**1. Introduction**

256 Practical Applications in Biomedical Engineering

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[26] Gudiña EJ, Teixeira JA, Rodrigues LR. Isolation and functional characterization of a biosurfactant produced by *Lactobacillus paracasei.* Colloids and Surfaces B: Biointerfaces

[27] Rodrigues LR, Banat IM, Van Der Mei HC, Teixeira JA, Oliveira R Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by

[30] Carrilo C, Teruel JA, Aranda FJ, Ortiz A Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochimica et Biophysica Acta

[31] Singh A, Van Hamme JD, Ward OP. Surfactants in microbiology and biotechnology:

[32] Elving GJ, Van Der Mei HC, Busscher HJ, Amerogen EC, Van Weissenbruch R, Albers FW. Antimicrobial activity of synthetic salivary peptides against voice prosthetic

[33] Lang S, Katsiwela E, Wagner F. Antimicrobial effects of biosurfactants. Fat Science

[34] Kitamoto D, Isoda H, Nakahara T. Functions and potential applications of glycolipid biosurfactants - from energy-saving materials to gene delivery carriers*.* Journal of

[35] Busscher HJ, Van Hoogmoed CG, Geertsema-Doornbusch GI, Van Der Kuijl-Booij M, Van Der Mei HC *Streptococcus thermophilus* and its biosurfactants inhibit adhesion by Candida spp. On silicone rubber. Applied Environ Microbiol 1997; 63: 3810-3817. [36] Nitschke M, Ferraz C, Pastore GM. Selection of microorganisms for biosurfactant production

[37] Falagas MF, Makris GC. Probiotic bacteria and biosurfactants for nosocomial infection

[38] Mireles JR, Toguchi A, Harshey RM. Salmonella enterica serovar Typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm

[39] Busscher HJ, Van Hoogmoed CG, Geertsema-Doornbusch GI, Van Der Kuijl-Booij M, Van Der Mei HC. *Streptococcus thermophilus* and its biosurfactants inhibit adhesion by Candida spp. On silicone rubber. Applied Environ Microbiol 1997; 63: 3810-3817. [40] Pratt- Terpstra IH, Busscher HJ. Microbial factors in a thermodynamic approach of oral streptococcal adhesion to solid substrata. Journal Colloid Interface Science 1989; 129: 568-574. [41] Fischer W. Molecular analysis of lipid macroamphiphiles by hydrophobic interaction

[42] Velraeds M, Van Der Mei HC, Reid G*.* Interference in initial adhesion of uropathogenic bacteria and yeasts to silicone rubber by a *Lactobacillus acidophilus* biosurfactant. J Med

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rhamnolipid biosurfactants. Journal of Applied Microbiology 2006 d; 100: 470-480. [28] Elving GJ, Van Der Mei HC, Busscher HJ, Van Weissenbruch R, Albers FW. Comparision of the microbial composition of voice prosthesis biofilms from patients requiring frequent versus infrequent replacement. Annals of Otology, Rihinol Laryngol 2002; 111: 200-203. [29] Calvo C, Manzanera M, Silva-Castro GA, González-Lopéz J. Application of bioemulsifiers in soil oil bioremediation processes. Future Prospects. Science of the

> Synthetic polymers are considered to be the promising materials for biomedical applications. Various polymer formulations have been employed to achieve the desired chemical, physical and biological properties. Recently, there has been much interest in the development of environmentally responsive polymers for use as biomaterials [1]. Such behavior is significant for the controlled release of drugs upon the application of a stimulus, such as pH, temperature, light or ionic strength. These mentioned properties are necessary for the utilization of polymeric materials for biomedical applications, such as drug and gene delivery, biomembrane technology and biocatalysis [2,3]. Polymer materials can be used in medicine as a part of implant, dialysis membranes, bone scaffolds or components of artificial organs. It means that polymers covers very broad range of biomedical applications. A critical point of the usage of synthetic polymers in living bodies is their utilization, accompanied with the interactions of the foreign material, with the living matter (cells, tissues etc.). The implantation of polymeric materials to a body is usually associated with the inflammation and biofouling. The inflammation is the first defense mechanism of the immune system followed by unspecific cell and protein adhesion and the formation of fibrotic tissue which leads to implant´s dysfunctions. The fundamental role in the implantation of these materials is to increase the tolerance of body to implanted material and to avoid the foreign body reaction [4,5].

