Modulation of macrophage‐mediated cardiac inflammation via targeted anionic liposomes

Abstract

Inflammatory response, via pro‐inflammatory (M1) infiltrating and resident macrophages, plays an important role in the development of various inflammatory cardiac pathologies, namely ischemic heart disease, myocardial infarction, and heart failure. Conversely, reparative (M2) macrophages have a vital inflammation resolution role following ischemia/reperfusion injury.Instead of mere inactivation of pathogenic M1 macrophages, as a general treatment approach, we better designed to actively polarizing them into reparative M2 phenotype ‐ via macrophage‐specific red blood cell (RBC) soluble receptor conjugated onto nanocarriers (NC). Liposomes (Lip) targeted via soluble CD47 immuno‐modulatory ligand of macrophage, can mitigate cardiac inflammation. Especially, when CD47‐Lip nano‐system further presents phosphatidylserine (PS)‐apoptotic cell signal ‐ for additional M2‐macrophage polarization.The RBC’s “self‐marker” signal, CD47, acts as a phagocytosis switch, through interactions with immuno‐receptor signal regulatory protein (SIRPα) and Thrombospondin‐1 (TSP1) on macrophages, to suppress and evade phagocytosis. Hence, we chemically coupled multiple recombinant human CD47 proteins onto anionic PS‐containing Lip surface, to produce hematopoietic and apoptotic cell dual‐mimicking CD47‐PSLip nano‐platform. Pharmaceutically, 67±11μg/mL of protein were covalently attached on CD47‐PSLip, which subsequently reduced overall anionic surface potential by approx. 9.3mV, while retaining at least 85% of original CD47 protein activity. Western blots and ELISA confirmed dose‐dependent binding of human TSP1: CD47‐PSLip.In addition, classically TNF‐α/LPS‐activated macrophages, of both human and murine origins, showed a substantial decrease in phagocytosis of labeled CD47‐PSLip, compared to unconjugated neutral or PSLip, and IgG‐PSLip controls (p<0.01, n=3–4), indicating their active self‐marker signal. Furthermore, anionic CD47‐PSLip, as a dual reprogramming strategy, induced substantial M2 polarization/reparative phenotype, evident through high levels of secreted anti‐inflammatory cytokines (IL‐10 and TGF‐β), concomitant with downregulation of pro‐inflammatory markers (IL‐12 and TNF‐α), following 24hr‐treatment of M1‐polarized human monocyte macrophages. In vivo RBC‐mimicry was imaged in C57 mice, using near infrared‐labeled Lip (av. 70–90nm), through minimal accumulation of CD47‐PSLip in liver and spleen (p<0.001 vs IgG‐Lip control), 48hr‐post intravenous injection. Non‐compartmental pharmacokinetic model analysis revealed marked increase in their mean residence plasma half‐life (MRT≈41.7hr), in comparison to standard Stealth™ Lip preparation (~15.6hr).This is the first report about CD47/PS presenting NC, selectively targeting pathogenic macrophage infiltration underlying inflammatory ischemic cardiac disorders. Our pre‐clinical data illustrate the potential of CD47‐PSLip as a dual reprogramming nanomedicine, to induce M2 polarization/reparative macrophage phenotype, as a novel therapeutic strategy to ameliorate exacerbated cardiac inflammation and hearth failure.Support or Funding InformationThe Cardiovascular Medical Research and Education Fund (CMREF).