*3.1. Synthesis and Labeling of PIPOx*

PIPOx represents a functional polymer with strong potential for different biomedical applications. We showed previously that PIPOx is non-cytotoxic up to a concentration of 10 mg/mL for fibroblasts and macrophages cell lines. For immunomodulation studies, we used splenocytes isolated from Balb/c mice [18]. This paper is a continuation of the work on immunomodulation properties studied on the splenocytes and mouse macrophages [18]. For better comparison, we used for all experiments the same batch of PIPOx as in the previous study. PIPOx was synthesized by the free-radical polymerization using AIBN as an initiator in bulk at 60 ◦C for 8 h. This type of polymerization provided PIPOx with broader dispersity (Ð = 1.85) and molar mass of 21,000 g/mol. Chemical structure of PIPOx was confirmed by NMR and FTIR (see Figures S1 and S2 in Supplementary Material).

Fluorescently labeled PIPOx containing FITC was prepared by two-step modification (Figure 1). The first step included thermally induced reaction of PIPOx with 4 aminobuttyric acid in DMAc at 120 ◦C for 6 h. The chemical structure was estimated by 1H NMR and FTIR (see Figures S3 and S2 in Supplementary Material). Second step comprised addition of FITC on amino groups of PIPOx-ABA according to standard procedure described in Experimental part. Similarly, the real content of fluorescein moieties in PIPOx-FITC was calculated from UV/Vis absorbance and found close to feeding ratio of 1 mol.% (Figure S5 in Supplementary Material). Fluorescence ability of PIPOx-FITC was studied by emission measurements in methanol in different concentrations ranged from 0.01 to 0.1 mg/mL (Figure 2). It can be seen from spectra that emission maximum was for all concentration same and equal to 520 nm. PIPOx-FITC was used for cell internalization and organelles tracking in macrophage cell lines.

**Figure 1.** Two-step synthesis of PIPOx labeled with fluorescein (PIPOx-FITC) comprising of the introduction of amino moiety and reaction with fluorescein isothiocyanate (FITC). (i) ABA, DMAc, 120 ◦C, 6 h, (ii) FITC, 0.1 M bicarbonate buffer, 2–8 ◦C, 8 h.

**Figure 2.** Emission spectra of PIPOx-FITC measured in methanol in three concentrations (0.1 mg/mL, 0.05 mg/mL and 0.01 mg/mL).
