**4. Methods for the assessment of immunocompatibility of poly(2-oxazolines)**

266 Practical Applications in Biomedical Engineering

hydrophobic character.

mg/ml (Fig. 7b).

O CH3

H3C N CH2 CH2 OH

**PMEOX**

n

amphiphilic poly(2-oxazolines) block copolymers that have different architectures, molar mass and chain termini were reported [62]. The relative cytotoxicity of poly(2-oxazolines) was tested on MCF7-ADR cells derived from human breast carcinoma cell line, MCF7 (ATCC HT-B22). All tests confirmed that these polymers are typically not cytotoxic even at high concentrations. The rate of endocytosis can be fine-tuned over a broad range by changing the polymer structure. The cellular uptake of the polymers increased with their

 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). 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), up to 15000 g/mol. The effect of incubation time and concentration was also studied. It was shown that cell viability is in the range of untreated control even after 48 hours and the polymer concentration up to 5

n

**Figure 7.** a) UV spectra of the reduced formazan dissolved in DMSO after incubation of rat fibroblasts with PETOX, b) relative cell viability of selected 2-oxazolines. PMEOX – poly(2-methyl-2-oxazoline),

The results obtained for the polymers with aliphatic side chains were compared with the analogues that possessed an aromatic moiety. It is known from the literature that the presence of benzene ring in many cases increases their toxicity as was showed for

PETOX - poly(2-ethyl-2-oxazoline), PETOX-py – pyrene labelled poly(2-ethyl-2-oxazoline).

CH2 N CH2 CH2 OH

n

O CH2CH3

**PETOX-py**

O

H3C N CH2 CH2 OH

**PETOX**

**Figure 6.** Structures of measured poly(2-alkyl-2-oxazolines)

O CH2CH3

Selected techniques as flow cytometric simultaneous evaluation of phagocytosis accompanied by oxidative burst, immunocytometric determination of TH1/TH2/TH17 cytokines using bead application and enzyme-linked spot evaluation of cytokine producing cells comprise novel approaches to characterise and consider immunomodulation of basic functions of immunocompetent cells targeted by biopolymers.

#### **4.1. Assessment of phagocyte functions by immunocytometry**

Macrophages are tissue-based cells belonging to the reticuloendothelial system, capable of phagocytosis and production of an array of immunomodulatory mediators. Foreign substance or invader triggered macrophage activation result in a release of proinflammatory interleukins, growth factors and chemokines and also in up regulation of induction of inducible oxide synthase and other potent reactive oxidants inflicting cell damage. Phagocytosis represents a complex process comprising several subsequent steps i.e. chemotaxis, attachment/adherence onto a phagocytotic membrane, internalization and fusion of the cell membrane, intra- and extracellular digestion of targets, and oxidative burst (Fig. 9). The rate of endocytosis can be fine-tuned over a broad range by changing the polymer structure. The cellular uptake increases with the hydrophobic character of the polymers and is observed even at nanomolar concentrations [62].

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 269

N+

Et

H2N NH2

exc= 520 nm

em = 610 nm

N

Et

**Figure 10.** Chemical structure of hydroethidine (HE) and ethidium (E+).

HE E+

To follow up the production of ROS throughout the oxidative burst of phagocyte, the fluorescence methodology, associated with the use of suitable fluorescence probes, is an excellent research approach applicable both for fluorescence microscopy and flow cytometry

Generally, flow cytometry measures optical and fluorescence characteristics of single cells or any other particles flowing in a single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity. Intensity of light scattered in the forward direction i.e. forward angle light scatter (FSC) roughly equates to the particle's size and can be used to distinguish between cellular debris and living cells. Light measured approximately at a 90° angle to the excitation line is called side scatter. The side scatter channel (SSC) provides information about the granular content within a particle. Fluorescence measurements at different wavelengths (λem) can provide qualitative and quantitative data about fluorochrome-labelled cell surface receptors or intracellular

**Figure 11.** Simultaneous phagocytosis and oxidative burst in mouse macrophage line P388D, by *S.aureus* BioParticles® FITC labeled (Molecular Probes) in conjunction with dihydroethidium.

Visualisation: Confocal Laser Scanning Microscopy LSM 710, Cell Observer (Zeiss).

H2N NH2

molecules such as DNA and cytokines [68,69].

H

(Fig. 11).