> Assessments of polymeric material biocompatibility and immunotoxicity are key issues to consider material to be suitable for biomedical applications. Biocompatibility assessment of a polymeric material includes adequate testing for undesired responses. To evaluate biocompatibility, examinations of acute and system toxicity, tissue cultures, cell growth inhibition, mutagenicity, carcinogenicity, teratogenicity and allergenic potential should be

© 2012 Kronek et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Kronek et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

conducted [6]. *In vitro* cytotoxicity can be tested by a number of methods that are mostly based on the colorimetric assay of dyes that are sensitive to viable or dead cells. Such assays include 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2 methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), water soluble tetrazolium salts (WST), trypan blue or sulforhodamine B as dyes.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 259

mucosal surfaces. Hence they are one of the most crucial cells acting in innate immune responses to exogenous challenge. They are equipped with restricted number of pattern recognition receptors (PRRs) to recognize foreign invader *via* their pathogen-associated molecular patterns (PAMPs). Pattern recognition receptors (PRRs) are membrane-bound or secreted microbial sensory receptors that include Toll-like receptors (TLRs), Nod-like receptors (NLRs), and C-type lectin receptor (CLRs). The main functions of PRRs´ include opsonization, activation of complement and coagulation cascades, phagocytosis, induction of inflammatory cytokines, and induction of apoptosis. Innate immune responses are triggered upon PAMPs recognition by the Toll-like receptors (TLR), type I transmembrane signalling cell surface molecules. Activation of TLRs induces, *via* the transcriptional activator nuclear factor kB (NF-kB), the triggering of a wide variety of inflammatory and immune-response genes and expression of a variety of molecules in macrophages including pro-inflammatory cytokines e.g. tumor necrosis factor - α (TNF-α) , interleukin-1 (IL-1), interleukin 6 (IL-6), interferons (IFN-γ, IFN β), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth

Host immune responses towards biomaterials and biocompatibility and immunocompatibility of polymeric materials are essentially important for their biomedical application. Recently, strategies of triggering appropriate immune responses by functional biomaterials and approaches of biomaterials that mimic the physiological extracellular matrix and modify cellular immune responses have been highlighted [14]. The targeting of dendritic cells with biopolymers [15] and targeting antigen to immune cells via PAMPmodified biomaterials [16] have received considerable attention and represents the novel strategy to control the subsequent immune responsiveness. Immunological inflammation comprising the temporal variation in the acute inflammatory response, chronic inflammatory response, granulation tissue development, and foreign-body reaction represents one of the most important reactions to biomaterials observed in a host [17,18]. Immunogenicity of synthetic biopolymers, their immunomodulative character and induction of cell and antibody immunities are crucial aspects of *in vivo* applications. The involvement of the immune system in incompatibility reactions towards biomaterials is not desirable in special cases, thus, various possibilities to shield the biomaterial and cell

The aim of this chapter is to summarize and evaluate the available methods for the biocompatibility and immunocompatibility assesment of 2-oxazoline-based polymers. 2- Oxazoline-based polymers serve a significant role in the field of polymers suitable for biological and medical use. Examples of their applications in field of biology and medicine

From a variety of synthetic polymers suitable for different biomedical applications, polymers based on 2-oxazoline chemistry represent very versatile and promising materials.

factor (TGF-β), macrophage inflammatory protein 1 (MIP-1) [11-13].

surfaces from recognition by the innate immune system are studied [19].

**2. Poly(2-oxazolines) as materials for biomedical applications** 

were also given.

The immunocompatibility of polymers is obviously compulsory for their potential medical application. Several aspects of targeted immune responses have to be taken into account. The immune system is an integrated system composed of two major sub-systems, the innate, non-specific immune system and the adaptive, specific immune system. The innate immune system represents a primary defense mechanism against invading microorganisms and foreign substances and the adaptive immune system operates as a second part of defense. Systemic host immune responses involve complex and multisided interactions between numerous mechanisms of innate and adaptive immunities and their cellular and humoral components. Apart from systemic immunity, common mucosal immune system is effective in all mucosal surfaces i.e. mucosa-associated lymphoid tissue (MALT), including aggregates and diffuse population of immune cells within mucosae; gut-associated lymphoid tissue (GALT), including isolated lymphoid follicles, Peyer´s patches, the appendix, and cells in the lamina propria; bronchus-associated lymphoid tissue (BALT), including defined aggregates and diffuse populations of lymphoid cells around the airways [7].

Several specifically programmed cells are involved in innate and adaptive immunity. These include neutrophiles and macrophages, which are involved in phagocytosis, basophils and mast cells, which are involved in inflammation and B and T lymphocytes which account for antibody mediated immunity and cell mediated immunity, respectively. Most of immune system's immunocompetent effector cells are derived from specific precursors in the bone marrow. In addition to red blood cells and platelets, pluripotent stem cells give rise to the lymphocytes, the monocytes and macrophages, and to the group of cells with collective term "the granulocytes", covering neutrophils, basophils and eosinophils.

