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

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,

and genetic factors, TH1, TH2, TH17 effector and/or memory cells and Tregs´ are generated by immune responses. Functionally-polarized CD4+ T cell subsets have been identified based on their exclusive patterns of various cytokine secretions. As a signature cytokine, TH1 cells selectively produce large amounts of interferon-gamma (IFN-γ). TH2 cells selectively produce IL-4, next characteristic TH2 cytokines are IL-5 and IL-13. The population of TH17 cells expresses high levels of IL-17A, followed by IL-17AF , IL-17F, IL-21 and IL-22. Unique cytokine expression for Tregs´ represents TGF-β and IL-10. The TH1/TH2/TH17/Treg paradigm provides a useful model system for investigating the cellular and molecular mechanisms that mediate protective as well as harmful immune responses.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 273

results at immediately after the PETOX100 treatment. When compared to the time-kinetics of untreated cell control, the significance was confirmed only for TNF-α after 24 hours of exposure. The observed secretion levels of cytokines were significantly (10-100 times, depending on cytokine) lower than the cytokine levels observed with classical bacterial inducer, such as LPS. Based on these results, it could be assumed that both polymers, AEOX10 and PETOX100, represent bioavailable materials with immunobiological properties without strong undesirable inflammatory effectiveness with respect to their capacity to

Apart from enzyme linked immunosorbent assay (ELISA) the more sensitive bead-based multiplex immunoassay for the flow cytometer and the enzyme linked immunosorbent spot assay (ELISpot) are frequently used for quantitative follow up of cytokine secretion by APC

**Figure 15.** Scheme of ELISpot assay for quantification of cytokine one cell-based production.

Ultra-sensitive ELISPOT assays (limit of detection less than 1 in 100,000) can provide accurate measurements of antigen-specific cell immune responses and cytokine release. ELISPOT assays can measure both the magnitude and the quality of the response, thus providing more information about the samples in comparison with other methods for determination of cytokines e.g. ELISA. The methodology of ELISpot (developed by Czerkinsky et al. [73]) is rational up-grade of previously used plaque forming cells (PFC) technique with combination of enzyme immunoassay. ELISPOT assay was originally

induce pro-inflammatory cytokines [70].

cells (Fig. 15).

**Figure 14.** APC-derived cytokines and TH cell polarization. Pattern-Recognition Receptors (PRR): Dectin-1, Dectin-2, mannose receptor (MR), Toll-like receptor, DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), Fc receptor (FcR) - binding specificity for a Fc region of immunoglobulins, complement receptor (CR) - receptor of the complement system

It has been documented that the surface with neutral hydrophilic surface properties was found to show the greatest inhibition of cell adhesion and IL-1, IL-6 and TNF-α release [72]. Recently, Brodbeck et al. have demonstrated that the surface chemistry (hydrophilicity and surface charge) of the biomaterial modulates the expression of anti- and pro-inflammatory TH1 and TH2 cytokines [71]. The influence of the PETOX100 and AEOX10 treatments of the mouse lymphoid line P388.D1 [clone 3124] on the changes of the IL-1 α, IL-6 and TNF-α was evaluated. The exposition of P388.D1 macrophages with both AEOX10 and PETOX100 resulted in significant enhancement of IL-1α IL-6 and TNF-α compared to the baseline results at immediately after the PETOX100 treatment. When compared to the time-kinetics of untreated cell control, the significance was confirmed only for TNF-α after 24 hours of exposure. The observed secretion levels of cytokines were significantly (10-100 times, depending on cytokine) lower than the cytokine levels observed with classical bacterial inducer, such as LPS. Based on these results, it could be assumed that both polymers, AEOX10 and PETOX100, represent bioavailable materials with immunobiological properties without strong undesirable inflammatory effectiveness with respect to their capacity to induce pro-inflammatory cytokines [70].