**Figure 9.** The sequential mechanism of phagocytosis.

Phagocytosing cells undergo a burst of oxygen consumption that is caused by an NADPH oxidase complex that assembles at the phagosomal membrane. Neutrophils and the other phagocytes create O2- (superoxide) by the one-electron reduction of oxygen at the expense of NADPH. A variety of soluble and particular stimuli induce extracellular superoxide production. Most of the oxygen consumed can be accounted for as hydrogen peroxide, which is formed from dismutation of superoxide radical. From these agents a large number of highly reactive microbicidal oxidants are formed, including HOCl (hypochlorous acid), which is produced by the myeloperoxidase-catalyzed oxidation of Cl by H2O2; OH (hydroxyl radical), produced by the reduction of H2O2 by Fe2+ or Cu+; ONOO (peroxynitrite), formed by the reaction between O2 and NO , and many others [66]. Reactive oxygen species (ROS) comprises not only free radicals, such as superoxide radical, hydroperoxyl radical, hydroxyl radical, peroxyl radical etc., but also non-radicals, namely hydrogen peroxide, singlet oxygen, and hypochlorous acid. These reactive oxidants are manufactured not only as microbicidal agents, but they also impose cell damage.

Hydroethidine (dihydroethidium; HE) is frequently used as fluorescence probe for the evaluation of cell oxidative burst by detection of ROS (Fig. 10). HE has the ability to cross-cellular membranes becoming therefore efficient in cell oxidative burst studies. When inside the cell the HE is oxidized by O2 - , it originates ethidium (E+), which becomes locked in the cell by virtue of its cationic nature. The ethidium intercalates into DNA and is red fluorescent when excited with either visible or UV light. HE can also be oxidized by different reactive species, being the relative reactivity ranked by ONOO- >HO- > O2 - >H2O2. Thus, HE provides an index of reactive oxygen and nitrogen species production [67].

**Figure 10.** Chemical structure of hydroethidine (HE) and ethidium (E+).

268 Practical Applications in Biomedical Engineering

**Figure 9.** The sequential mechanism of phagocytosis.

(peroxynitrite), formed by the reaction between O2-

When inside the cell the HE is oxidized by O2

phagocytes create O2-

>HO-

> O2 -

production [67].

Phagocytosing cells undergo a burst of oxygen consumption that is caused by an NADPH oxidase complex that assembles at the phagosomal membrane. Neutrophils and the other

NADPH. A variety of soluble and particular stimuli induce extracellular superoxide production. Most of the oxygen consumed can be accounted for as hydrogen peroxide, which is formed from dismutation of superoxide radical. From these agents a large number of highly reactive microbicidal oxidants are formed, including HOCl (hypochlorous acid),

(hydroxyl radical), produced by the reduction of H2O2 by Fe2+ or Cu+; ONOO

oxygen species (ROS) comprises not only free radicals, such as superoxide radical, hydroperoxyl radical, hydroxyl radical, peroxyl radical etc., but also non-radicals, namely hydrogen peroxide, singlet oxygen, and hypochlorous acid. These reactive oxidants are

Hydroethidine (dihydroethidium; HE) is frequently used as fluorescence probe for the evaluation of cell oxidative burst by detection of ROS (Fig. 10). HE has the ability to cross-cellular membranes becoming therefore efficient in cell oxidative burst studies.

becomes locked in the cell by virtue of its cationic nature. The ethidium intercalates into DNA and is red fluorescent when excited with either visible or UV light. HE can also be oxidized by different reactive species, being the relative reactivity ranked by ONOO-

which is produced by the myeloperoxidase-catalyzed oxidation of Cl-

manufactured not only as microbicidal agents, but they also impose cell damage.

(superoxide) by the one-electron reduction of oxygen at the expense of

and NO


>H2O2. Thus, HE provides an index of reactive oxygen and nitrogen species

by H2O2; OH

, and many others [66]. Reactive

, it originates ethidium (E+), which

To follow up the production of ROS throughout the oxidative burst of phagocyte, the fluorescence methodology, associated with the use of suitable fluorescence probes, is an excellent research approach applicable both for fluorescence microscopy and flow cytometry (Fig. 11).

Generally, flow cytometry measures optical and fluorescence characteristics of single cells or any other particles flowing in a single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity. Intensity of light scattered in the forward direction i.e. forward angle light scatter (FSC) roughly equates to the particle's size and can be used to distinguish between cellular debris and living cells. Light measured approximately at a 90° angle to the excitation line is called side scatter. The side scatter channel (SSC) provides information about the granular content within a particle. Fluorescence measurements at different wavelengths (λem) can provide qualitative and quantitative data about fluorochrome-labelled cell surface receptors or intracellular molecules such as DNA and cytokines [68,69].

**Figure 11.** Simultaneous phagocytosis and oxidative burst in mouse macrophage line P388D, by *S.aureus* BioParticles® FITC labeled (Molecular Probes) in conjunction with dihydroethidium. Visualisation: Confocal Laser Scanning Microscopy LSM 710, Cell Observer (Zeiss).