Dendritic cells (DCs) comprise a subset of cells with different characteristics, derived from bone marrow precursor cells. Dendritic cells are professional antigen presenting cells that play a central role in the running and regulation of immune responses, as well as control the innate (NK cells, NKT cells, T cells) and adaptive (T and B cells) arm of immunity DCs have a crucial role in initiating T-cell mediated immunity. DCs´can control a substantial part of the adaptive immune response by internalizing and processing antigen through MHCclass I and class II pathways and, finally, presenting antigenic peptides to CD4+ and CD8+ T lymphocytes [8-10].

The specific adaptive immune system requires some time period to act in a response to an invader (microorganism or foreign substance), whereas the non-specific innate immune system comprises fundamental defenses mostly constitutively present and mobilized immediately upon the invasion and recognition of foreign invader. Macrophages and the progeny of granulocyte differentiation, tissue-based mast cells and NK cells are decisive cellular components of the innate immune response. Monocyte–derived macrophages represent the first line of host defence after the epithelial barrier against invader that reaches mucosal surfaces. Hence they are one of the most crucial cells acting in innate immune responses to exogenous challenge. They are equipped with restricted number of pattern recognition receptors (PRRs) to recognize foreign invader *via* their pathogen-associated molecular patterns (PAMPs). Pattern recognition receptors (PRRs) are membrane-bound or secreted microbial sensory receptors that include Toll-like receptors (TLRs), Nod-like receptors (NLRs), and C-type lectin receptor (CLRs). The main functions of PRRs´ include opsonization, activation of complement and coagulation cascades, phagocytosis, induction of inflammatory cytokines, and induction of apoptosis. Innate immune responses are triggered upon PAMPs recognition by the Toll-like receptors (TLR), type I transmembrane signalling cell surface molecules. Activation of TLRs induces, *via* the transcriptional activator nuclear factor kB (NF-kB), the triggering of a wide variety of inflammatory and immune-response genes and expression of a variety of molecules in macrophages including pro-inflammatory cytokines e.g. tumor necrosis factor - α (TNF-α) , interleukin-1 (IL-1), interleukin 6 (IL-6), interferons (IFN-γ, IFN β), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF-β), macrophage inflammatory protein 1 (MIP-1) [11-13].

258 Practical Applications in Biomedical Engineering

conducted [6]. *In vitro* cytotoxicity can be tested by a number of methods that are mostly based on the colorimetric assay of dyes that are sensitive to viable or dead cells. Such assays include 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2 methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), water soluble

The immunocompatibility of polymers is obviously compulsory for their potential medical application. Several aspects of targeted immune responses have to be taken into account. The immune system is an integrated system composed of two major sub-systems, the innate, non-specific immune system and the adaptive, specific immune system. The innate immune system represents a primary defense mechanism against invading microorganisms and foreign substances and the adaptive immune system operates as a second part of defense. Systemic host immune responses involve complex and multisided interactions between numerous mechanisms of innate and adaptive immunities and their cellular and humoral components. Apart from systemic immunity, common mucosal immune system is effective in all mucosal surfaces i.e. mucosa-associated lymphoid tissue (MALT), including aggregates and diffuse population of immune cells within mucosae; gut-associated lymphoid tissue (GALT), including isolated lymphoid follicles, Peyer´s patches, the appendix, and cells in the lamina propria; bronchus-associated lymphoid tissue (BALT), including defined aggregates

Several specifically programmed cells are involved in innate and adaptive immunity. These include neutrophiles and macrophages, which are involved in phagocytosis, basophils and mast cells, which are involved in inflammation and B and T lymphocytes which account for antibody mediated immunity and cell mediated immunity, respectively. Most of immune system's immunocompetent effector cells are derived from specific precursors in the bone marrow. In addition to red blood cells and platelets, pluripotent stem cells give rise to the lymphocytes, the monocytes and macrophages, and to the group of cells with collective term

Dendritic cells (DCs) comprise a subset of cells with different characteristics, derived from bone marrow precursor cells. Dendritic cells are professional antigen presenting cells that play a central role in the running and regulation of immune responses, as well as control the innate (NK cells, NKT cells, T cells) and adaptive (T and B cells) arm of immunity DCs have a crucial role in initiating T-cell mediated immunity. DCs´can control a substantial part of the adaptive immune response by internalizing and processing antigen through MHCclass I and class II pathways and, finally, presenting antigenic peptides to CD4+ and

The specific adaptive immune system requires some time period to act in a response to an invader (microorganism or foreign substance), whereas the non-specific innate immune system comprises fundamental defenses mostly constitutively present and mobilized immediately upon the invasion and recognition of foreign invader. Macrophages and the progeny of granulocyte differentiation, tissue-based mast cells and NK cells are decisive cellular components of the innate immune response. Monocyte–derived macrophages represent the first line of host defence after the epithelial barrier against invader that reaches

tetrazolium salts (WST), trypan blue or sulforhodamine B as dyes.

and diffuse populations of lymphoid cells around the airways [7].

