Diels-Alder Hydrogels for Controlled Antibody Release: Correlation between Mesh Size and Release Rate.

Eight-armed PEG, molecular mass 10 kDa, was functionalized with furyl and maleimide groups, respectively; the obtained macromonomers were cross-linked via Diels-Alder chemistry. The mesh size (ξ) of the prepared hydrogels was determined by swelling studies, rheology, and low field NMR spectroscopy. The in vitro release of fluorescein isothiocyanate labeled dextrans (FDs) and bevacizumab was investigated. The average mesh size (ξavg) increased from 5.8 ± 0.1 nm to 56 ± 13 nm during degradation, as determined by swelling studies. The result of the rheological measurements (8.0 nm) matched the initial value of ξavg. Low field NMR spectroscopy enabled the determination of the mesh size distribution; the most abundant mesh size was found to be 9.2 nm. In combination with the hydrodynamic radius of the molecule (Rh), the time-dependent increase of ξavg was used to predict the release profiles of incorporated FDs applying an obstruction-scaling model. The predicted release profiles matched the experimentally determined release profiles when Rh < ξavg. However, significant deviations from the theoretical predictions were observed when Rh ≥ ξavg, most likely due to the statistical distribution of ξ in real polymer networks. The release profile of bevacizumab differed from those of equivalently sized FDs. The delayed release of bevacizumab was most likely a result of the globular structure and rigidity of the protein. The observed correlation between ξ and the release rate could facilitate the design of controlled release systems for antibodies.

Biodegradable hydrogels for time-controlled release of tethered peptides or proteins.

Tethering drug substances to a gel network is an effective way of controlling the release kinetics of hydrogel-based drug delivery systems. Here, we report on in situ forming, biodegradable hydrogels that allow for the covalent attachment of peptides or proteins. Hydrogels were prepared by step-growth polymerization of branched poly(ethylene glycol). The gel strength ranged from 1075 to 2435 Pa; the degradation time varied between 24 and 120 h. Fluorescence recovery after photobleaching showed that fluorescently labeled bovine serum albumin (FITC-BSA) was successfully bound to the gel network during gel formation. Within 168 h, the mobility of the tethered molecules gradually increased due to polymer degradation. Using FITC-BSA and lysozyme as model proteins, we showed the potential of the developed hydrogels for time-controlled release. The obtained release profiles had a sigmoidal shape and matched the degradation profile very well; protein release was complete after 96 h.

Diels-Alder hydrogels with enhanced stability: First step toward controlled release of bevacizumab.

Eight-armed PEG was functionalized with furyl and maleimide groups (8armPEG20k-Fur and 8armPEG20k-Mal); degradable hydrogels were obtained by cross-linking via Diels-Alder chemistry. To increase the stability to degradation, the macromonomers were modified by introducing a hydrophobic 6-aminohexanoic acid spacer between PEG and the reactive end-groups (8armPEG20k-Ahx-Fur and 8armPEG20k-Ahx-Mal). In an alternative approach, the number of reactive groups per macromonomer was increased by branching the terminal ends of eight-armed PEG with lysine (Lys) and Ahx residues (8armPEG20k-Lys-Ahx-Fur2 and 8armPEG20k-Lys-Ahx-Mal2). The hydrolytic resistance of the synthesized macromonomers was determined by UV spectroscopy; the obtained hydrogels were characterized by rheology and degradation studies. The degradation time of 5% (w/v) 8armPEG20k-Ahx hydrogels (28days) was twice as long as the degradation time of 5% (w/v) 8armPEG20k hydrogels (14days); this is explained by increased hydrolytic resistance of the maleimide group. Using dendritic 8armPEG20k-Lys-Ahx macromonomers substantially increased the stability of the resulting hydrogels; degradation of 5% (w/v) 8armPEG20k-Lys-Ahx hydrogels occurred after 34 weeks. 8armPEG20k hydrogels had the largest mesh size of all tested hydrogels, while hydrogels made from dendritic 8armPEG20k-Lys-Ahx macromonomers showed the smallest value. To evaluate their potential for the controlled release of therapeutic antibodies, the hydrogels were loaded with bevacizumab. The incorporated bevacizumab was released over 10 days (8armPEG20k) and 42days (8armPEG20k-Ahx), respectively; release from 8armPEG20k-Lys-Ahx hydrogels was not completed after 105 days. In summary, we believe that 8armPEG20k-Ahx or 8armPEG20k-Lys-Ahx hydrogels could serve as controlled release system for therapeutic antibodies such as bevacizumab.