272 Practical Applications in Biomedical Engineering

and genetic factors, TH1, TH2, TH17 effector and/or memory cells and Tregs´ are generated by immune responses. Functionally-polarized CD4+ T cell subsets have been identified based on their exclusive patterns of various cytokine secretions. As a signature cytokine, TH1 cells selectively produce large amounts of interferon-gamma (IFN-γ). TH2 cells selectively produce IL-4, next characteristic TH2 cytokines are IL-5 and IL-13. The population of TH17 cells expresses high levels of IL-17A, followed by IL-17AF , IL-17F, IL-21 and IL-22. Unique cytokine expression for Tregs´ represents TGF-β and IL-10. The TH1/TH2/TH17/Treg paradigm provides a useful model system for investigating the cellular and molecular

mechanisms that mediate protective as well as harmful immune responses.

**Figure 14.** APC-derived cytokines and TH cell polarization. Pattern-Recognition Receptors (PRR): Dectin-1, Dectin-2, mannose receptor (MR), Toll-like receptor, DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), Fc receptor (FcR) - binding specificity for a Fc region of immunoglobulins, complement receptor (CR) - receptor of the complement system

It has been documented that the surface with neutral hydrophilic surface properties was found to show the greatest inhibition of cell adhesion and IL-1, IL-6 and TNF-α release [72]. Recently, Brodbeck et al. have demonstrated that the surface chemistry (hydrophilicity and surface charge) of the biomaterial modulates the expression of anti- and pro-inflammatory TH1 and TH2 cytokines [71]. The influence of the PETOX100 and AEOX10 treatments of the mouse lymphoid line P388.D1 [clone 3124] on the changes of the IL-1 α, IL-6 and TNF-α was evaluated. The exposition of P388.D1 macrophages with both AEOX10 and PETOX100 resulted in significant enhancement of IL-1α IL-6 and TNF-α compared to the baseline Apart from enzyme linked immunosorbent assay (ELISA) the more sensitive bead-based multiplex immunoassay for the flow cytometer and the enzyme linked immunosorbent spot assay (ELISpot) are frequently used for quantitative follow up of cytokine secretion by APC cells (Fig. 15).

**Figure 15.** Scheme of ELISpot assay for quantification of cytokine one cell-based production.

Ultra-sensitive ELISPOT assays (limit of detection less than 1 in 100,000) can provide accurate measurements of antigen-specific cell immune responses and cytokine release. ELISPOT assays can measure both the magnitude and the quality of the response, thus providing more information about the samples in comparison with other methods for determination of cytokines e.g. ELISA. The methodology of ELISpot (developed by Czerkinsky et al. [73]) is rational up-grade of previously used plaque forming cells (PFC) technique with combination of enzyme immunoassay. ELISPOT assay was originally developed to enumerate B lymphocytes secreting antigen-specific antibodies. Nowadays, this technique has been adapted also for the identification and enumeration of cytokineproducing cells at the single cell level. ELISPOT assay allows visualization of the secretory product of individual activated or stimulated cells. Each spot represents an antigen-induced response of single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of cytokine, antibody etc.) and quantitative (number of responding cells) information [74].

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 275

makes it possible to distinguish up to 20 bead sets in one fluorescent channel. Reporter fluorochrome PE bound to strepavidin, binds to the biotin conjugate, emits at 578 nm and is