"the granulocytes", covering neutrophils, basophils and eosinophils.

CD8+ T lymphocytes [8-10].

Host immune responses towards biomaterials and biocompatibility and immunocompatibility of polymeric materials are essentially important for their biomedical application. Recently, strategies of triggering appropriate immune responses by functional biomaterials and approaches of biomaterials that mimic the physiological extracellular matrix and modify cellular immune responses have been highlighted [14]. The targeting of dendritic cells with biopolymers [15] and targeting antigen to immune cells via PAMPmodified biomaterials [16] have received considerable attention and represents the novel strategy to control the subsequent immune responsiveness. Immunological inflammation comprising the temporal variation in the acute inflammatory response, chronic inflammatory response, granulation tissue development, and foreign-body reaction represents one of the most important reactions to biomaterials observed in a host [17,18]. Immunogenicity of synthetic biopolymers, their immunomodulative character and induction of cell and antibody immunities are crucial aspects of *in vivo* applications. The involvement of the immune system in incompatibility reactions towards biomaterials is not desirable in special cases, thus, various possibilities to shield the biomaterial and cell surfaces from recognition by the innate immune system are studied [19].

The aim of this chapter is to summarize and evaluate the available methods for the biocompatibility and immunocompatibility assesment of 2-oxazoline-based polymers. 2- Oxazoline-based polymers serve a significant role in the field of polymers suitable for biological and medical use. Examples of their applications in field of biology and medicine were also given.

#### **2. Poly(2-oxazolines) as materials for biomedical applications**

From a variety of synthetic polymers suitable for different biomedical applications, polymers based on 2-oxazoline chemistry represent very versatile and promising materials.

Poly(2-oxazolines) can be prepared by the living cationic polymerization of 2-oxazolines containing aliphatic or aromatic substituent [20] (Fig. 1). This means that polymers with defined structure, size and narrow dispersity can be prepared. Living character of polymerization of 2-oxazolines can be employed for the preparation of polymers with complex architecture, such as star-shaped polymers [21,22] or polymer combs and brushes [23], and also for the synthesis of block copolymers.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 261

Due to their versatility and ability to form functional materials and nanostructures, 2 oxazoline based polymers have a promising use in biomedical applications. One of the principal application areas for polymers in biological and medicinal contexts is drug and gene delivery. The concept of drug delivery is based on either encapsulation of a drug in polymer and its delivery through diffusion or covalent attachment onto functional polymer matrix through labile bond [31]. The majority of polymers that have been investigated for use in drug and gene delivery applications have focused on linear polymers such as poly(ethylene oxide) (PEO), poly(N-2-hydroxypropyl methacrylamide) (HPMA) and others. One possibility is based on the preparation of liposome-polyoxazoline conjugates [32,33]. Prepared poly(2-methyl-2-oxazoline) (PMEOX) and poly(2-ethyl-2-oxazoline) (PETOX) containing the carboxylic group positioned at either the initiation or termination ends of the polymer chains. Distearoylphosphatidylethanolamine was covalently attached to the polymers through the carboxyl end groups, resulting in conjugates which incorporate readily into liposomes. Both polymers exhibit long plasma lifetimes and low hepatosplenic

Another possibility is the preparation of amphiphilic block copolymers consisted of either blocks of hydrophilic and hydrophobic 2-oxazoline monomers or blocks of hydrophilic poly(2-oxazoline) and another hydrophobic polymer. In such manner, amphiphilic block copolymers containing poly(2-ethyl-2-oxazoline) as hydrophilic block and polycaprolactone as hydrophobic block have been prepared (Fig. 3b). Such block copolymers are able to form micelles in aqueous solution and can be exploited for delivery of paclitaxel (Fig. 3a) [34]. Similarly, sustained release of the antibody bevacizumab (Avastin) from a biocompatible material based on triblock copolymer of poly(2-ethyl-2-oxazoline)-block- poly(epsiloncaprolactone)-block- poly(2-ethyl-2-oxazoline) has been reported by Wang et al. [35]. Bevacizumac has been used clinically to treat intraocular neovascular diseases based on its

**Figure 3.** Structure of paclitaxel (a) and poly(2-ethyl-2-oxazoline)-block- poly(epsilon-caprolactone) (b).

Amphiphilic graft copolymers with a thermosensitive poly(N-isopropylacrylamide) (PNiPAAM) backbone and pH-sensitive hydrophilic poly(2-carboxyethyl-2-oxazoline) graft chains were prepared and used for the formation of stable micelle-like aggregates with reversible thermosensitive and pH dependent swelling behaviour [36]. The temperatureswitchable hydrophilicity or swelling of the core as well as the pHdependent stretching of

antivascular endothelial growth factor (VEGF) character.

uptake.

