10 wt% gelatin type B hydrogels

1. IMICWP 6.1In vitro immunologicalproperties of polyplexes and polymer membranesWP 6.2In vivoimmunological properties of polyplexes and polymermembranes

2. 10 wt% gelatin type B hydrogels - preapared by polymerization of methacrylamide modified gelatin using e-beam - treatment also enables the simultaneous sterilization of the scaffolds 96-well plates and 24-well plates for tissue culture (Nunc, Denmark) Cationic polymers: preliminary results Poly(dimethylaminoethyl-L-glutamine) = PDMAEG (V01) molecular weight: 44000 g/mol mass/charge: 235 g/mol (V03) molecular weight: 242000 g/mol mass/charge: 235 g/mol biocompatibility assay: no spontaneous hemolysis during 24 and 48 h incubation

3. In vitro screening of biocomatibility and immunocompatibilityof hydrogels • Hemolysis • Proliferation of mouse spleen cells • Proliferation of human peripheral blood mononuclear cells • Cultivation of mouse peritoneal cells • Growth of permanent cell lines • endothelial • fibroblast • monocyte/macrophage

4. OD 545 nm incubation Preincubation of hydrogel-coated plates Optimal protocol: 24 hours in PBS or serum-free medium 2 hours in medium containing 5 – 10% FCS for hemolysis: 3x wash (PBS) Hemolysis spontaneous(PBS) positive control(H2O)

5. 60,000 gelatin-treated 50,000 untreated 40,000 30,000 fluorescence (U) 20,000 10,000 0 ctrl Con A PWM PHA LPS 8 7 6 5 stimulation index 4 3 2 1 0 ctrl Con A PWM PHA LPS Proliferation of normal mouse spleen cells strains BALB/c, C57BL/6 Mitogens: Con A ........T cells PHA ...........T cells PWM ..........T and B cells LPS ............B cells Detection (viability) XTT ....... colorimetric assay Alamar blue ........fluorescence in vitro stimulation

6. Proliferation of peripheral blood mononuclear cells (PBMC) XTT detection 0.4 gelatin B 0.3 untreated 0.2 OD 0.1 0 ctrl Con A PWM PHA LPS in vitro stimulation

7. no inflammatory stimulus by the hydrogel Mouse peritoneal cells (PEC) detection of nitric oxide (NO) in supernatants as a measure of early inflammatory changes unstimulated PEC do not produce NO positive control: lipopolysaccharide (LPS), interferon g IFNg IFNg LPS

8. Permanent cell lines fibroblasts: mouse 3T3 endothelial: EAHY 926 monocyte/macrophage: mouse RAW 274.6 human THP-1 Gelatin B hydrogel did not induced any significant toxic damage to cells of different origin The growth of the cell was modulated due to the shape of the hydrogel surface - cells grow in clusters Number of cells seeded to hydrogel-coated plates was important

9. SUMMARY: Preincubation of hydrogels is important: optimal protocol was found Gelatin B does not induce spontaneous hemolysis of human ery Spontaneous proliferation of mouse spleen cells and human PBMC is not increased within 3 days of cultivation on hydrogel Gelatin B hydrogel does not induce NO production by mouse macrophages - no inflammatory stimulus Gelatin B does not induce any significant toxic damage to cells of different origin (normal and permanent cell lines) Growth and viability of cells (normal, permanent cell lines) is modulated due to hydrogel surface (clusters)

10. Biocompatibility and immunocompatibility of the gelatin B hydrogel Cytokine detection in supernatants FLOW CYTOMIX: IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNFα, TNF β samples: supernatants of mouse spleen cells, unstimulated and mitogen-stimulated; PEC mRNA quantfication (Riboquant) for selected cytoknes Other types of hydrogels could be studied using the same protocols

11. Cationic polymers for preparation of polyplexes, plasmids • in vitro assays (preliminary results) • hemolysis (control of biocompatibility) • proliferation of normal cells • polyplexes - in vitro: proliferation of normal cells, permanent cell lines • in vivo study: immunization (i.p, s.c.) • PEC - cellularity, phenotype, cytokines, heat shock proteins • antibody production (anti-DNA, anti-polymer, anti-polyplex) • genotype – Th1/Th2 prone (HLA-DR4, DR5)