Macrophages play crucial roles in various immune-related responses, such as host defense, wound healing, disease progression, and tissue regeneration. Macrophages perform distinct and dynamic functions in vivo, depending on their polarization states, such as the pro-inflammatory M1 phenotype and pro-healing M2 phenotype. Remote manipulation of the adhesion of host macrophages to the implants and their subsequent polarization in vivo can be an attractive strategy to control macrophage polarization-specific functions but has rarely been achieved. In this study, we grafted RGD ligand-bearing superparamagnetic iron oxide nanoparticles (SPIONs) to a planar matrix via a long flexible linker. We characterized the nanoscale motion of the RGD-bearing SPIONs grafted to the matrix, in real time by in situ magnetic scanning transmission electron microscopy (STEM) and in situ atomic force microscopy. The magnetic field was applied at various oscillation frequencies to manipulate the frequency-dependent ligand nano-oscillation speeds of the RGD-bearing SPIONs. We demonstrate that a low oscillation frequency of the magnetic field stimulated the adhesion and M2 polarization of macrophages, whereas a high oscillation frequency suppressed the adhesion of macrophages but promoted their M1 polarization, both in vitro and in vivo. Macrophage adhesion was also temporally regulated by switching between the low and high frequencies of the oscillating magnetic field. To the best of our knowledge, this is the first demonstration of the remote manipulation of the adhesion and polarization phenotype of macrophages, both in vitro and in vivo. Our system offers the promising potential to manipulate host immune responses to implanted biomaterials, including inflammation or tissue reparative processes, by regulating macrophage adhesion and polarization.

Cellular adhesion is regulated by the dynamic ligation process of surface receptors, such as integrin, to adhesive motifs, such as Arg-Gly-Asp (RGD). Remote control of adhesive ligand presentation using external stimuli is an appealing strategy for the temporal regulation of cell-implant interactions in vivo and was recently demonstrated using photochemical reaction. However, the limited tissue penetration of light potentially hampers the widespread applications of this method in vivo. Here, we present a strategy for modulating the nanoscale oscillations of an integrin ligand simply and solely by adjusting the frequency of an oscillating magnetic field to regulate the adhesion and differentiation of stem cells. A superparamagnetic iron oxide nanoparticle (SPION) was conjugated with the RGD ligand and anchored to a glass substrate by a long flexible poly(ethylene glycol) linker to allow the oscillatory motion of the ligand to be magnetically tuned. In situ magnetic scanning transmission electron microscopy and atomic force microscopy imaging confirmed the nanoscale motion of the substrate-tethered RGD-grafted SPION. Our findings show that ligand oscillations under a low oscillation frequency (0.1 Hz) of the magnetic field promoted integrin-ligand binding and the formation and maturation of focal adhesions and therefore the substrate adhesion of stem cells, while ligands oscillating under high frequency (2 Hz) inhibited integrin ligation and stem cell adhesion, both in vitro and in vivo. Temporal switching of the multimodal ligand oscillations between low- and high-frequency modes reversibly regulated stem cell adhesion. The ligand oscillations further induced the stem cell differentiation and mechanosensing in the same frequency-dependent manner. Our study demonstrates a noninvasive, penetrative, and tunable approach to regulate cellular responses to biomaterials in vivo. Our work not only provides additional insight into the design considerations of biomaterials to control cellular adhesion in vivo but also offers a platform to elucidate the fundamental understanding of the dynamic integrin-ligand binding that regulates the adhesion, differentiation, and mechanotransduction of stem cells.