(W1330-01-03) Deciphering the Role of Hydrogel Network Topology in Programmably Degradable Hydrogels

Purpose: Hydrogel network topology provides critical insights into the structure and chain arrangement of injectable hydrogels, pivotal for the controlled release of biomolecules. We present a hydrogel system that utilizes oxanorbornadiene (OND)-modified dextran in conjunction with multivalent polyethylene glycol thiols (PEG-SH), exploiting the OND's reactivity with thiol nucleophiles to initiate bond cleavage with variable half-lives. This facilitates a programmable release of virus-like particles (VLPs), nano-sized vaccine candidates, with diverse release patterns such as burst, linear, positive exponential, and delayed release, spanning up to two weeks. While the impact of OND chemistry on material properties has been explored, the role of hydrogel network topology in the degradation process and subsequent cargo release is yet to be fully elucidated. The degree of crosslinking (DC) is a tunable factor, dependent on the substitution degree of OND on dextran (ranging from 7% to 32%) and the valence of PEG-SH (from 2-arm to 8-arm), which together influence the release kinetics. The architecture of the polymer, considering both dextran size (5 kDa to 40 kDa) and PEG chain branching patterns (star-like or branched), also plays a crucial role. The insights obtained from this study, along with previous research, are instrumental in devising mathematical models to quantitatively describe the release behavior from degradable hydrogels.

Methods: Three classes of OND linkages with various fragmentation half-lives were synthesized by [4+2] cycloaddition of furans with alkynes. These ONDs were connected to the dextran side-chains via esterification reactions. Synthetic procedures were based on previous publications. The degree of substitution (DS) of ONDs on dextran was analyzed via ¹H NMR spectroscopy. In addition, the free SH% of the commercially available multivalent PEG-thiols was quantified by a standard Ellman's assay. Then, for fabricating hydrogels entrapping VLP cargo, dextran-OND and PEG-SH samples were dissolved separately in VLP-containing PBS buffer (1X, pH 6.0) and mixed in a 1:1 ratio of OND and SH, resulting in gels each with 10 wt% of polymers and 100 μL size. The gelation was performed at either room temperature or on ice and confirmed by an inversion test of the vials. After gelation, 1 mL of blank PBS buffer (1X, pH 7.4) was added above the gel; the amount of cargo released in vitro was determined by measuring the fluorescence of the supernatant.

Results: OND linkers with differing rDA rates were attached to dextran at a DS of 7 to 32%. Ellman's assay reported the SH% of PEG-2-SH, PEG-4-SH, PEG-6-SH, and PEG-8-SH as 90-95%. The injectability of the gel materials was validated using a clinically approved syringe-to-syringe transfer technique followed by injection through a needle. Certain formulations were cooled to 4°C to slow down the gelation process to avoid clogging the needle. Once the gels were formed, they began to degrade spontaneously in a programmable manner by applying different types and combinations of OND linkers, allowing the entrapped VLPs to release according to pre-set kinetics. The in vitro release profiles included first-order release with an initial lag over 1.5, 3, and 7 days, linear release over 2.5 and 8 days, positive-exponential (slow then faster) release over 10 days, and delayed release after 1.5, 5.5, 8.5, and 12 days. The delayed-burst pattern was achieved due to aqueous hydrolysis of the ester bonds connecting ONDs and the dextran backbone, while other release patterns relied on OND rDA fragmentation triggered by reacting with thiol nucleophiles. In addition, our initial findings indicate that higher degree of crosslinking level is associated with more extended release periods, with the longest release exceeding four weeks using a 32% OND substitution and 8-arm PEG-SH.

Conclusion: The dextran-OND/PEG-SH hydrogels were formulated for convenient injectability, and the release of entrapped VLPs was found to be programmable with a wide range of profiles, including first-order release with an initial lag, linear, positive-exponential, and delayed-burst release over a two-week period. In addition, longer period of release was achieved by increasing the degree of crosslinking in the hydrogel system.

References: (1) D.J. Irvine et al. Advanced Drug Delivery Reviews 158 (2020) 91–115.
(2) Y. Sun et al. Polymer Testing 81 (2020) 106283.
(3) M.D. Johnson et al. ACS Appl. Bio Mater. 2020, 3, 4779−4788
(4) C.J. Higginson et al. J. Am. Chem. Soc. 2015, 137, 15, 4984–4987
(5) S. Das et al. Biomacromolecules 2020, 21, 6, 2432–2439.

Acknowledgements: We thank Dr. Soumen Das (Georgia Tech, Finn group) for the Qβ virus-like particles.

Figure 1. Schematic description of the hydrogel design and OND chemistry. (a) Dextran-OND/PEG-SH hydrogels. (b) Principle of OND chemistry and synthesis results for various OND linkers (fragmentation half-lives were tested in chloroform at room temperature).

Figure 2. In vitro VLP release profiles. (a) Illustration of the entrapped cargo release mechanism. (b)-(e) Four distinct VLP release patterns (first-order with initial lag, linear, positive-exponential, and delayed-burst) at 37°C. All gels in c-e used the PEG-4-SH component. Data are represented as mean ± S.D. (n = 3).