Brendan P. Purcell

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 remodeling. While MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application due 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 over-expression following a myocardial infarction (MI) significantly reduced MMP activity and attenuated adverse left ventricular remodeling in a porcine model of MI. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.

Injectable acellular hydrogels for cardiac repair.

Injectable hydrogels are being developed as potential translatable materials to influence the cascade of events that occur after myocardial infarction. These hydrogels, consisting of both synthetic and natural materials, form through numerous chemical crosslinking and assembly mechanisms and can be used as bulking agents or for the delivery of biological molecules. Specifically, a range of materials are being applied that alter the resulting mechanical and biological signals after infarction and have shown success in reducing stresses in the myocardium and limiting the resulting adverse left ventricular (LV) remodeling. Additionally, the delivery of molecules from injectable hydrogels can influence cellular processes such as apoptosis and angiogenesis in cardiac tissue or can be used to recruit stem cells for repair. There is still considerable work to be performed to elucidate the mechanisms of these injectable hydrogels and to optimize their various properties (e.g., mechanics and degradation profiles). Furthermore, although the experimental findings completed to date in small animals are promising, future work needs to focus on the use of large animal models in clinically relevant scenarios. Interest in this therapeutic approach is high due to the potential for developing percutaneous therapies to limit LV remodeling and to prevent the onset of congestive heart failure that occurs with loss of global LV function. This review focuses on recent efforts to develop these injectable and acellular hydrogels to aid in cardiac repair.

Synergistic effects of SDF-1α chemokine and hyaluronic acid release from degradable hydrogels on directing bone marrow derived cell homing to the myocardium

Poor cell engraftment in the myocardium is a limiting factor towards the use of bone marrow derived cells (BMCs) to treat myocardial infarction (MI). In order to enhance the engraftment of circulating BMCs in the myocardium following MI, we have developed in situ forming hyaluronic acid (HA) hydrogels with degradable crosslinks to sustain the release of recombinant stromal cell-derived factor-1 alpha (rSDF-1α) and HA to the injured myocardium. Both rSDF-1α and the crosslinkable HA macromer stimulate BMC chemotaxis up to 4-fold in vitro through CXCR4 and CD44 receptor signaling, respectively. Moreover, the HA macromer binds rSDF-1α with a dissociation constant of 36 ± 5 μM through electrostatic interaction. When formed into hydrogels via photoinitiated crosslinking, release of encapsulated rSDF-1α and crosslinked HA was sustained for over 7 days, and these molecules significantly increased BMC chemotaxis in vitro. When applied to the heart following experimental MI in mice, the HA gel containing rSDF-1α significantly increased the number of systemically infused BMCs in the heart by ~8.5 fold after 7 days, likely through both systemic and local effects of released molecules. We conclude that sustained release of rSDF-1α and HA from our engineered HA hydrogels enhances BMC homing to the remodeling myocardium better than delivery of rSDF-1α alone.

Sustained Release of Engineered Stromal Cell–Derived Factor 1-α From Injectable Hydrogels Effectively Recruits Endothelial Progenitor Cells and Preserves Ventricular Function After Myocardial Infarction

Background— Exogenously delivered chemokines have enabled neovasculogenic myocardial repair in models of ischemic cardiomyopathy; however, these molecules have short half-lives in vivo. In this study, we hypothesized that the sustained delivery of a synthetic analog of stromal cell–derived factor 1-&agr; (engineered stromal cell–derived factor analog [ESA]) induces continuous homing of endothelial progenitor cells and improves left ventricular function in a rat model of myocardial infarction. Methods and Results— Our previously designed ESA peptide was synthesized by the addition of a fluorophore tag for tracking. Hyaluronic acid was chemically modified with hydroxyethyl methacrylate to form hydrolytically degradable hydrogels through free-radical–initiated crosslinking. ESA was encapsulated in hyaluronic acid hydrogels during gel formation, and then ESA release, along with gel degradation, was monitored for more than 4 weeks in vitro. Chemotactic properties of the eluted ESA were assessed at multiple time points using rat endothelial progenitor cells in a transwell migration assay. Finally, adult male Wistar rats (n=33) underwent permanent ligation of the left anterior descending (LAD) coronary artery, and 100 µL of saline, hydrogel alone, or hydrogel+25 µg ESA was injected into the borderzone. ESA fluorescence was monitored in animals for more than 4 weeks, after which vasculogenic, geometric, and functional parameters were assessed to determine the therapeutic benefit of each treatment group. ESA release was sustained for 4 weeks in vitro, remained active, and enhanced endothelial progenitor cell chemotaxis. In addition, ESA was detected in the rat heart >3 weeks when delivered within the hydrogels and significantly improved vascularity, ventricular geometry, ejection fraction, cardiac output, and contractility compared with controls. Conclusions— We have developed a hydrogel delivery system that sustains the release of a bioactive endothelial progenitor cell chemokine during a 4-week period that preserves ventricular function in a rat model of myocardial infarction.

