In vitro bio-immunological and cytotoxicity studies of poly(2-oxazolines)

Abstract

Poly(2-oxazolines) with varying alkyl chain lengths (e.g., methyl, ethyl, aryl) and molar masses have been tested for cell cytotoxicity in vitro. A standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for the estimation of cell viability. Two monomers, 2-methyl-2-oxazoline and 2-ethyl-2-oxazoline, were found to provide polymers with non-cytotoxic properties. The dependence of cell viability on molar mass confirmed the expected trend; the viability increased with the higher molar mass of poly(2-ethyl-2-oxazoline) (PETOX), up to 15,000 g/mol. The results obtained for the polymers with aliphatic side chains were compared with the analogues that possessed an aromatic moiety. All results confirmed low cytotoxicity of the polymers prepared by cationic polymerization of 2-alkyl- and 2-aryl-2-oxazolines, which supports their utilization in biomedical applications. Fluorescence microscopy and steady-state fluorescence were used to observe pyrene-labeled polymer interactions with living cells. Polymer accumulated within the cells was found to be dependent on polymer concentration in media. The immunoefficiency of aromatic and aliphatic oxazoline polymers and copolymers was also studied. Phagocytic and metabolic activities of macrophages were used to assess the immunosuppressive effects of the selected copolymers for possible applications in drug delivery and immunobiology. Overall, the tested polymers demonstrated no significant influences on the cellular immunological parameters.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines)

“…% aromatic moiety (AEOX10) (synthesis see Fig. 8) has no affect on the growth and proliferation of cells and that the values of cytotoxicity measured in the MTT test were on the same level as in the case of PETOX and PMEOX [27,63]. All results confirmed low cytotoxicity of the polymers prepared by cationic polymerization of 2-alkyl-and 2-aryl-2-oxazolines, which supports their utilization in biomedical applications.…”

“…Low cytotoxicity of aliphatic poly(2-alkyl-2-oxazoline)s was confirmed also in in vitro studies with Rat-2 fibroblasts using the MTT test [63] (Figs. 6 and 7).…”

“…Biodistribution of PETOX labeled with pyrene moiety in cells has been monitored by the Carl-Zeiss confocal laser-scanning microscopy [63]. The cuts of the treated cells were used for constructing 3D model (Fig.…”

“…Fig.19 shows the fluorescence microscopy of Rat-2 fibroblasts incubated in the presence of PETOX-py in PBS with the concentration of 5 mg/ml. It can be seen that macromolecules are accumulated in the cells in specific organelles, most probably in peroxisomes [63]. (b) (a) Peroxisomes are small membrane-enclosed organelles that contain enzymes involved in a variety of metabolic reactions, including several aspects of energy metabolism.…”

“…Fluorescence measurements at different wavelengths ( em) can provide qualitative and quantitative data about fluorochrome-labelled cell surface receptors or intracellular molecules such as DNA and cytokines [68,69]. In our experiments we analyzed the time and concentration dependent influence of poly(2-oxazoline)s: AEOX10 and PETOX100 (PETOX with theoretical degree of polymerization equal to 100) on phagocytosis and oxidative burst of macrophages as representative professional phagocytic cells belonging to innate immune system compartments [63]. The treatment was performed for 1, 3, 6 and 24 hours with selected concentrations 0.5 mg/ml and 5mg/ml, respectively.…”