**Figure 1.** Scheme of living cationic polymerization of 2-substituted 2-oxazolines.

Polymers containing functional groups represent an important part of polymer therapeutics, especially in the drug delivery systems. Polymers containing functional groups in the side chain can be prepared directly from the 2-oxazoline monomer containing the required group, or by polymer analogous reaction of polymer precursors. Preparation of polymers containing amino, carboxyl, mercapto, and aldehyde groups in the side chain was reported [24-26]. In all cases, monomers containing a protected functional group were used for the polymer synthesis. The protecting group was removed after the polymerization process was completed. Copolymers containing a free amino group were prepared directly by the cationic copolymerization of 2-ethyl-2-oxazoline and 2-(4-aminophenyl)-2-oxazoline [27].

Poly(2-alkyl-2-oxazolines) with shorter alkyl chain are water soluble and exhibit thermosensitive behaviour (Fig. 2). Poly(2-ethyl-2-oxazoline) (PETOX) represents a thermoresponsive polymer with the lower critical solution temperature (LCST) equal to 69 °C [28]. Poly(2-isopropyl-2-oxazoline) (PIPOX) also exhibits thermosensitive behavior, and its LCST is near 37 °C, which is necessary for its utilization in drug and gene delivery [29]. Poly(2-propyl-2-oxazoline) (PPOX) has LCST equal to 25 °C [30]. These results show that the length of alkyl chain has a great impact on solution properties of poly(2-alkyl- 2-oxazolines) and phase transition can be adjusted also by copolymerization of monomers with different alkyl chain.

**Figure 2.** Structure of thermosensitive poly(2-oxazolines).

Due to their versatility and ability to form functional materials and nanostructures, 2 oxazoline based polymers have a promising use in biomedical applications. One of the principal application areas for polymers in biological and medicinal contexts is drug and gene delivery. The concept of drug delivery is based on either encapsulation of a drug in polymer and its delivery through diffusion or covalent attachment onto functional polymer matrix through labile bond [31]. The majority of polymers that have been investigated for use in drug and gene delivery applications have focused on linear polymers such as poly(ethylene oxide) (PEO), poly(N-2-hydroxypropyl methacrylamide) (HPMA) and others. One possibility is based on the preparation of liposome-polyoxazoline conjugates [32,33]. Prepared poly(2-methyl-2-oxazoline) (PMEOX) and poly(2-ethyl-2-oxazoline) (PETOX) containing the carboxylic group positioned at either the initiation or termination ends of the polymer chains. Distearoylphosphatidylethanolamine was covalently attached to the polymers through the carboxyl end groups, resulting in conjugates which incorporate readily into liposomes. Both polymers exhibit long plasma lifetimes and low hepatosplenic uptake.

260 Practical Applications in Biomedical Engineering

<sup>R</sup><sup>1</sup> <sup>X</sup>

N O

R

alkyl chain.

CH2 CH2 N

C H2C O CH3

**Figure 2.** Structure of thermosensitive poly(2-oxazolines).

n

[23], and also for the synthesis of block copolymers.

R

N+ <sup>O</sup>

R

**Figure 1.** Scheme of living cationic polymerization of 2-substituted 2-oxazolines.

X

Poly(2-oxazolines) can be prepared by the living cationic polymerization of 2-oxazolines containing aliphatic or aromatic substituent [20] (Fig. 1). This means that polymers with defined structure, size and narrow dispersity can be prepared. Living character of polymerization of 2-oxazolines can be employed for the preparation of polymers with complex architecture, such as star-shaped polymers [21,22] or polymer combs and brushes

> N O R

Polymers containing functional groups represent an important part of polymer therapeutics, especially in the drug delivery systems. Polymers containing functional groups in the side chain can be prepared directly from the 2-oxazoline monomer containing the required group, or by polymer analogous reaction of polymer precursors. Preparation of polymers containing amino, carboxyl, mercapto, and aldehyde groups in the side chain was reported [24-26]. In all cases, monomers containing a protected functional group were used for the polymer synthesis. The protecting group was removed after the polymerization process was completed. Copolymers containing a free amino group were prepared directly by the cationic copolymerization of 2-ethyl-2-oxazoline and 2-(4-aminophenyl)-2-oxazoline [27].