The *in vivo* applications of polymers require investigating the intracellular metabolic pathways of distribution of polymer in cells and tissues. This can be examined by employing labeled systems. One way is the utilization of fluorescent probes for the visualization through labelling or staining. Labelled polymeric materials can be used for monitoring of cell uptake of desired materials using fluorescence microscopy or confocal laser scanning microscopy (CLSM). CLSM is a technique for obtaining high-resolution optical images with high depth resolution. This technique is widely used in cell biology, genetics, microbiology or developmental biology. Clinically, CLSM is an important method for the evaluation of different diseases, in endoscopic procedures and also in pharmaceutics for imaging the drug distribution. In the field of biocompatible polymers, this technique can be employed for the imaging of polymer uptake and distribution in cell, tissues or whole organs. For example, MCF-7 cancer cell uptake of coumarin-6-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles was visualized using CLSM technique by overlaying the images obtained through the FITC filter and the PI filter [75]. The described system was used for sustainable release of Taxol in the treatment of cancer. CLSM was used for the examination of cell uptake and gene release from the ternary polyplexes of the plasmid DNA with B-PEI and pH responsive diblock copolymer poly(2-ethyl-2-oxazoline)-*block*-poly(methacrylic acid) (PETOX-*b*-PMAA) [76]. Time-dependent observations in three channels (green fluorescence of LysoTracker, blue fluorescence of 4',6-diamidino-2-phenylindole –DAPI andred channel of Cyanin 5.5) revealed the cell uptake process of the polyplex started after 3 hours and accelerated after 6 hours of incubation. Nanoparticles composed of statistical copolymers prepared by cationic copolymerization 2-ethyl-2-oxazoline and 2-decenyl-2 oxazoline were prepared for encapsulation of various biomolecules [77]. Their cellular uptake of the prepared nanoparticles labeled with fluorescein was monitored with CLSM. Confocal micrographs revealed a high loading of mouse connective tissue fibroblasts with nanoparticles although no transfection agents were used to promote particle uptake. The overlay of the transmission and the fluorescence image shows the uniform distribution of the particles in intracellular compartments after endocytosis, which is the common way for the nanoparticle uptake. The results obtained by confocal microscopy were confirmed by flow cytometry. It was showed that the particles did not show toxic effects during the time

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. 17). The confocal micrographs supported the hypothesis of the polymer accumulation in specific organelles as observed also in the case of fluorescence

detected in the FL-2 channel, allowing the quantification of the cytokine.

**5.** *In vitro* **and** *in vivo* **visualization methods** 

of incubation.

microscopy measurements.

**Figure 16.** Example of FlowCytomix™ simultaneous quantification of Th1, Th2, Th17 and Treg cytokines in mice splenocytes (unpublished data)

Bead-based multiplex immunoassays for the immunocytometry are applied for the multiple detection of cytokines, chemokines, growth factors and related proteins. The FlowCytomix™ Multiple Analyte Detection System is designed to simultaneously quantify up to 20 protein targets. The methodology significantly reduces sample volumes and time compared to traditional ELISA and Western blot techniques. The FlowCytomix technology is based on the sandwich immunoassay technology where microspheres are used as a solid phase instead of a 96-well plate (Fig. 16). Within two bead size populations 4 & 5 μm latex beads coated with specific antibodies are multiple bead subsets, differentiated by varying intensities of an internal fluorescent dye Starfire Red. This dye can be excited at 488 nm by an Argon, He-Ne, or UV laser, and emits at 690 nm (the far red spectrum), which is detected in the FL-3/FL-4 channel. The combination of the two different bead sizes and different internal dye intensities makes it possible to distinguish up to 20 bead sets in one fluorescent channel. Reporter fluorochrome PE bound to strepavidin, binds to the biotin conjugate, emits at 578 nm and is detected in the FL-2 channel, allowing the quantification of the cytokine.

#### **5.** *In vitro* **and** *in vivo* **visualization methods**

274 Practical Applications in Biomedical Engineering

developed to enumerate B lymphocytes secreting antigen-specific antibodies. Nowadays, this technique has been adapted also for the identification and enumeration of cytokineproducing cells at the single cell level. ELISPOT assay allows visualization of the secretory product of individual activated or stimulated cells. Each spot represents an antigen-induced response of single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of cytokine, antibody etc.) and quantitative (number of responding cells) information [74].

**Figure 16.** Example of FlowCytomix™ simultaneous quantification of Th1, Th2, Th17 and Treg

Bead-based multiplex immunoassays for the immunocytometry are applied for the multiple detection of cytokines, chemokines, growth factors and related proteins. The FlowCytomix™ Multiple Analyte Detection System is designed to simultaneously quantify up to 20 protein targets. The methodology significantly reduces sample volumes and time compared to traditional ELISA and Western blot techniques. The FlowCytomix technology is based on the sandwich immunoassay technology where microspheres are used as a solid phase instead of a 96-well plate (Fig. 16). Within two bead size populations 4 & 5 μm latex beads coated with specific antibodies are multiple bead subsets, differentiated by varying intensities of an internal fluorescent dye Starfire Red. This dye can be excited at 488 nm by an Argon, He-Ne, or UV laser, and emits at 690 nm (the far red spectrum), which is detected in the FL-3/FL-4 channel. The combination of the two different bead sizes and different internal dye intensities

cytokines in mice splenocytes (unpublished data)

