Introduction: Diels-Alder (DA) cross-linked poly(ethylene glycol) (PEG) hydrogels have the potential to become a promising delivery system for therapeutic proteins. The aim of this study was to combine chemical cross-linking via DA “click chemistry” with physical interactions in a tandem approach to afford hydrogels with improved properties. For this purpose, hydrophobic spacers of different length were inserted between the PEG chains and the functional end-groups required for cross-linking (i.e., maleimide and furan). The hydrophobic interactions between the polymers were studied. The influence of hydrophobic interactions on the gel formation of DA hydrogels was investigated by rheology; the in vitro release of bevacizumab, which served as a model antibody, was determined.
Materials and Methods: Eight-armed PEG-amine with a molecular weight of 40 kDa (8armPEG40-NH2 ) was functionalized with furyl (8armPEG40k-Fur) and maleimide groups (8armPEG40k-Mal) as previously described[1],[2]. 8armPEG40-NH2 was reacted with Boc-6-aminohexanoic acid (8armPEG40k-C6-NH-Boc) or Boc-12-aminododecanoic acid (8armPEG40k-C12-NH-Boc). The obtained polymers were then functionalized with furyl (8armPEG40k-C6-Fur, 8armPEG40k-C12-Fur) and maleimide groups (8armPEG40k-C6-Mal, 8armPEG40k-C12-Mal).
The critical micelle concentration of the polymers was determined by fluorescence spectroscopy using pyrene as a probe molecule (λex = 339 nm, λem = 390 nm). The viscosity (shear rate from 0.10 to 10 s-1) was determined on a TA Instruments Discovery HR-2 rheometer. For hydrogel preparation, both polymer components (e.g., 8armPEG40k-Fur/-Mal) were dissolved in purified water. Gel point was determined at 37 °C as previously described [1][2]. In vitro release of bevacizumab was studied as previously described[2].
Results and Discussion: Hydrophobic interactions between the polymers were probed using pyrene fluorescence. In contrast to 8armPEG40k-OH, a sharp increase of pyrene fluorescence was observed for 8armPEG40k-C6-NH-Boc and 8armPEG40k-C12-NH-Boc above a certain concentration. This suggests the formation of micelles. Regarding viscosity, no differences were observed at low polymer concentrations. At higher concentrations, the association of polymers due to hydrophobic interactions led to an increased inner friction of the liquid. As a consequence, 20% aqueous solutions of 8armPEG40k-C6-NH-Boc and 8armPEG40k-C12-NH-Boc showed a much higher viscosity than a solution of 8armPEG40k-OH. Due to these hydrophobic interactions, gel formation occurred considerably faster for C6 and C12 modified hydrogels (Figure 1A). Interestingly, no effect was observed when only one of the two components was modified. From this observation it can be concluded that interactions between macromonomers are the underlying mechanism for accelerated gel formation. Release of bevacizumab from 8armPEG40k hydrogels was completed after only 15 days (Figure 1B). In contrast to this, release of bevacizumab was delayed when using C6 or C12 modified hydrogels; the release was completed after 30 and 60 days, respectively.
Conclusion: Hydrophobic interactions between the polymers can accelerate gel formation and delay the release of antibodies from DA hydrogels. As a consequence, modification of polymers with hydrophobic spacers represents a powerful tool to improve the characteristics of DA hydrogels and bring them closer to pharmaceutical application.
References:
[1] S. Kirchhof, F.P. Brandl, N. Hammer, A.M. Goepferich, Investigation of the Diels–Alder reaction as a cross-linking mechanism for degradable poly(ethylene glycol) based hydrogels, J. Mater. Chem. B. 1 (2013) 4855–4864. doi:10.1039/c3tb20831a.
[2] S. Kirchhof, M. Gregoritza, V. Messmann, N. Hammer, A.M. Goepferich, F.P. Brandl, Diels-Alder hydrogels with enhanced stability: First step toward controlled release of bevacizumab, Eur. J. Pharm. Biopharm. (2015) 217-225. doi: http://dx.doi.org/10.1016/j.ejpb.2015.07.024.