**1. Introduction**

Poly(2-alkyl-2-oxazolines) represent biocompatible and non-cytotoxic polymeric materials [1] with a high potential in different biomedical applications such as controlled drug and gene release, tissue engineering, hydrogel technologies, etc. [2–4]. Usually, they are achieved in the cationic ring-opening polymerization (CROP) of 2-alkyl-2-oxazolines leading to polymers with defined structure, predicted molar mass, and narrow dispersity [5,6]. Using different 2-oxazoline monomers, the wide library of hydrophilic, thermosensitive [7,8], hydrophobic, amphiphilic [9], or functional [10] polymers has been prepared. Apart from 2-alkyl-2-oxazolines, 2-alkenyl-2-oxazolines can provide, depending on polymerization conditions, several polymerization reactions leading to polymers with various structures and architectures [11–14].

From this group of monomers, 2-isopropenyl-2-oxazoline represents a monomer with dual orthogonal functionality able to polymerize by CROP of 2-oxazoline unit providing polymers with free double bonds [15,16]. On the other hand, an isopropenyl unit enables the preparation of poly(2-isopropenyl-2-oxazolines) (PIPOx) containing a free 2-oxazoline group in the side chain. Free-radical polymerization belongs to the most common polymerizations of 2-isopropenyl-2-oxazolines leading to polymers with broader dispersity (Ð) and lower control over molar mass and architecture [17,18]. Similarly, polymers with broader dispersity were achieved in the frustrated Lewis-pair polymerization of 2-isopropenyl-2-oxazoline [19]. Better-defined polymers have been achieved using various methods of living or controlled polymerizations. Living polymerizations of 2-isopropenyl-2-oxazoline typically involve living anionic polymerization initiated by

**Citation:** Pauloviˇcová, E.; Kroneková, Z.; Pauloviˇcová, L.; Majerˇcíková, M.; Kronek, J. Cell-Mediated Immunoreactivity of Poly(2-isopropenyl-2-oxazoline) as Promising Formulation for Immunomodulation. *Materials* **2021**, *14*, 1371. https://doi.org/10.3390/ ma14061371

Academic Editors: Montserrat Colilla and Yury A. Skorik

Received: 20 December 2020 Accepted: 8 March 2021 Published: 12 March 2021

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diphenylmethylpotassium/diethylzinc [20] or n-butyllithium [21]. Polymers with low dispersity were also achieved in the rare-earth metal-mediated group transfer polymerization of 2-isopropenyl-2-oxazoline [22]. On the other hand, reversible addition−fragmentation chain transfer polymerization of 2-isopropenyl-2-oxazoline provided PIPOx with molar mass under 3000 g/mol and relatively high dispersity around 1.35 [23]. Moreover, polymerizations proceeded in only 30% of conversions. Recently, Raus at al. for the first time successfully prepared PIPOx through aqueous Cu(0)-mediated atom-transfer radical polymerization of 2-isopropenyl-2-oxazoline in a controlled way initiated and catalyzed by the 2-chloropropionitrile/CuCl(CuCl2)/TPMA system [24].

PIPOx containing a free 2-oxazoline ring can be further modified employing a reaction of 2-oxazoline moiety with compounds containing thiol [25] or carboxylic groups [17,18,23,25]. Due to these unique chemical features, PIPOx is currently attractive for different fields of biotechnology and medicine due to possible preparation of thermosensitive polymers [26,27], hydrogels [27–30], or (bio)conjugates with peptides, saccharides, or drugs [31]. Therefore, their rising importance is expected, especially in drug delivery, gene delivery, tissue engineering, or vaccine technology. The basic requirement for the perspective use in biomedical applications is the tolerance of cells to the used polymer materials. Therefore, the assessment of biocompatibility using tissue cultures evaluating acute and system toxicity, examining of the inhibition of cell growth, mutagenicity, carcinogenicity, teratogenicity, and pro-allergenic potential must be included.

Immunomodulative activities of polymeric biomaterials have become the most relevant ones concerning their bioavailability and biocompatibility. Different strategies of triggering the appropriate immune system responses by functional biomaterials and various applications of biomaterials mimicking the physiological extracellular matrix and modifications of cell-mediated immune responses are of interest [32,33]. Spleen-derived cells are represented by a mixture of immune cells including macrophages, monocytes, dendritic cells (DCs), and T-lymphocytes that possess different functions in immune system. Myeloid phagocytes such as macrophages or DCs representing a complex network of cells with protective functionalities are also involved in mechanisms of homoeostasis such as tissue remodeling and wound healing [34]. Macrophages, comparable with Th-lymphocytes, have been divided into main subsets: pro-inflammatory M1-classically activated macrophages and anti-inflammatory M2-alternatively activated macrophages with distinct functional and phenotypical characteristics [35,36]. In general, inflammatory cytokines such as TNF-α and IFN-γ induce the M1 phenotype. On the contrary, antiinflammatory interleukins such as IL-10, IL-4, and IL-13 induce the M2 phenotype [33]. Macrophages play an important role in orchestrating immune responses to biomaterials used in the construction of implantable devices and drug-delivery systems [37].

It was shown recently that positively charged polymers such as polyethyleneimine and cationic dextran have the potential to modulate macrophages and change their phenotype from tumor growth-promoting M2 macrophages to anti-tumor M1 macrophages [38]. In the last study, we showed that PIPOx is also not cytotoxic to the cells up to 10 mg/mL and significantly stimulates in vitro and ex vivo proliferation of macrophages [18]. We have shown that co-stimulation of non-adherent cells (T-lymphocyte-enriched splenocytes) with PIPOx-stimulated adherent cells (enriched in DCs) leads to their induced proliferation. These results suggest that PIPOx may play a role in various immunomodulatory processes [18].

In this work, we focused on the cell-mediated bioimmunological behavior of PIPOx and its cellular compatibility as promising matrix biomaterial. The sequential adherence of splenocytes and isolated cell populations have been used to ascertain their immunobiological activity following PIPOx exposure. The aim of study suggested the sensitization of population of adherent CD11c<sup>+</sup> and CD14+ spleen cells and adherent spleen cells more enriched in CD11c<sup>+</sup> antigen-presenting cells with PIPOx and follow up the polarization of immune response towards Th1, Th2, Th17, or Treg evaluated by measuring the production of selected signature cytokines. The cytotoxicity has been determined via phagocytosis

process. Next, the RAW 264.7 macrophage internalization of PIPOx-FITC followed by intracellular localization has been assayed based on colocalization in vesicular structures of the cells resembling organelles of phagocytic and/or endocytic pathway.