Poly(2-alkyl-2-oxazolines) with shorter alkyl chain are water soluble and exhibit thermosensitive behaviour (Fig. 2). Poly(2-ethyl-2-oxazoline) (PETOX) represents a thermoresponsive polymer with the lower critical solution temperature (LCST) equal to 69 °C [28]. Poly(2-isopropyl-2-oxazoline) (PIPOX) also exhibits thermosensitive behavior, and its LCST is near 37 °C, which is necessary for its utilization in drug and gene delivery [29]. Poly(2-propyl-2-oxazoline) (PPOX) has LCST equal to 25 °C [30]. These results show that the length of alkyl chain has a great impact on solution properties of poly(2-alkyl- 2-oxazolines) and phase transition can be adjusted also by copolymerization of monomers with different

CH2 CH2 N

C HC O

CH3

**PETOX PIPOX PPOX**

H3C <sup>n</sup>


1

R N CH2 CH2

CH2 CH2 N

H3C

C H2C O CH2

n

R O

R

<sup>X</sup> <sup>n</sup> -

Another possibility is the preparation of amphiphilic block copolymers consisted of either blocks of hydrophilic and hydrophobic 2-oxazoline monomers or blocks of hydrophilic poly(2-oxazoline) and another hydrophobic polymer. In such manner, amphiphilic block copolymers containing poly(2-ethyl-2-oxazoline) as hydrophilic block and polycaprolactone as hydrophobic block have been prepared (Fig. 3b). Such block copolymers are able to form micelles in aqueous solution and can be exploited for delivery of paclitaxel (Fig. 3a) [34]. Similarly, sustained release of the antibody bevacizumab (Avastin) from a biocompatible material based on triblock copolymer of poly(2-ethyl-2-oxazoline)-block- poly(epsiloncaprolactone)-block- poly(2-ethyl-2-oxazoline) has been reported by Wang et al. [35]. Bevacizumac has been used clinically to treat intraocular neovascular diseases based on its antivascular endothelial growth factor (VEGF) character.

**Figure 3.** Structure of paclitaxel (a) and poly(2-ethyl-2-oxazoline)-block- poly(epsilon-caprolactone) (b).

Amphiphilic graft copolymers with a thermosensitive poly(N-isopropylacrylamide) (PNiPAAM) backbone and pH-sensitive hydrophilic poly(2-carboxyethyl-2-oxazoline) graft chains were prepared and used for the formation of stable micelle-like aggregates with reversible thermosensitive and pH dependent swelling behaviour [36]. The temperatureswitchable hydrophilicity or swelling of the core as well as the pHdependent stretching of the side chains in these core–shell nanogels offers a great potential for applications such as drug delivery systems or nanoreactors/carriers in biotechnology. Nanoparticles based on block copolymers of 2-ethyl-2-oxazoline and 2-phenyl-2-oxazolines were studied as promising tools for targeted delivery of drugs in the treatment of neuropsychiatric disorders [37].

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 263

polymers containing alkyl ammonia functions with alkyl chains of twelve carbon atoms or

 Polymers based on 2-oxazoline chemistry were used in biotechnology for different applications. Biofouling release properties are important for the development of coatings exhibiting marine biofouling resistance. It was found that copolymers prepared from perfluoroalkyl acrylates and poly(2-isopropenyl-2-oxazoline) exhibited unprecedented resistance to marine biofouling [48]. Another example is the development of an optical biosensor using an electrically controlled-release system for measurement of peroxide concentration. The response is based on the hydrogen bond of carboxylic group of currentsystem and 2-oxazoline group of a polymer complex [49]. Activating enzymes in organic solvents is of great interest in current biocatalysis. Amphiphilic and liposome-like polymers have exploitation also in membrane technology. Amphiphilic polymer tether consisted of linear hydrophilic poly(2-methyl-2-oxazoline) chains with defined length with surfacecoupling silane groups on one chain-end and hydrophobic n-alkyl chains on second end [50]. These polymers represent linear polymer spacers between supported lipid membranes and solid substrates. Lipid-tehtered poly(2-methyl-2-oxazoline) lipopolymer chains can be used as Langmuir monolayers at the air-water interface [51]. Recently, novel alpha,omegafunctionalized amphiphilic lipopolymers based on a proximal lipid moiety and hydrophilic poly(2-oxazoline) were used in asymmetric functionalized model lipid membranes for transmembrane transport and cell adhesion/recognition [52]. Amphiphilic co-networks composed of more hydrophilic poly(2-hydroxyethyl acrylate) and more hydrophobic, telechelic poly(2-ethyl-2-oxazoline) segments were prepared as free-standing membranes for entrapment of an enzyme molecule [53]. Amphiphilic miktoarm star copolymers containing cyclodextrin core can be used as nanocarriers in drug delivery but also in biocatalysis. Amphiphilic copolymers were consisted of poly(2-ethyl-2-oxazoline) and polylactide arms

All reported applications of poly(2-oxazolines) confirm a great potential of 2-oxazoline based polymers as biomaterials. Detailed study of their biocompatibility and immunocompatibility is essential and necessary to understand their interactions with the

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,

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

Sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

longer exhibit antimicrobial activity.

covalently linked to cyclodextrine core [54].

living systems (biomolecules, cells, tissues etc.).

