Synthesis and characterization of enzymatically degradable PEG-based peptide-containing hydrogels.

Biodegradable hydrogels were synthesized by the click reaction of 4-arm azido-terminated PEG differing in molecular weight (2 100 and 8 800) and two alkyne-terminated peptides: [alkyne]-GFLGK-[alkyne] and ([alkyne]-GFLG)2K. The physical properties of in situ formed hydrogels were examined. The hydrogels were highly elastic as determined by rheological and micro-rheological studies. Swelling degree and enzymatic degradation by papain were dependent on the molecular weight of the PEG, but not the peptide. For PEG8800-based hydrogels, time-course analysis of degradation showed that the molecular weight of the soluble fraction quickly reached the PEG precursor value. These findings may guide future design of hydrogels with controllable mechanical properties and enzymatic degradability. Keywords: biodegradable, click chemistry, hydrogels, peptide crosslinks, poly(ethylene glycol) Introduction Hydrogels were the first biomaterials designed for clinical use.[1] They have found applications in numerous areas, including tissue engineering, drug delivery, sensing, diagnostics, and microfluidics.[2,3] Several factors need to be considered when designing biocompatible hydrogel biomaterials including controlled chemistry for the formation of reproducible, reversible 3D networks with precisely defined structures, and biodegradability. Living radical polymerization has been used for the control of the molecular weight distribution of the primary chains of the network;[4,5] copper catalyzed[6,7] or copper-free [8] Huisgen cycloaddition of azides and alkynes (click chemistry) and Michael addition[9] have been used to control crosslinking and/or attachment of biologically active molecules. The combination of synthetic polymers with sequences/chains of natural polymers is a promising route for the design and synthesis of enzymatically degradable hybrid polymers/hydrogels.[2,10] To render a hybrid polymer/hydrogel degradable, the structure of the degradable sequences should match the active site of respective enzyme(s); oligopeptide sequences have been frequently used as degradable crosslinks in hydrogels. Hydrogels, containing oligopeptide crosslinks susceptible to chymotrypsin-catalyzed hydrolysis were synthesized by crosslinking N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing reactive side-chains (terminated in p-nitrophenoxy groups) with oligopeptide-containing diamines.[10] The degradability of hydrogels was dependent on the length and detailed structure of the oligopeptide sequence and on the equilibrium degree of swelling (network density); the higher the degree of swelling, the faster the rate of degradation. The degree of swelling also has an impact on surface versus bulk degradation of the hydrogel. If the enzyme cannot diffuse into the hydrogel interior, only surface degradation takes place.[11] Similar HPMA-based hydrogels degradable by cathepsin B, a lysosomal thiol proteinase, have also been evaluated.[12] In further experiments, HPMA-based hydrogels with degradable crosslinks were shown to release FITC-dextran and daunomycin (covalently bound via oligopeptide spacer) during incubation with a mixture of lysosomal enzymes (Tritosomes) or chymotrypsin.[13] Recently, Plunkett et al. studied acrylamide-based hydrogels containing oligopeptides degradable by chymotrypsin.[14] Poly(ethylene glycol) (PEG) is a hydrophilic polymer that has been used in several clinical applications. PEG-based hydrogels have been extensively studied. Hubbell’s laboratory used multiarm-PEG and Michael-type addition to synthesize extracellular matrix mimicking hydrogels degradable by cell-excreted matrix metalloproteinases.[9,15] PEG-based hydrogels containing Schiff base linkages were designed for low molecular weight (doxorubicin) drug delivery.[16] Azide-alkyne click chemistry was also used for the synthesis of PEG-based hydrogels.[6,17] Hawker and coworkers[6] synthesized hydrogels by reacting a tetraazide-modified tetraethylene glycol with telechelic alkyne terminated PEGs of varying molecular weight. Yang and coworkers employed 4-arm alkyne terminated PEG and crosslinked it with a series of RGD containing dialkyne-peptides. However, both alkyne groups were attached to the N-terminal lysine. Consequently, the side-chains were degradable, but the hydrogel crosslinked backbone remained stable.[17] In this study we synthesized two 4-arm azide-terminated PEGs differing in molecular weight (2 100 and 8 800) and two alkyne-terminated peptides, [alkyne]-GFLGK-[alkyne] (GFLG1) and ([alkyne]-GFLG)2K (GFLG2). The hydrogels were synthesized by Huisgen cycloaddition (click chemistry) of PEG azides with peptide alkynes and contained enzymatically degradable crosslinks. The kinetics of the crosslinking reaction was monitored by dynamic rheology. The hydrogels were characterized by the equilibrium degree of swelling, their morphology, and bulk and micro-rheology. The relationship between the structure of the hydrogels and their degradability by papain, a thiol proteinase, was also evaluated.