Local Hydrogel Release of Recombinant TIMP-3 Attenuates Adverse Left Ventricular Remodeling After Experimental Myocardial Infarction

Delivery of a hydrogel providing sustained release of recombinant TIMP-3 attenuated adverse ventricular remodeling after myocardial infarction in pigs. Hydrogel-Inhibitor Combo Stops Heart Damage After a heart attack, or myocardial infarction (MI), the heart tries to repair itself. This natural process is well intentioned but results in infarct expansion, scar formation, and, in turn, reduced heart function. To prevent such adverse remodeling, Eckhouse and colleagues designed an injectable hydrogel that inhibits the activity of enzymes directly involved in tissue repair. Matrix metalloproteinases (MMPs) are enzymes that are activated in heart tissue after MI. The authors encapsulated TIMP-3 (tissue inhibitor of metalloproteinase 3) in hyaluronic acid hydrogels. The gel/TIMP-3 combo or a control gel without the inhibitor was injected into the hearts of pigs after a heart attack. Weeks later, heart function, inflammation, and remodeling were evaluated. Animals administered the hydrogel with TIMP-3 had improved heart function [as determined by the left ventricular ejection fraction (LVEF)], improved LV geometry, and reduced infarct size. This local delivery mechanism could be used in the context of surgery, such as during coronary revascularization after a heart attack. Because it has been tested in pigs—which have similar heart anatomy to humans—and because other hydrogels, like alginate, have been tested in the human heart before, it is possible that this gel-inhibitor combination therapy could advance to clinical trials in the near future. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) contributes to the left ventricle (LV) remodeling that occurs after myocardial infarction (MI). However, translation of these observations into a clinically relevant, therapeutic strategy remains to be established. The present study investigated targeted TIMP augmentation through regional injection of a degradable hyaluronic acid hydrogel containing recombinant TIMP-3 (rTIMP-3) in a large animal model. MI was induced in pigs by coronary ligation. Animals were then randomized to receive targeted hydrogel/rTIMP-3, hydrogel alone, or saline injection and followed for 14 days. Instrumented pigs with no MI induction served as referent controls. Multimodal imaging (fluoroscopy/echocardiography/magnetic resonance imaging) revealed that LV ejection fraction was improved, LV dilation was reduced, and MI expansion was attenuated in the animals treated with rTIMP-3 compared to all other controls. A marked reduction in proinflammatory cytokines and increased smooth muscle actin content indicative of myofibroblast proliferation occurred in the MI region with hydrogel/rTIMP-3 injections. These results provide the first proof of concept that regional sustained delivery of an MMP inhibitor can effectively interrupt adverse post-MI remodeling.

Incorporation of sulfated hyaluronic acid macromers into degradable hydrogel scaffolds for sustained molecule delivery

Synthetically sulfated hyaluronic acid (HA) has been shown to bind proteins with high affinity through electrostatic interactions. While HA-based hydrogels have been used widely in recent years for drug delivery and tissue engineering applications, incorporation of sulfated HA into these networks to attenuate the release of proteins has yet to be explored. Here, we developed sulfated and methacrylate-modified HA macromers and incorporated them into HA hydrogels through free radical-initiated crosslinking. The sulfated HA macromers bound a heparin-binding protein (i.e., stromal cell-derived factor 1-α, SDF-1α) with an affinity comparable to heparin and did not alter the gelation behavior or network mechanics when copolymerized into hydrogels at low concentrations. Further, these macromers were incorporated into electrospun nanofibrous hydrogels to introduce sulfate groups into macroporous scaffolds. Once incorporated into either uniform or fibrous HA hydrogels, the sulfated HA macromers significantly slowed encapsulated SDF-1α release over 12 days. Thus, these macromers provide a useful way to introduce heparin-binding features into radically-crosslinked hydrogels to alter protein interactions for a range of applications.

Ischemia induces P-selectin-mediated selective progenitor cell engraftment in the isolated-perfused heart.

