Shear-Thinning Supramolecular Hydrogels with Secondary Autonomous Covalent Crosslinking to Modulate Viscoelastic Properties In Vivo.

Clinical percutaneous delivery of synthetically engineered hydrogels remains limited due to challenges posed by crosslinking kinetics - too fast leads to delivery failure, too slow limits material retention. To overcome this challenge, we exploit supramolecular assembly to localize hydrogels at the injection site and introduce subsequent covalent crosslinking to control final material properties. Supramolecular gels were designed through the separate pendant modifications of hyaluronic acid (HA) by the guest-host pair cyclodextrin and adamantane, enabling shear-thinning injection and high target site retention (>98%). Secondary covalent crosslinking occurred via addition of thiols and Michael-acceptors (i.e., methacrylates, acrylates, vinyl sulfones) on HA and increased hydrogel moduli (E=25.0±4.5kPa) and stability (>3.5 fold in vivo at 28 days). Application of the dual-crosslinking hydrogel to a myocardial infarct model showed improved outcomes relative to untreated and supramolecular hydrogel alone controls, demonstrating its potential in a range of applications where the precise delivery of hydrogels with tunable properties is desired.

Protease-degradable electrospun fibrous hydrogels.

Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

Nanofibrous hydrogels with spatially patterned biochemical signals to control cell behavior.

The ability to spatially pattern biochemical signals into nanofibrous materials using thiol-ene reactions of thiolated molecules to presented norbornene groups is demonstrated. This approach is used to pattern three molecules independently within one scaffold, to pattern molecules through the depth of a scaffold, and to spatially control cell adhesion and morphology.

Selective Proteolytic Degradation of Guest-Host Assembled, Injectable Hyaluronic Acid Hydrogels.

There have been significant advances in the past decades toward the engineering of materials with biomimetic properties. In particular, hydrogels covalently cross-linked with protease degradable peptides have demonstrated the importance of protease mediated degradation for targeted therapeutic cargo delivery and controlling cell-material interactions. However, the incorporation of such degradation mechanisms into synthetic shear-thinning hydrogels has yet to be accomplished. Herein, we utilize supramolecular self-assembly mediated by the guest-host interaction of hyaluronic acid (HA) separately modified by adamantane (Ad) or cyclodextrin (CD) to form shear-thinning and self-healing hydrogels. In this design, Ad is bound to HA via a proteolytically degradable peptide tether (attached via Michael-addition of a cysteine residue in an Ad-terminated peptide with maleimide modified HA), enabling subsequent proteolytic degradation of the assembly. Upon mixing of the Ad-peptide modified HA and the CD modified HA, a supramolecular hydrogel was formed (G' ≈ 300 Pa at 1 Hz), which displayed shear-thinning (>80% viscosity reduction at 0.5 s-1) and near-instantaneous self-healing properties. Rational, selective modification of amino acid residues near the proteolytic site enabled control over peptide cleavage kinetics, specifically with either collagenases or MMP-2. Hydrogel degradation, mediated by a combination of stochastically governed erosion and proteolytic degradation, was influenced by peptide susceptibility to proteolysis both in vitro and in vivo (>2 fold difference at 3 weeks in vivo) when injected subcutaneously. This material system provides unique opportunities for therapeutic delivery (e.g., growth factors, cells) through facile material formation, ease of injection, and bioresponsive material degradation.

Ordered, adherent layers of nanofibers enabled by supramolecular interactions.

Aligned nanofibrous substrates can be created by electrospinning, but methods for creating multilamellar structures of aligned fibers are limited. Here, apposed nanofibrous scaffolds with pendant ß-cyclodextrin (CD) were adhered together by adamantane (Ad) modified hyaluronic acid, exploiting the guest-host interactions of CD and Ad for macroscopic assembly. Stable user-defined multi-layered scaffolds were formed for cell culture or tissue engineering.

Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition.

Inhibitors of matrix metalloproteinases (MMPs) have been extensively explored to treat pathologies where excessive MMP activity contributes to adverse tissue remodelling. Although MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application owing to the dose-limiting side effects following systemic administration of the drugs. Here, we describe the synthesis of a polysaccharide-based hydrogel that can be locally injected into tissues and releases a recombinant tissue inhibitor of MMPs (rTIMP-3) in response to MMP activity. Specifically, rTIMP-3 is sequestered in the hydrogels through electrostatic interactions and is released as crosslinks are degraded by active MMPs. Targeted delivery of the hydrogel/rTIMP-3 construct to regions of MMP overexpression following a myocardial infarction significantly reduced MMP activity and attenuated adverse left ventricular remodelling in a porcine model of myocardial infarction. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.