Injectable Hydrogels for Mechanically Active Tissues

Injectable hydrogels have great potential in healthcare, as they can locally deliver therapeutics to target tissue in a minimally invasive manner, overcoming the negative side-effects of systemic delivery. However, injectable delivery to mechanically active tissues, such as the heart, has been notoriously difficult, as the injected material is often pushed out of the tissue, leading to loss of the therapeutic cargo. Therefore, there is a pressing need to develop hand-injectable hydrogels that are biocompatible and mechanically stiff enough to be retained within mechanically active tissue.<br/> <br/>To address this demand, our group hypothesized that a hydrogel crosslinked with dynamic covalent bonds; which are strong like primary bonds, yet dynamic like secondary bonds; would result in the desired mechanical properties. For clinical translation, the ideal material would be cell-adhesive, biodegradable, and fully chemically defined. Thus, we chose two recombinant biopolymers to comprise our gel system: (1) an engineered elastin-like protein (ELP) modified with hydrazines and (2) a hyaluronic acid (HA) modified with aldehydes. Upon mixing, the two components spontaneously form a network, which we term HELP gels, as the hydrazines react with the aldehydes to form dynamic covalent hydrazone bonds.<br/> <br/>To investigate the role of molecular-level material design choices on the macroscopic mechanical properties, we synthesized a family of HELP gels with control over the HA molecular weight (20 - 100 kDa), the degree of HA-aldehyde modification (6-30%), and the kinetics of the dynamic hydrazone bond (a faster aldehyde moiety and a slower benzaldehyde moiety). The ELP component was kept constant with a molecular weight of 37 kDa and 14 hydrazines per molecule. These gels achieved plateau shear storage moduli (<i>G’</i>) ranging from 10 - 1,000 Pa. At the lowest HA molecular weight, gel stiffness was predominately dictated by the ratio of hydrazine to aldehyde groups, with a theoretical ratio of 1:1 predicted to have the highest stiffness. In general, as HA molecular weight was increased, this led to higher gel stiffness, presumably due to enhanced chain entanglements.<br/> <br/>Injectability of each sample was assessed empirically following two criteria important for clinical translation: injectability with one hand post complete gelation through a 30-gauge syringe and the absence of burst injection, which can produce local tissue damage. We determined that the molecular weight of the HA had a direct impact on the injectability of the resulting hydrogel, with all gels containing HA of 60 kDa or less being easily injectable through a 30-gauge syringe. Further, the kinetics of the hydrazone bond was also found to impact injectability, with the 100-kDa, benzaldehyde-modified HELP system not being injectable, while the aldehyde-modified version was injectable. To demonstrate potential clinical translatability, we observed that some gel formulations were injectable through a 5-ft catheter, allowing for potential minimally-invasive delivery to different systems in the body.<br/> <br/>In summary, we have demonstrated a reproducible synthetic strategy to produce injectable HELP gels with tunable molecular-level design parameters. Our analyses of gel mechanics and preclinical injectability show that both HA molecular weight and kinetics of the dynamic covalent hydrazone bond impact gel injectability. This biocompatible, catheter-injectable HELP gel has great potential to significantly improve the retention of therapeutic cargo within the beating heart, opening a door to direct prolonged delivery of pharmaceuticals and gene therapies.