**3. Methods for the assessment of cytotoxicity of poly(2-oxazolines)**

Generally, biocompatibility is the ability of a material to perform with an appropriate host response in a specific application [55]. It was already mentioned that biocompatibility assessment includes different assays of acute and system toxicity, tissue cultures, cell growth inhibition, mutagenicity, carcinogenicity, teratogenicity and allergenic potential. *In vitro* cytotoxicity can be measured by different laboratory assays, such as the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 2,3-bis-(2-methoxy-4 nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, Trypan blue (TB) assay, Sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

The MTT assay is a laboratory test and a standard colorimetric assay for measuring cellular growth. It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials [56]. Yellow MTT is reduced to purple formazan in the mitochondria of living cells (Fig. 4). A solubilization solution (usually dimethyl sulfoxide, an acidified ethanol solution or a solution of the detergent sodium dodecyl sulfate in dilute hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a UV-Vis spectrophotometer. The absorption maximum is dependent on the solvent employed. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of cells can be deduced (Fig. 4.).

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 265

S O

O

HO

S O

O

N N N N

**trypan blue**

A clonogenic assay is a microbiology technique for studying the effectiveness of specific agents on survival and proliferation of cells. It is frequently used in cancer research laboratories to determine the effect of drugs or radiation on proliferating tumor cells [59]. Although this technique can provide accurate results, the assay is time-consuming to set up and analyse and can only provide data on tumor cells that can grow in culture. The word

At the conclusion of the experiment, the percentage of cells that survived the treatment is measured. A graphical representation of survival versus drug concentration or dose of

Although a number of assays can be employed for the estimation of *in vitro* cytotoxicity of different materials, the MTT assay was used almost exclusively for the assessment of *in vitro*  cytotoxicity of poly(2-oxazoline)s and 2-oxazoline related polymers. The cytotoxicity of poly(2-ethyl-2-oxazoline) (PETOX) as a function of hydrolysis to linear poly(ethylene imine) (PEI) was studied by conducting an MTT assay [60]. It was found that cell viability decreased with an increasing degree of hydrolysis, and above 50 % hydrolysis the cell viability decreased to 20 %. Another example of this polymer as a suitable material for medical use is employing polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(εcaprolactone) copolymer (PETOX-PCLs) as a carrier for paclitaxel as mentioned above. It was showed that the viability of KB human epidermoid carcinoma cells in the presence of PETOX-PCL copolymers were in the range 80 to 100 % depending on the concentration of the copolymer solution and also on the ratio of both building blocks [34]. Similarly, polymeric micelles prepared from poly(2-ethyl-2-oxazoline)-*b*-poly(l-lactide) diblock copolymers (PETOX–PLLA) have been considered biomaterials for drug delivery. PETOX– PLLA polymeric micelles had low cytotoxicity in human normal fibroblast HFW cells for 72 h by using MTT assay. It was found that cell viability was higher than 80 % for the concentration range below 1 mg/ml [61]. Structure-uptake relationships of a series of

"clonogenic" refers to the fact that these cells are clones of one another.

2. The cells are "plated" in a tissue culture vessel and allowed to grow.

OH HO NH2 H2N

O

S O

<sup>O</sup> OH

The experiment involves three major steps:

1. The treatment is applied to a sample of cells.

ionizing radiation is called a *cell survival curve*.

3. The colonies produced are fixed, stained, and counted.

**Figure 5.** Structure of Trypan blue

O

HO

S OH

**Figure 4.** Scheme of reduction of the MTT to fozmazan.

The principle of the WST assay is the same as in the case of MTT assay. In contrast to the MTT assay, no extraction step was necessary due to the water solubility of the reduced form of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) so that absorbance could be determined directly at 450 nm after conversion [57]. The same principle is used in the XTT assay which employs 2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2Htetrazolium-5-carboxanilide (XTT) as reagent.

Trypan blue is a vital stain used to selectively color dead tissues or cells blue (Fig. 5). Live cells or tissues with intact cell membranes are impermeable for Trypan bluesince cells are very selective in the compounds that pass through the membrane. Trypan blue is not absorbed. In dead cells, the dye traverses the membrane and dead cells are stained blue under a microscope.

Sulforhodamine B or Kiton Red is a fluorescent dye with uses spanning from L.I.F.(Laser Induced Fluorescense) to the quantification of cellular proteins of cultured cells. The red, solid, water soluble dye is primarily used as a polar tracer. The dye absorbs 565 nm light and emits 586 nm light. It does not exhibit pH dependent absorption or fluorescence over the range of 3 to 10 [58].

**Figure 5.** Structure of Trypan blue

deduced (Fig. 4.).

N

N N+

Br-

N

**Figure 4.** Scheme of reduction of the MTT to fozmazan.

tetrazolium-5-carboxanilide (XTT) as reagent.

CH3

CH3

S

N

under a microscope.

the range of 3 to 10 [58].

