Poly(2-alkyl-2-oxazoline)-Heparin Hydrogels-Expanding the Physicochemical Parameter Space of Biohybrid Materials

Abstract

Poly(ethylene glycol) (PEG)-glycosaminoglycan (GAG) hydrogel networks are established as very versatile biomaterials. Herein, the synthetic gel component of the biohybrid materials is systematically varied by combining different poly(2-alkyl-2-oxazolines) (POx) with heparin applying a Michael-type addition crosslinking scheme: POx of gradated hydrophilicity and temperature-responsiveness provides polymer networks of distinctly different stiffness and swelling. Adjusting the mechanical properties and the GAG concentration of the gels to similar values allows for modulating the release of GAG-binding growth factors (VEGF165 and PDGF-BB) by the choice of the POx and its temperature-dependent conformation. Adsorption of fibronectin, growth of fibroblasts, and bacterial adhesion scale with the hydrophobicity of the gel-incorporated POx. In vitro hemocompatibility tests with freshly drawn human whole blood show advantages of POx-based gels compared to the PEG-based reference materials. Biohybrid POx hydrogels can therefore enable biomedical technologies requiring GAG-based materials with customized and switchable physicochemical characteristics.

Keywords: heparin; hydrogels; poly(2-alkyl-2-oxazolines); thermoresponsiveness.

Figure 1

Formation of the compared POx‐HEP and PEG‐HEP gel types. A) Chemical structures of the POx, PEG, and heparin units. B) Network structure resulting from the reactive conversion of POx (or PEG) dithiol with HEP‐maleimide by Michael‐type addition, the crosslinking degree can be adjusted by the maleimidation of heparin and by the concentration of the synthetic polymer. C) Biofunctional key properties of the developed materials.

Figure 2

Mechanical properties of POx‐HEP and PEG‐HEP gels. A) Storage moduli of gels with differing crosslinking degrees and constant solid content of 5% (determined at room temperature using rheometry). B) Swelling degrees Q (gel volume after equilibrium swelling compared to dried gel volume) of samples as in (A). C) Concentrations of both HEP and the synthetic polymer component in the respective swollen hydrogel type dependent on the crosslinking degree as determined by the HEP‐maleimide functionalization (all concentrations were determined in hydrogels with 10% solid content). D) Storage moduli of gels at constant heparin concentration of 4.5 × 10⁻³ m and adjusted hydrogel parameters as in Table 2 (determined at room temperature using rheometry).

Figure 3

Physicochemical properties of soluble POx and PEG, and of POx‐HEP and PEG‐HEP hydrogels. A) Retention time of dissolved POx and PEG in C18 reverse phase chromatography, using a linear AcN gradient. B) Calorimetric curves in the range of 20–60 °C for POx and PEG in aqueous solution (PBS; 1 mg mL⁻¹ polymer concentration adjusted in the solution and in the hydrogel; transition temperatures highlighted). C) Collapsed diameter of hydrogel disks upon transition from room temperature to 37 °C. D) Stiffness of the hydrogels (Young's Modulus corresponding to ≈3 × G’ assuming a Poison ratio of 0.5) measured at room temperature and at 37 °C by AFM (p < 0.001) of nonresponsive P(EtOx)‐HEP and temperature‐responsive P(iPrOx)‐ and P(nPrOx)‐HEP hydrogels.

Figure 4

Growth factor release from POx‐HEP and PEG‐HEP hydrogels. A) Percentage release of VEGF165 at room temperature. B) Percentage release of PDGF‐BB at room temperature. C) Ratio of VEGF165 release at 37 °C to release at room temperature in percent. D) Ratio of PDGF‐BB release at 37 °C to release at room temperature in percent.

Figure 5

Fibroblast culture and fibronectin adsorption on POx‐HEP hydrogels and PEG‐HEP hydrogels at 37 °C. A) Fibroblast (L929) adhesion after 5 h of culture on POx‐HEP and PEG‐HEP layers (cell count based on light microscopy). B) Adsorption of TAMRA‐labeled fibronectin on POx‐HEP and PEG‐HEP layers (quantification based on mean gray value).

Figure 6

Bacteria adhesion on POx‐HEP and PEG‐HEP hydrogels at 37 °C. A) Average number of adhering P. aeruginosa (strain PAO1) on POx‐HEP and PEG‐HEP layers ((10⁴ µm²)⁻¹). B) Average number of adhering S. aureus (strain ATCC12600) on POx‐HEP and PEG‐HEP layers ((10⁴ µm²)⁻¹). C) Representative fluorescent microscopy images of DAPI‐stained nucleic acid for S. aureus (strain ATCC12600) on glass, P(MeOx)‐, P(nPrOx)‐, and PEG‐HEP layers.

Figure 7

Hemocompatibility of POx‐HEP and PEG‐HEP materials as determined by human whole blood incubation in vitro. A) F1+2 concentration after incubation. B) C5a concentration after incubation. * indicates significant difference (p < 0.05) against P(nPrOx), P(EtOx), and Teflon AF in ANOVA on ranks (n  =  9).