The *in vivo* applications of polymers require investigating the intracellular metabolic pathways of distribution of polymer in cells and tissues. This can be examined by employing labeled systems. One way is the utilization of fluorescent probes for the visualization through labelling or staining. Labelled polymeric materials can be used for monitoring of cell uptake of desired materials using fluorescence microscopy or confocal laser scanning microscopy (CLSM). CLSM is a technique for obtaining high-resolution optical images with high depth resolution. This technique is widely used in cell biology, genetics, microbiology or developmental biology. Clinically, CLSM is an important method for the evaluation of different diseases, in endoscopic procedures and also in pharmaceutics for imaging the drug distribution. In the field of biocompatible polymers, this technique can be employed for the imaging of polymer uptake and distribution in cell, tissues or whole organs. For example, MCF-7 cancer cell uptake of coumarin-6-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles was visualized using CLSM technique by overlaying the images obtained through the FITC filter and the PI filter [75]. The described system was used for sustainable release of Taxol in the treatment of cancer. CLSM was used for the examination of cell uptake and gene release from the ternary polyplexes of the plasmid DNA with B-PEI and pH responsive diblock copolymer poly(2-ethyl-2-oxazoline)-*block*-poly(methacrylic acid) (PETOX-*b*-PMAA) [76]. Time-dependent observations in three channels (green fluorescence of LysoTracker, blue fluorescence of 4',6-diamidino-2-phenylindole –DAPI andred channel of Cyanin 5.5) revealed the cell uptake process of the polyplex started after 3 hours and accelerated after 6 hours of incubation. Nanoparticles composed of statistical copolymers prepared by cationic copolymerization 2-ethyl-2-oxazoline and 2-decenyl-2 oxazoline were prepared for encapsulation of various biomolecules [77]. Their cellular uptake of the prepared nanoparticles labeled with fluorescein was monitored with CLSM. Confocal micrographs revealed a high loading of mouse connective tissue fibroblasts with nanoparticles although no transfection agents were used to promote particle uptake. The overlay of the transmission and the fluorescence image shows the uniform distribution of the particles in intracellular compartments after endocytosis, which is the common way for the nanoparticle uptake. The results obtained by confocal microscopy were confirmed by flow cytometry. It was showed that the particles did not show toxic effects during the time of incubation.

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. 17). The confocal micrographs supported the hypothesis of the polymer accumulation in specific organelles as observed also in the case of fluorescence microscopy measurements.

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 277

**PETOX-py**

n

CH2 N CH2 CH2 OH

CH2CH3

O C

experiments on CLSM. Studied polymers were labeled with fluorescent probe. For these purposes, a pyrene moiety was introduced into the macromolecule of poly(2-ethyl-2 oxazoline) (PETOX-py) using 1-(bromoacetyl)pyrene as initiator of cationic polymerization

Pyrene-based probes are in a class of hydrophobic dyes and can be used not only for the visualization and analysis of polymer concentrations within cells but also for the investigation of the environment polarity and the formation of agglomerates. 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

**Figure 19.** Fluorescence micrographs of rat fibroblasts loaded with pyrene-labeled PETOX for 24 hours in DMEM medium in the concentration equal to 5 mol/l (a) and a detail of polymer uptake in cells (b).

CH2Br

O

**Figure 18.** Synhesis of pyrene-labeled poly(2-ethyl-2-oxazoline) (PETOX-py).

in the cells in specific organelles, most probably in peroxisomes [63].

(b) (a)

O

of 2-ethyl-2-oxazoline (Fig. 18).

N O

CH2CH3

**Figure 17.** The cuts of the mouse macrophages treated loaded with pyrene-labeled PETOX conducted on the Carl-Zeiss confocal laser-scanning microscopy.

