Macrophage-Targeted Therapeutics to Attenuate the Development of Ischemic Heart Failure
Authors:   , Soni Shreya S. , Rodell Christopher B. 
Published: December 2024

Heart failure (HF) remains the leading cause of death worldwide and is predicted to afflict over 8 million Americans by 2030. Nearly 70% of HF cases are the direct result of myocardial infarction (MI), causing a cascade of cell death, loss of muscle contraction, and maladaptive tissue remodeling. Inflammation has recently been recognized as a critical regulator of adverse left ventricular remodeling (LVR) post-MI. Following injury, classically activated (inflammatory) macrophages initially dominate the immune microenvironment, necessary for early tissue remodeling. With injury resolution, alternatively activated (reparatory) macrophages later dominate the local immune landscape, mitigating inflammation and promoting repair. In many cases, this transition is impeded by the establishment of a hyper-inflammatory milieu and resulting chronic inflammation. The hyper-inflammatory environment is driven by multiple parallel mechanisms, including the local cytokine toxicity, the continual recruitment of inflammatory cells to the injury, an autoimmune reaction against the myocardium, and even genetic factors. Therapeutic strategies have sought to leverage immune modulation to improve outcomes and have shown promise in both pre-clinical and clinical studies. However, phenotypic modulation of macrophages by small-molecule drugs remains under-explored, and specific delivery of these agents to immune cells at the injury site remains challenging. To address these critical needs, we i) identify small-molecule drugs that directly combat the hyper-inflammatory environment and ii) aim to prevent HF development through delivery of the chosen drug via a biomaterial-based vehicle. First, we sought to identify an anti-inflammatory drug that could inhibit the damaging inflammatory macrophage phenotype and promote the healing reparatory phenotype. Using a two-step drug screening process, which involved a developed reporter assay, as well as follow-up quantitative polymerase chain reaction (qPCR) and nanoString analysis, we identified celastrol as a potent inhibitor of inflammatory signaling (IC50 < 100 nM, NF-[kappa]B inhibition) that likewise promoted a pro-healing phenotype. We then developed cyclodextrin nanoparticles (CDNPs) that could bind to and sequester celastrol (Keq = 0.474 mM) via non-covalent guest-host interactions. [beta]-Cyclodextrin is widely used to improve drug solubility and bioavailability. Here, we observed that the CDNPs likewise enable macrophage targeting, as the particles are recognized by cell surface receptors (scavenger and mannose receptors), resulting in rapid macrophage-targeted delivery that further improved the anti-inflammatory effects of celastrol in vitro. In a murine model of ischemia reperfusion injury, macrophage populations were observed to increase over the first two days after MI (>2-fold), dominated by inflammatory (Nos2+) cells that precipitate HF. Intravenous injection of CDNPs two days post-injury resulted in in selective myocardial accumulation of the nanoparticles (a >2.5-fold increase relative to sham controls) due to macrophage uptake. At 24 hours post-treatment, CDNP+celastrol delivery re-oriented macrophages towards a reparatory (Arg1+CD206+) phenotype. Histological, geometric, and functional assessment of the LV at day 28 demonstrated reduced fibrosis, prevention of ventricular dilation (end-diastolic volume, end-systolic volume), and retained function (ejection fraction) after treatment of CDNP+celastrol, all similar to that of the sham controls. While CDNPs can be administered systemically (intravenously), we also developed a locally delivered injectable polymer-nanoparticle (iPNP) hydrogel, composed of CDNPs dynamically crosslinked by adamantane-modified hyaluronic acid (Ad-HA). The iPNP hydrogel possesses shear-thinning (>90% loss in G' at 500% strain) and self-healing (>98% recovery within seconds) capabilities, allowing for local delivery via minimally invasive injection. After optimizing hydrogel properties (i.e., polymer concentration, polymer-to-nanoparticle ratio), the chosen formulation, loaded with celastrol, demonstrated sustained knockdown of inflammatory pathway activity (>80%) over 14 days in vitro. Additionally, the hydrogel demonstrated prolonged degradation over 28 days after subcutaneous injection in a control mouse. This local delivery system is a promising strategy to achieve local immune modulation, such as by intramyocardial injection, to concentrate drug effects at the site of injury. In conclusion, we have identified celastrol as a potent immunomodulatory small molecule drug, capable of modulating macrophage phenotypes in vitro. For macrophage-targeted delivery of celastrol, we have developed two drug-delivery platforms: CDNPs for systemic delivery and iPNP hydrogels for local delivery. Both drug-loaded vehicles knock down inflammatory signaling significantly in vitro. Furthermore, treatment in a mouse model of myocardial injury demonstrated the ability of celastrol-loaded CDNPs to locally modulate macrophage phenotype, preventing LVR. Thus, the CDNP and iPNP hydrogel are versatile drug-delivery platforms for macrophage-targeted therapy to modulate the post-injury immune microenvironment, including for the prevention of ischemic HF.