Protein compatibility of selected cross-linking reactions for hydrogels.

The compatibility of selected cross-linking reactions with lysozyme is investigated. Michael-type additions of nucleophilic amino acids to maleimide, vinyl sulfone and acrylamide groups are detected by gel electrophoresis. The degree of modification depends on the polymer and the pH. Complete modification with more than five PEG chains is observed after incubation with mPEG5k-vinyl sulfone at pH 9, whereas 96% of the protein remains unmodified after incubation with mPEG5k-acrylamide at pH 4. Incubation with mPEG5k-thiol results in thiol-disulfide exchange reactions. Hydrogel preparation is simulated by using polymer mixtures. Protein modifications are detected, which may affect the protein structure, decrease activity and bioavailability, and increase the risk for immune responses.

New insights into the cross-linking and degradation mechanism of Diels-Alder hydrogels.

Eight-armed poly(ethylene glycol) was functionalized with furyl and maleimide groups. The two macromonomers were cross-linked by Diels-Alder (DA) reactions and the degradation behavior of the formed hydrogels was investigated. UV spectroscopy showed that maleimide groups were subject to ring-opening hydrolysis above pH 5.5, with the reaction rate depending on the pH and temperature. As a result of this, the gelation kinetics and stiffness of DA hydrogels were dependent on the temperature and the pH of the cross-linking medium, as demonstrated by rheological experiments. The gel time varied between 87.8 min (pH 3.0, 37 °C) and 374.7 min (pH 7.4, 20 °C). Values between 420 Pa (pH 9.0, 37 °C) and 3327 Pa (pH 3.0, 37 °C) were measured for the absolute value of the complex shear modulus. Hydrogel swelling and degradation were influenced by the same parameters. With increasing pH and temperature the degradation time was reduced from 98 days (pH 7.4, 20 °C) to 2 days (pH 7.4, 50 °C); no degradation was observed at pH 3.0 and 5.5. Molecular modeling studies of the DA and retro-Diels-Alder (rDA) moieties revealed that hydrogel degradation occurred by rDA reaction followed by OH--catalyzed ring-opening hydrolysis of maleimide groups to unreactive maleamic acid derivatives.

Cleavable carbamate linkers for controlled protein delivery from hydrogels.

The reversible attachment of proteins to polymers is one potential strategy to control protein release from hydrogels. In this study, we report the reversible attachment of lysozyme to poly(ethylene glycol) (PEG) by degradable carbamate linkers. Phenyl groups with different substituents were used to control the rate of carbamate hydrolysis and the resulting protein release. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed modification with 1-3 PEG chains per lysozyme molecule. Protein PEGylation and PEG chain elimination occurred without changes in secondary protein structure, as demonstrated by circular dichroism spectroscopy. The lytic activity of lysozyme was restored to 73.4±1.7%-92.5±1.2% during PEG chain elimination. Attached PEG chains were eliminated within 24h to 28days, depending on the used linker molecule. When formulated into hydrogels, a maximum of about 60% of the initial dose was released within 7days to 21days. Linker elimination occurs 'traceless', so that the protein is released in its native, unmodified form. Altogether, we believe that tethering proteins by degradable carbamate linkers is a promising strategy to control their release from hydrogels.

Investigation of the Diels-Alder reaction as a cross-linking mechanism for degradable poly(ethylene glycol) based hydrogels.

The Diels-Alder (DA) reaction was investigated as a cross-linking mechanism for poly(ethylene glycol) (PEG) based hydrogels. Two complementary macromonomers were synthesized by functionalizing star-shaped PEG with furyl and maleimide groups. Gel formation occurred in water at 37 °C; the gelation time ranged between 171 ± 25 min and 14 ± 1 min depending on the used hydrogel formulation. The complex shear modulus was dependent on the concentration, branching factor and molecular weight of the macromonomers; values between 2821 ± 1479 Pa and 37097 ± 6698 Pa were observed. Hydrogel swelling and degradation were influenced by the same parameters; the degradation time varied between a few days and several weeks. Gel dissolution was found to occur by retro-DA reaction and subsequent hydrolysis of maleimide groups. Calculations of the network mesh size revealed that the prepared hydrogels would be suitable for the controlled release of therapeutic proteins.