Assessment of phagocyte functions i.e phagocytosis and oxidative burst using *S. aureus*-FITC and fluorescent probe HE by flow cytometry is an approach based on the complex imunocytometric analysis of multiple characteristic of individual cells i.e neutrophils, monocyte-derived macrophages (Fig. 12). Analyses were performed with a BeckmanCoulter FC 500 flow cytometer with 488-nm excitation and filters for the detection of green fluorescence (525 nm) of *S. aureus* BioParticles® FITC labelled and red fluorescence of ethidium (610 nm). Two parameters, MFI (mean fluorescence intensity) and percentage of fluorescence-positive cells were determined separately from approx. 5000 macrophages with a flow rate of 200-300 events/s by gating the cell population according to the forward scatter (FS)/side scatter (SS) histogram. To exclude cell debris and non-ingested *S. aureus* BioParticles from FS/SS histogram, discriminator was set into the FS channel.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 271

**Figure 13.** The influence of AEOX10 and PETOX100 (5 mg/ml) on phagocytic effectiveness, oxidative

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. In all experiments the metabolic activity, including phagocytosis and oxidative burs, treated macrophage cell line P388D remained comparable with untreated control cells

**4.2. Assessment of triggered cytokine release and TH polarisation using ELISpot** 

Cytokines are proteins secreted by different cells of the immune system and serve as molecular messengers between cells. Cytokines interact with cells of the immune system in order to regulate the host response to pathological disorders; apart from this they also mediate physiological cellular processes. Different types of cytokines as colony stimulating factors, growth and differentiation factors, immunoregulatory and proinflammatory cytokines are involved in direct cell stimulation, differentiation, development and polarisation of immune response (Fig. 14). CD4+ T lymphocytes are crucial mediators of the cellular immune response. The early response of naive CD4+ T cells to antigenic stimulation (via pattern-recognition receptors) is characterized by induced proliferation. Further differentiation give arise of cells with a significant potential for cytokine expression. Depending upon the balance of local cytokines, co-stimulatory molecules, antigen levels,

burst and mtabolic activity (phagocytosis and oxidative burst) of macrophages.

without any significant immunosuppressive effect of poly(2-oxazoline)s (Fig. 13).

**and FlowCytomix™ assays** 

**Figure 12.** Simultaneous flow cytometric evaluation (Beckman Coulter FC 500) of phagocytosis and oxidative burst in mouse macrophage line P388D, by *S.aureus* BioParticles® FITC labeled (Molecular Probes), (SPA-FITC) in the conjunction with dihydroethidium (HE). Mean % of phagocytic cells represents the percentage of granulocytes ingesting at least one SPA-FITC particle; mean % of oxidative burst represents the percentage of granulocytes tagged by ethidium; mean metabolic activity % represents the percentage of granulocytes ingested at least one SPA-FITC and were tagged by ethidium.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 271

Assessment of phagocyte functions i.e phagocytosis and oxidative burst using *S. aureus*-FITC and fluorescent probe HE by flow cytometry is an approach based on the complex imunocytometric analysis of multiple characteristic of individual cells i.e neutrophils, monocyte-derived macrophages (Fig. 12). Analyses were performed with a BeckmanCoulter FC 500 flow cytometer with 488-nm excitation and filters for the detection of green fluorescence (525 nm) of *S. aureus* BioParticles® FITC labelled and red fluorescence of ethidium (610 nm). Two parameters, MFI (mean fluorescence intensity) and percentage of fluorescence-positive cells were determined separately from approx. 5000 macrophages with a flow rate of 200-300 events/s by gating the cell population according to the forward scatter (FS)/side scatter (SS) histogram. To exclude cell debris and non-ingested *S. aureus*

BioParticles from FS/SS histogram, discriminator was set into the FS channel.

**Figure 12.** Simultaneous flow cytometric evaluation (Beckman Coulter FC 500) of phagocytosis and oxidative burst in mouse macrophage line P388D, by *S.aureus* BioParticles® FITC labeled (Molecular Probes), (SPA-FITC) in the conjunction with dihydroethidium (HE). Mean % of phagocytic cells represents the percentage of granulocytes ingesting at least one SPA-FITC particle; mean % of oxidative burst represents the percentage of granulocytes tagged by ethidium; mean metabolic activity % represents the percentage of granulocytes ingested at least one SPA-FITC and were tagged by ethidium.

**Figure 13.** The influence of AEOX10 and PETOX100 (5 mg/ml) on phagocytic effectiveness, oxidative burst and mtabolic activity (phagocytosis and oxidative burst) of macrophages.

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. In all experiments the metabolic activity, including phagocytosis and oxidative burs, treated macrophage cell line P388D remained comparable with untreated control cells without any significant immunosuppressive effect of poly(2-oxazoline)s (Fig. 13).
