Injectable Granular Hydrogels Enable Avidity-Controlled Biotherapeutic Delivery

Introduction: Biotherapeutics exhibit excellent biocompatibility and target specificity, and are used to treat a range of diseases from cancer to autoimmune dysfunction. Affinity-based drug delivery systems overcome challenges of rapid burst release and afford prolonged presentation of biotherapeutics by enabling the selective retention of biomolecules in hydrogel scaffolds. Here, we develop a generalizable approach to achieve sustained and local biomolecule delivery (Fig 1A) made possible by injectable granular hydrogels with a favorable ‘host’ capacity. Interaction of these hydrogels with ‘guest’-modified proteins allows for sustained release through synthetically-tunable hydrogel avidity (i.e., the cumulative strength of multiple physical bonds acting cooperatively). We sought to enable local biomolecule release across different time scales by tuning the avidity of the biomolecule-hydrogel interaction via synthetically introduced guest-host bonds.

Materials and

Methods: β-Cyclodextrin and dextran were methacrylated via esterification with glycidyl methacrylate and co-polymerized (5mM LAP, 10mW/cm2) to yield methacrylated β-cyclodextrin (MeCD) and dextran (DexMA) hydrogels (G’~15kPa) with a high host concentration. MeCD/DexMA bulk hydrogels (5%w/v DexMA, 10%w/v MeCD) were processed into microgels (34.0±16.7µm) by extrusion fragmentation and jammed via centrifugation to form injectable granular hydrogels, the physical properties of which were assessed by rheology. Bovine serum albumin (BSA, a model biomolecule) and cytokines were conjugated to supramolecular guests (adamantane, Ad) via EDC chemistry in near-quantitative yield (1-10 molar ratio of Ad:BSA); guest–host affinity was assessed by isothermal calorimetry, while avidity was assessed by surface plasmon resonance. Protein release was monitored in vitro, including for fluorescein-BSA and modified cytokines. Bone marrow derived macrophages (BMDMs) were treated with unmodified and guest-modified cytokines, as well as final hydrogels at the conclusion of 14-day release studies; gene expression was measured by qPCR. Biocompatibility was assessed in healthy BalbC mice (15µL kidney subcapsular injection) relative to saline injection controls; kidney function was assessed (transdermal glomerular filtration rate, blood urea nitrogen, serum creatinine, fibrosis) over 28 days.

Results, Conclusions, and Discussions: Granular hydrogels were strain-yielding and shear-thinning (to enable injectable delivery) and rapidly recovered their solid properties to allow for retention after injection (Fig 1B). The avidity of Ad-BSA conjugates increased with the extent of guest-modification (kd=1.58±0.36×10-4 to 3.95±0.94×10-5 s-1), while maintaining guest–host affinity (12.0±1.81µM). Guest-modification attenuated burst release (>5-fold) and sustained BSA release for over one month (Fig 1C), as well as IFNγ (>2-fold less cumulative release, relative to unmodified controls, Fig 1D) for 14 days, with release rates inversely related to avidity. Adamantane conjugation to cytokines did not alter their bioactivity; BMDMs treated with IFNγ and Ad-IFNγ similarly downregulated anti-inflammatory (Mrc1) and upregulated pro-inflammatory (Nos2, Il1b) gene expression, while treatment with IL-4 and Ad-IL4 similarly upregulated anti-inflammatory (Arg1) and downregulated (Nos2, Il1b) expression levels (Fig 1E). Further, cytokine-loaded hydrogels remained bioactive at the study endpoint, indicating guest-modified cytokines were well-retained within the host hydrogels. After bilateral subcapsular kidney injection of unloaded granular hydrogels, transdermal glomerular filtration rate over the course of a month indicated normal kidney function, relative to saline controls (Fig 1F). At endpoint, blood urea nitrogen, serum creatinine, and fibrosis (picrosirius red, PSR) levels exhibited no difference (Fig 1G), demonstrating that overall, granular hydrogel injection did not negatively impact kidney function.

The delivery of biotherapeutics via guest-host interactions allows for retention and sustained release, where release is tunable via protein avidity. The bioconjugation proceeds under mild aqueous conditions, is non-selective, and is therefore applicable to a range of biotherapeutics (e.g., chemokines, cytokines, and antibodies) that may benefit from sustained local delivery. These immunomodulatory proteins can be harnessed to play a pivotal therapeutic role in chronic disease, including for tissue repair following acute kidney injury.

Acknowledgements (Optional):