Similarly, cell uptake of polymers can be examined by fluorescence microscopy. A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence using two filters. An illumination (or excitation) filter ensures almost monochromatic excitation of a sample and a second emission (or detection) filter which ensures none of the excitation light source reaches the detector. The technique of fluorescence microscopy has become an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorochromes has made it possible to identify cells and submicroscopic cellular components with a high degree of specificity amid non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the presence of a single molecule. Different probes can simultaneously identify several target molecules through the use of multiple fluorescence labeling. Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of specific specimen features, the detection of fluorescing molecules below such limits is readily achieved. Recently, infrared microscopy became very powerful method for optical visualization in the fields of biology, biochemistry, microbiology or genetics. Fluorescence microscopy was used for the visualization of cellular uptake of 2-oxazoline polymers. The principle was related to experiments on CLSM. Studied polymers were labeled with fluorescent probe. For these purposes, a pyrene moiety was introduced into the macromolecule of poly(2-ethyl-2 oxazoline) (PETOX-py) using 1-(bromoacetyl)pyrene as initiator of cationic polymerization of 2-ethyl-2-oxazoline (Fig. 18).

**Figure 18.** Synhesis of pyrene-labeled poly(2-ethyl-2-oxazoline) (PETOX-py).

276 Practical Applications in Biomedical Engineering

on the Carl-Zeiss confocal laser-scanning microscopy.

**Figure 17.** The cuts of the mouse macrophages treated loaded with pyrene-labeled PETOX conducted

Similarly, cell uptake of polymers can be examined by fluorescence microscopy. A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence using two filters. An illumination (or excitation) filter ensures almost monochromatic excitation of a sample and a second emission (or detection) filter which ensures none of the excitation light source reaches the detector. The technique of fluorescence microscopy has become an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorochromes has made it possible to identify cells and submicroscopic cellular components with a high degree of specificity amid non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the presence of a single molecule. Different probes can simultaneously identify several target molecules through the use of multiple fluorescence labeling. Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of specific specimen features, the detection of fluorescing molecules below such limits is readily achieved. Recently, infrared microscopy became very powerful method for optical visualization in the fields of biology, biochemistry, microbiology or genetics. Fluorescence microscopy was used for the visualization of cellular uptake of 2-oxazoline polymers. The principle was related to Pyrene-based probes are in a class of hydrophobic dyes and can be used not only for the visualization and analysis of polymer concentrations within cells but also for the investigation of the environment polarity and the formation of agglomerates. 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].

**Figure 19.** Fluorescence micrographs of rat fibroblasts loaded with pyrene-labeled PETOX for 24 hours in DMEM medium in the concentration equal to 5 mol/l (a) and a detail of polymer uptake in cells (b).

Peroxisomes are small membrane-enclosed organelles that contain enzymes involved in a variety of metabolic reactions, including several aspects of energy metabolism. Peroxisomes contain at least 50 different enzymes, which are involved in a variety of biochemical pathways in different types of cells. Peroxisomes were defined as organelles that carry out oxidation reactions leading to the production of hydrogen peroxide. A variety of substrates are broken down by such oxidative reactions in peroxisomes, including uric acid, amino acids, and fatty acids [78].

Biocompatibility and Immunocompatibility Assessment of Poly(2-Oxazolines) 279

**Author details** 

**Acknowledgement** 

the acronym GLYCOMED.

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*Institute of Chemistry, Centre of Excellence GLYCOMED, Slovak Academy of Sciences, Bratislava,* 

The authors appreciate the financial support of the Slovak Scientifical Agency VEGA in the Project nr. 2/0151/12 and the Centre of Excellence of the Slovak Academy of Sciences with

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Biodistribution, targeting and excretion can also be investigated by radiolabeled poly(2 alkyl-2-oxazolines). Authors used 111In-labeled polymers to study the distribution and excretion of such polymers in mice [79]. Similarly, 125I-labeled 2-oxazoline-based copolymers were examined after their intravenous administration to mice, and their biodistribution was assessed [80]. Beside the described visualization techniques, other modern methods such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) or tomography are available for the analysis of biodistribution.
