Collagen targeting using protein-functionalized micelles: the strength of multiple weak interactions.

Collagen is an important marker for the assessment of tissue remodeling, both in normal tissue maturation and in a variety of prevalent disease processes. Given the importance of multivalency in the natural interactions of collagen, multivalent ligands provide unique opportunities to target collagen architectures. Here, we explored the use of micelles as dynamic self-assembling multivalent scaffolds for the collagen binding protein CNA35. Despite the increased popularity of micelles as nanosized carriers in targeted drug delivery and molecular imaging, few studies have actually directly addressed the importance of multivalent interactions for micelle-based targeting. Native chemical ligation was used as a chemoselective and efficient method to prepare relatively well-defined and stable micelles with a tunable average protein content between 0 and 20 copies of CNA35 per micelle. The thermodynamics and kinetics of CNA35 micelle binding to collagen was studied using solid-phase and surface plasmon resonance assays. Multivalent interactions between the micelles and collagen had a remarkable effect on micellar stability, since no dissociation of collagen-bound micelles was observed even after extensive washing. In addition, an impressive enhancement of collagen affinity was observed both in vitro and ex vivo resulting from multivalent display of a so-called "nonbinding" variant of CNA35. This "restoration" of collagen affinity was subsequently also observed for liposomes displaying the same low-affinity CNA35 variant at a sufficient density. These results demonstrate the importance of multivalent interactions for micelle-based targeting and illustrate the strength of multiple weak interactions when targeting intrinsically multivalent extracellular matrix (ECM) proteins such as collagen.