]. Although PIPOx is formally a member of the poly(2 oxazoline) family, it is a structurally different polymer with free 2-oxazoline rings in the side chain, compared to previously studied polymers of this class.

It was of high importance to subject PIPOx, as a promising biomedical formulation, for evaluation of immunomodulative effectivity in primary immunocytes and its capability to effectively polarize immune responses, thus to reach the best possible strategy to construct new biocompatible delivery systems. Further research in this area is therefore highly appreciated.

### **5. Conclusions**

We have shown that PIPOx stimulation of different immunocompetent cells accelerates cell-specific immune responses. PIPOx-sensitized adherent CD11c+ and CD14+ spleen cells induced statistically significantly enhanced production of IFN-γ and IL-17, indicating polarization of immune cell response towards Th1/Th17 over Th2 and Treg immune responses. Adherent spleen cell-derived CD11c+-enriched APCs produced statistically significantly higher secretion of IL-10, the signature cytokine of Treg phenotype. The time and concentration-dependent PIPOx-FITC RAW 264.7 cell-processing revealed sequential intracellular accumulation of PIPOx. The complex phagocytosis of RAW 264.7 macrophages following PIPOx exposure did not exert significant down-regulation of engulfment and internalization throughout effective cell phagocytosis. Using the colocalization of fluorescently labeled PIPOx and organelle tracking dyes, it was shown that PIPOx after internalization to cell occurs in lysosomes and other vesicular structures of endocytic pathway. The results of this study suggest PIPOx as a biocompatible polymer enhancing protective Th1/Th17 immunity over the Treg immune responses.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1996-194 4/14/6/1371/s1, Figure S1: 1H NMR spectrum of PIPOx measured in CDCl3. Figure S2: ATR-FTIR spectrum of PIPOx, PIPOx-ABA, and PIPOx-FITC. Figure S3: 1H NMR spectrum of PIPOx-ABA measured in CDCl3. Figure S4: 1H NMR spectrum of PIPOx-FITC measured in DMSO-d6. Figure S5. UV/Vis measurements of fluorescein isothiocyanate and PIPOx-FITC. (a) UV/Vis spectra of fluorescein isothiocyanate measured in methanol in the concentration range from <sup>5</sup> <sup>×</sup> <sup>10</sup>−<sup>6</sup> mol dm−<sup>3</sup> to 10−<sup>4</sup> mol dm−3. (b) Calibration curves of fluorescein isothiocyanate at 278 and 452 nm. (c) UV/Vis spectra of PIPOx-FITC measured in methanol in the concentrations of 0.5 and 1 mg/mL. Concentration of fluorescein unit in PIPOx-FITC calculated from calibration curve was equal to 1 mol.%.

**Author Contributions:** Conceptualization: E.P., J.K. and Z.K.; Funding acquisition: J.K., Z.K. and E.P.; Synthesis and material preparation: M.M. and J.K.; Bioimmunological experiments: E.P. and L.P., Colocalization experiments: Z.K.; Writing—original draft preparation: E.P., L.P., J.K. and Z.K.; Writing—review and editing: E.P., Z.K. and J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Slovak Grant Agency VEGA in the project 2/0124/18 and Slovak Research and Development Agency in the project APVV 19-0487. This study was performed during the implementation of the project Building-up Centre for advanced materials application of the Slovak Academy of Sciences, ITMS project code 313021T081 supported by Research & Innovation Operational Program funded by the ERDF.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Ethics Committee of Research Base of Slovak Medical University, Institute of Preventive and Clinical Medicine and State veterinary and Food Administration of the Slovak Republic (protocol code 2939/09-221, 30.12.2009).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in Supplementary Materials.

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

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