Injectable Hydrogels for the Delivery of Minicircle-based Myocardial Infarction Therapy

Introduction: Every year, approximately 805,000 people in America suffer from myocardial infarction (MI). After an MI, tissue death and scar formation impair cardiac function, often leading to heart failure (HF). While current treatments focus on slowing down cardiac worsening,3 new regenerative approaches have emerged. Stromal cell-derived factor-1α (SDF-1α), an endothelial progenitor cell (EPC) chemokine, has shown promising results in improving microvasculature formation and heart function. Unfortunately, direct delivery of SDF-1α has limited benefits as it (1) quickly diffuses away, (2) is degraded by proteases, and (3) requires prolonged activity to be therapeutically effective. Localized gene therapy encoding SDF-1α would assist in overcoming these challenges. Minicircle-DNA (MC) therapies are nonviral vectors lacking the bacterial plasmid backbone, leading to longer protein expression and lower immune response. However, locally delivering therapies to the heart remains notoriously difficult, as the injected material is often pushed out of the tissue, leading to loss of therapeutic cargo. Thus, there is a need to develop hand-injectable hydrogels that are biocompatible and mechanically stiff enough to be retained within the beating heart. To address these demands, we propose the development of a catheter-injectable gel to deliver SDF-1α encoding MC to the infarcted myocardium. Combining catheter-injectable gel with MC therapy will enable sustained SDF-1α expression for tissue repair in a stable, minimally invasive manner, improving heart function in a preclinical MI model.

Materials and

Methods: Our injectable hydrogel is comprised of two recombinant biopolymers: (1) a hyaluronic acid (HA) modified with benzaldehyde and (2) an engineered elastin-like protein (ELP) modified with hydrazine through biorthogonal chemistry techniques (Fig. 1A,B). Upon mixing, the two components spontaneously form a network, termed HELP gel, as the moieties react to form dynamic covalent hydrazone bonds. To find the optimal formulation that allows for non-invasive catheter delivery and improved cargo retention within the myocardium, we synthesized a family of HELP gels with control over the HA molecular weight (20, 40, 60, 100 kDa), and the degree of HA-benzaldehyde modification (6, 12, 20, 30 %). The ELP component was kept constant with a molecular weight of 37 kDa and 14 hydrazines per molecule. The mechanical properties of the HELP gels were characterized through rheological studies and extrusion force measurements. To investigate the therapeutic effects of SDF-1α, we inserted the gene of interest into the parental minicircle cloning plasmid (MN530A-1, System Biosciences - SBI), which also encodes for green fluorescent protein (GFP). Validation of SDF-1α MC bioactivity was performed by transfection into HEK-293T cells and human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, with and without lipofectamine. SDF-1α levels were quantified through Enzyme Linked Immunosorbent Assay (ELISA) and immunocytochemistry (ICC) followed by fluorescence microscopy.

Results, Conclusions, and Discussions: The HELP gels achieved plateau shear storage moduli (G’) ranging from 10 - 1,000 Pa. In general, increasing the HA molecular weight led to higher gel stiffness, presumably due to enhanced chain entanglements. Further, the increase in degree of crosslinker modification reduced the gelation time, leading to the creation of gels with defects that had different impacts on final stiffness depending on HA molecular weight. Aiming to match the stiffness of the extracellular matrix (ECM), the injectability of gels with G’ closer to 1,000 Pa was evaluated (Fig.1C). Each sample was assessed empirically following two criteria important for clinical translation: (1) injectability with one hand post complete gelation through a 31-gauge syringe needle and (2) absence of burst injection, which can produce local tissue damage. Extrusion force measurements showed that formulations with HA under 60 kDa required on average 10 - 15 N for extrusion, and 100 kDa HA formulations required 25 - 30 N (Fig.1D). This is consistent with our rheological analysis, as this higher force is likely a direct effect of entanglements that are more prevalent in networks with higher molecular weight HA. To demonstrate potential clinical translatability, we identified gel formulations that were injectable through a 2-m catheter, which would enable minimally invasive delivery to myocardial tissue (Fig.1E). MC production yielded a concentration of ~500 ng/µL, and sequencing confirmed the presence of the gene of interest. Fluorescence microscopy confirmed the transfection of HEK-293T and cardiomyocytes through the expression of GFP after 2 and 8 days respectively. ELISA analysis indicated measurable levels of SDF-1α at ~100 ng/µL by day 2 in collected media from cells transfected with lipofectamine, while about ~2.5 ng/µL was produced in cells transfected without any agent, validating the successful creation of this novel gene therapy. Thus, we have developed a catheter-injectable gel for non-invasive sustained local delivery of SDF-1α expressing MC therapeutic after MI. Ongoing studies involve the analysis of MC release profile from HELP gel and in vitro bioactivity analysis. Furthermore, we have started preclinical studies, delivering hydrogel-therapy treatment to the heart in a rat MI model of left anterior descending artery ligation.

Acknowledgements (Optional): American Heart Association Predoctoral Fellowship
Stanford Cardiovascular Institute