Polymers based on 2-oxazoline chemistry were also used in gene delivery. Polyethylene imine (PEI) is one of widely used vectors for nucleic acid delivery. It was found that linear PEI is more effective than branched PEI prepared by conventional polymerization of aziridine. Therefore, DNA complex with fully deacylated PEI can be used in gene therapy for lung diseases [38]. On the other hand, partially hydrolyzed poly(2-ethyl-2-oxazoline) showed lower cytotoxicity comparing to PEI and hasstill high transfection efficiency in the gene expression [39]. Linear PEI can be prepared by basic or acidic hydrolysis of poly(2 ethyl-2-oxazoline). Recently, microwave synthesizer induced hydrolysis of poly(2-ethyl-2 oxazoline) was described as powerful method for the preparation of "pharmagrade" PEI [40]. A block copolymer of a hyperbranched poly(ethylene glycol)-like core and linear polyethylenimine (HBP) was prepared by cationic polymerization of 2-ethyl-2-oxazoline initiated by hyperbranched initiator bearing p-chloromethylbenzoyl initiating groups and subsequent hydrolysis [41]. Linear PEI-bearing hyperbranched polycations (HBP) is able to form a complex with DNA with an average diameter of 50 nm that is stable for several weeks and showed resistance to DNAse I-mediated degradation. The 'inverted' block copolymers showed several orders higher transfection efficiency than block copolymers of PEG-b-LPEI *in vitro*. Comb-like copolymers suitable for DNA transfection consisted of linear poly(ethyleneimine)-graft-poly(2-ethyl-2-oxazoline) (LPEI-comb-PETOX) and (ethyleneimine)-graft-poly(ethyleneimine) (LPEI-comb-LPEI) [42]. Successful gene delivery systems with high transfection efficiency was prepared also by condensation of therapeutic DNA with different copolymers of poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) of variable grafting densities [43]. Amphiphilic poly[(propylene glycol)-block-(2-methyl-2 oxazoine)] block copolymers were studied for gene transfer in skeletal muscle [44].

Poly(2-oxazolines) have found utilization also in vaccine technology. Recently, a novel glycoconjugates consisting of detoxified lipopolysaccharide of *Vibrio cholerae* O135, linear poly(2-oxazoline) copolymer containing free amino groups as a carrier, and an immunogenic protein, BSA, has been prepared. Multiple attachments of small effector antigen molecules to a suitable matrix is considered a novel technique for creating efficient glycoconjugate vaccines [45,46].

Antimicrobial polymers are becoming increasingly important materials in the face of spreading microbial infections and increasing microbial resistance to antibiotics. Waschinski et al. recently reported the synthesis of poly(2-methyl-2-oxazolines) and poly(2-ethyl-2 oxazolines) with quarternary ammonium terminating groups [47]. The polymers were prepared via standard cationic ring-opening polymerisation and terminated using a series of N-alkyl-N,N-dimethyl amines as well as pyridine (Fig. 4). The study of antimicrobial properties toward *Staphylococcus aurea* showed that only poly(2-methyl-2-oxazoline)-based polymers containing alkyl ammonia functions with alkyl chains of twelve carbon atoms or longer exhibit antimicrobial activity.

262 Practical Applications in Biomedical Engineering

glycoconjugate vaccines [45,46].

[37].

the side chains in these core–shell nanogels offers a great potential for applications such as drug delivery systems or nanoreactors/carriers in biotechnology. Nanoparticles based on block copolymers of 2-ethyl-2-oxazoline and 2-phenyl-2-oxazolines were studied as promising tools for targeted delivery of drugs in the treatment of neuropsychiatric disorders

