Matrix Metalloproteinase-9-Responsive Lipid-Core Micelles for Proof-of-Concept Enzyme-Triggered Cardiomyocyte Association

Cardiovascular diseases (CVDs) remain the leading cause of global mortality and are frequently associated with increased matrix metalloproteinase-9 (MMP-9) activity during cardiac remodeling and inflammation. Here, we developed lipid-core micelles (LCMs) functionalized with a multifunctional peptide incorporating a cardiomyocyte-targeting sequence (PCM-1) and an MMP-9 cleavable linker (GPQGIAGQ), as a proof-of-concept for enzyme-responsive targeting platform. The peptide was conjugated to a maleimide-terminated PEGylated lipid and incorporated into LCMs prepared by low-energy hot emulsification. The resulting nanoparticles were spherical, monodisperse (12–16 nm), and exhibited near-neutral surface charge, with > 80% rhodamine entrapment efficiency. In vitro assays demonstrated that the surface-displayed peptide remained enzymatically accessible, although cleavage kinetics were reduced compared to the free peptide, indicating partial but functionally relevant responsiveness under controlled conditions. Cleavage was associated with enhanced cargo and altered nanoparticle behavior. Fluorescence-based assays demonstrated that CLV-functionalized LCM exhibited significantly higher cardiomyocyte association than non-functionalized particles, although lower than the full targeting sequence, indicating preserved sequence-dependent recognition despite residual linker fragments. Importantly, the present study evaluates cardiomyocyte binding rather than internalization and does not address receptor specificity or intracellular trafficking. Overall, these findings provide mechanistic proof-of-concept evidence that MMP-9 responsive LCMs can undergo an enzyme-triggered transition that modulate targeting and release behavior. While translational applicability requires additional validation in complex biological systems, these results support further investigation of enzyme-responsive targeting strategies for CVDs.