The MTT assay is a laboratory test and a standard colorimetric assay for measuring cellular growth. It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials [56]. Yellow MTT is reduced to purple formazan in the mitochondria of living cells (Fig. 4). A solubilization solution (usually dimethyl sulfoxide, an acidified ethanol solution or a solution of the detergent sodium dodecyl sulfate in dilute hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a UV-Vis spectrophotometer. The absorption maximum is dependent on the solvent employed. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of cells can be

> mitochondrial reductase

The principle of the WST assay is the same as in the case of MTT assay. In contrast to the MTT assay, no extraction step was necessary due to the water solubility of the reduced form of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) so that absorbance could be determined directly at 450 nm after conversion [57]. The same principle is used in the XTT assay which employs 2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-

Trypan blue is a vital stain used to selectively color dead tissues or cells blue (Fig. 5). Live cells or tissues with intact cell membranes are impermeable for Trypan bluesince cells are very selective in the compounds that pass through the membrane. Trypan blue is not absorbed. In dead cells, the dye traverses the membrane and dead cells are stained blue

Sulforhodamine B or Kiton Red is a fluorescent dye with uses spanning from L.I.F.(Laser Induced Fluorescense) to the quantification of cellular proteins of cultured cells. The red, solid, water soluble dye is primarily used as a polar tracer. The dye absorbs 565 nm light and emits 586 nm light. It does not exhibit pH dependent absorption or fluorescence over

**MTT formazan**

N

NH

<sup>N</sup> <sup>N</sup>

S

CH3

CH3

N

A clonogenic assay is a microbiology technique for studying the effectiveness of specific agents on survival and proliferation of cells. It is frequently used in cancer research laboratories to determine the effect of drugs or radiation on proliferating tumor cells [59]. Although this technique can provide accurate results, the assay is time-consuming to set up and analyse and can only provide data on tumor cells that can grow in culture. The word "clonogenic" refers to the fact that these cells are clones of one another.

The experiment involves three major steps:


At the conclusion of the experiment, the percentage of cells that survived the treatment is measured. A graphical representation of survival versus drug concentration or dose of ionizing radiation is called a *cell survival curve*.

Although a number of assays can be employed for the estimation of *in vitro* cytotoxicity of different materials, the MTT assay was used almost exclusively for the assessment of *in vitro*  cytotoxicity of poly(2-oxazoline)s and 2-oxazoline related polymers. The cytotoxicity of poly(2-ethyl-2-oxazoline) (PETOX) as a function of hydrolysis to linear poly(ethylene imine) (PEI) was studied by conducting an MTT assay [60]. It was found that cell viability decreased with an increasing degree of hydrolysis, and above 50 % hydrolysis the cell viability decreased to 20 %. Another example of this polymer as a suitable material for medical use is employing polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(εcaprolactone) copolymer (PETOX-PCLs) as a carrier for paclitaxel as mentioned above. It was showed that the viability of KB human epidermoid carcinoma cells in the presence of PETOX-PCL copolymers were in the range 80 to 100 % depending on the concentration of the copolymer solution and also on the ratio of both building blocks [34]. Similarly, polymeric micelles prepared from poly(2-ethyl-2-oxazoline)-*b*-poly(l-lactide) diblock copolymers (PETOX–PLLA) have been considered biomaterials for drug delivery. PETOX– PLLA polymeric micelles had low cytotoxicity in human normal fibroblast HFW cells for 72 h by using MTT assay. It was found that cell viability was higher than 80 % for the concentration range below 1 mg/ml [61]. Structure-uptake relationships of a series of

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 hydrophobic character.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 267

HO CH2 CH2 N CH2CH2 N CH3

C O

CH2CH3

**AEOX10**

C

90 10

O

NH2

different aromatic drugs [64,65]. In spite of these results, our measurements indicated that the presence of a benzene ring in poly[2-ethyl-2-oxazoline-co-2-(4-aminophenyl)-2 oxazoline] with 10 mol. % 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

O2N SO3Me

**Figure 8.** Synthesis of poly[2-ethyl-2-oxazoline-co-2-(4-aminophenyl)-2-oxazoline] (9:1).

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

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

**4. Methods for the assessment of immunocompatibility** 

functions of immunocompetent cells targeted by biopolymers.

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

polymers and is observed even at nanomolar concentrations [62].

applications.

CH2CH3

**of poly(2-oxazolines)** 

+ O N

NH2

N O

 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 mg/ml (Fig. 7b).

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

**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), PETOX - poly(2-ethyl-2-oxazoline), PETOX-py – pyrene labelled poly(2-ethyl-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 different aromatic drugs [64,65]. In spite of these results, our measurements indicated that the presence of a benzene ring in poly[2-ethyl-2-oxazoline-co-2-(4-aminophenyl)-2 oxazoline] with 10 mol. % 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.

**Figure 8.** Synthesis of poly[2-ethyl-2-oxazoline-co-2-(4-aminophenyl)-2-oxazoline] (9:1).