Polymers based on 2-oxazoline chemistry were also used in gene delivery. Polyethylene imine (PEI) is one of widely used vectors for nucleic acid delivery. It was found that linear PEI is more effective than branched PEI prepared by conventional polymerization of aziridine. Therefore, DNA complex with fully deacylated PEI can be used in gene therapy for lung diseases [38]. On the other hand, partially hydrolyzed poly(2-ethyl-2-oxazoline) showed lower cytotoxicity comparing to PEI and hasstill high transfection efficiency in the gene expression [39]. Linear PEI can be prepared by basic or acidic hydrolysis of poly(2 ethyl-2-oxazoline). Recently, microwave synthesizer induced hydrolysis of poly(2-ethyl-2 oxazoline) was described as powerful method for the preparation of "pharmagrade" PEI [40]. A block copolymer of a hyperbranched poly(ethylene glycol)-like core and linear polyethylenimine (HBP) was prepared by cationic polymerization of 2-ethyl-2-oxazoline initiated by hyperbranched initiator bearing p-chloromethylbenzoyl initiating groups and subsequent hydrolysis [41]. Linear PEI-bearing hyperbranched polycations (HBP) is able to form a complex with DNA with an average diameter of 50 nm that is stable for several weeks and showed resistance to DNAse I-mediated degradation. The 'inverted' block copolymers showed several orders higher transfection efficiency than block copolymers of PEG-b-LPEI *in vitro*. Comb-like copolymers suitable for DNA transfection consisted of linear poly(ethyleneimine)-graft-poly(2-ethyl-2-oxazoline) (LPEI-comb-PETOX) and (ethyleneimine)-graft-poly(ethyleneimine) (LPEI-comb-LPEI) [42]. Successful gene delivery systems with high transfection efficiency was prepared also by condensation of therapeutic DNA with different copolymers of poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) of variable grafting densities [43]. Amphiphilic poly[(propylene glycol)-block-(2-methyl-2-

oxazoine)] block copolymers were studied for gene transfer in skeletal muscle [44].

Poly(2-oxazolines) have found utilization also in vaccine technology. Recently, a novel glycoconjugates consisting of detoxified lipopolysaccharide of *Vibrio cholerae* O135, linear poly(2-oxazoline) copolymer containing free amino groups as a carrier, and an immunogenic protein, BSA, has been prepared. Multiple attachments of small effector antigen molecules to a suitable matrix is considered a novel technique for creating efficient

Antimicrobial polymers are becoming increasingly important materials in the face of spreading microbial infections and increasing microbial resistance to antibiotics. Waschinski et al. recently reported the synthesis of poly(2-methyl-2-oxazolines) and poly(2-ethyl-2 oxazolines) with quarternary ammonium terminating groups [47]. The polymers were prepared via standard cationic ring-opening polymerisation and terminated using a series of N-alkyl-N,N-dimethyl amines as well as pyridine (Fig. 4). The study of antimicrobial properties toward *Staphylococcus aurea* showed that only poly(2-methyl-2-oxazoline)-based  Polymers based on 2-oxazoline chemistry were used in biotechnology for different applications. Biofouling release properties are important for the development of coatings exhibiting marine biofouling resistance. It was found that copolymers prepared from perfluoroalkyl acrylates and poly(2-isopropenyl-2-oxazoline) exhibited unprecedented resistance to marine biofouling [48]. Another example is the development of an optical biosensor using an electrically controlled-release system for measurement of peroxide concentration. The response is based on the hydrogen bond of carboxylic group of currentsystem and 2-oxazoline group of a polymer complex [49]. Activating enzymes in organic solvents is of great interest in current biocatalysis. Amphiphilic and liposome-like polymers have exploitation also in membrane technology. Amphiphilic polymer tether consisted of linear hydrophilic poly(2-methyl-2-oxazoline) chains with defined length with surfacecoupling silane groups on one chain-end and hydrophobic n-alkyl chains on second end [50]. These polymers represent linear polymer spacers between supported lipid membranes and solid substrates. Lipid-tehtered poly(2-methyl-2-oxazoline) lipopolymer chains can be used as Langmuir monolayers at the air-water interface [51]. Recently, novel alpha,omegafunctionalized amphiphilic lipopolymers based on a proximal lipid moiety and hydrophilic poly(2-oxazoline) were used in asymmetric functionalized model lipid membranes for transmembrane transport and cell adhesion/recognition [52]. Amphiphilic co-networks composed of more hydrophilic poly(2-hydroxyethyl acrylate) and more hydrophobic, telechelic poly(2-ethyl-2-oxazoline) segments were prepared as free-standing membranes for entrapment of an enzyme molecule [53]. Amphiphilic miktoarm star copolymers containing cyclodextrin core can be used as nanocarriers in drug delivery but also in biocatalysis. Amphiphilic copolymers were consisted of poly(2-ethyl-2-oxazoline) and polylactide arms covalently linked to cyclodextrine core [54].

All reported applications of poly(2-oxazolines) confirm a great potential of 2-oxazoline based polymers as biomaterials. Detailed study of their biocompatibility and immunocompatibility is essential and necessary to understand their interactions with the living systems (biomolecules, cells, tissues etc.).