Clinical trials infusing Bone Marrow Cells (BMCs) into injured hearts have produced measureable improvements in cardiac performance, but were insufficient to improve patient outcomes. Low engraftment rates are cited as probable contributor to limited improvements. To understand the mechanisms that control myocardial engraftment of BMCs following ischemia-reperfusion injury, in isolated-perfused mouse hearts, stop-flow ischemia was followed by variable-duration reperfusion (0-60 min) before addition of labeled syngenic BMCs to the perfusate. After a buffer-only wash, the heart was disaggregated. Retained BMCs (digest) and infused BMCs (aliquot) were compared by flow cytometry for c-kit and CD45 expression to determine the proportion of cell subtypes engrafted versus delivered (selectivity ratio). In these studies, a time-dependent selective retention of c-kit(+) cells was apparent starting at 30 min of reperfusion, at which time c-kit(+)/CD45(+) BMCs showed a selectivity ratio of 18 ± 2 (versus 2 ± 1 in sham-ischemic controls). To study the underlying mechanism for this selective retention, neutralizing antibodies for P-selectin or L-selectin were infused into the heart preparation and incubated with BMCs prior to BMC infusion. Blocking P-selectin in ischemic hearts ablated selectivity for c-kit(+)/CD45(+) BMCs at 30 min reperfusion (selectivity ratio of 3 ± 1) while selectivity persisted in the presence of L-selectin neutralization (selectivity ratio of 17 ± 2). To corroborate this finding, a parallel plate flow chamber was used to study capture and rolling dynamics of purified c-kit(+) versus c-kit- BMCs on various selectin molecules. C-kit(+) BMCs interacted weakly with L-selectin substrates (0.03 ± 0.01% adhered) but adhered strongly to P-selectin (0.28±0.04% adhered). C-kit- BMCs showed intermediate binding regardless of substrate (0.18 ± 0.04% adhered on L-selectin versus 0.17 ± 0.04% adhered on P-selectin). Myocardial ischemia-reperfusion stress induces selective engraftment of c-kit(+) bone marrow progenitor cells via P-selectin activation.

Delivery of a matrix metalloproteinase-responsive hydrogel releasing TIMP-3 after myocardial infarction: effects on left ventricular remodeling

Although improvements in timing and approach for early reperfusion with acute coronary syndromes have occurred, myocardial injury culminating in a myocardial infarction (MI) remains a common event. Although a multifactorial process, an imbalance between the induction of proteolytic pathways, such as matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinase (TIMPs), has been shown to contribute to this process. In the present study, a full-length TIMP-3 recombinant protein (rTIMP-3) was encapsulated in a specifically formulated hyaluronic acid (HA)-based hydrogel that contained MMP-cleavable peptide cross-links, which influenced the rate of rTIMP-3 release from the HA gel. The effects of localized delivery of this MMP-sensitive HA gel (HAMMPS) alone and containing rTIMP-3 (HAMMPS/rTIMP-3) were examined in terms of the natural history of post-MI remodeling. Pigs were randomized to one of the following three different groups: MI and saline injection (MI/saline group, 100-μl injection at nine injection sites, n = 7), MI and HAMMPS injection (MI/HAMMPS group; 100-μl injection at nine injection sites, n = 7), and MI and HAMMPS/rTIMP-3 injection (MI/HAMMPS/rTIMP-3 group; 20-μg/100-μl injection at nine injection sites, n = 7). Left ventricular (LV) echocardiography was serially performed up to 28 days post-MI. LV dilation, as measured by end-diastolic volume, and the degree of MI wall thinning were reduced by ~50% in the HAMMPS/rTIMP-3 group ( P < 0.05). Furthermore, indexes of heart failure progression post-MI, such as LV filling pressures and left atrial size, were also attenuated to the greatest degree in the HAMMPS/rTIMP-3 group. At 28 days post-MI, HAMMPS/rTIMP-3 caused a relative reduction in the transcriptional profile for myofibroblasts as well as profibrotic pathways, which was confirmed by subsequent histochemistry. In conclusion, these findings suggest that localized delivery of a MMP-sensitive biomaterial that releases a recombinant TIMP holds promise as a means to interrupt adverse post-MI remodeling. NEW & NOTEWORTHY The present study targeted a myocardial matrix proteolytic system, matrix metalloproteinases (MMPs), through the use of a recombinant tissue inhibitor of MMPs incorporated into a MMP-sensitive hydrogel, which was regionally injected using a large animal model of myocardial infarction. Left ventricular geometry and function and indexes of myocardial remodeling were improved with this approach and support the advancement of localized therapeutic strategies that specifically target the myocardial matrix.