Abstract3D culture of cells in designer biomaterial matrices provides a biomimetic cellular microenvironment and can yield critical insights into cellular behaviours not available from conventional 2D cultures. Hydrogels with dynamic properties, achieved by incorporating either degradable structural components or reversible dynamic crosslinks, enable efficient cell adaptation of the matrix and support associated cellular functions. Herein we demonstrate that given similar equilibrium binding constants, hydrogels containing dynamic crosslinks with a large dissociation rate constant enable cell force-induced network reorganization, which results in rapid stellate spreading, assembly, mechanosensing, and differentiation of encapsulated stem cells when compared to similar hydrogels containing dynamic crosslinks with a low dissociation rate constant. Furthermore, the static and precise conjugation of cell adhesive ligands to the hydrogel subnetwork connected by such fast-dissociating crosslinks is also required for ultra-rapid stellate spreading (within 18 h post-encapsulation) and enhanced mechanosensing of stem cells in 3D. This work reveals the correlation between microscopic cell behaviours and the molecular level binding kinetics in hydrogel networks. Our findings provide valuable guidance to the design and evaluation of supramolecular biomaterials with cell-adaptable properties for studying cells in 3D cultures.

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Cells sense and respond to the surrounding microenvironment through binding of membranous integrin to ligands such as the Arg-Gly-Asp (RGD) peptide. Previous studies show that the RGD tether properties on substrate influence cell adhesion and spreading, but few studies have reported strategies to control the tether mobility of RGD on substrate via a physical and noncontact approach. Herein, we demonstrate a novel strategy to tune the tether mobility of RGD on substrate via magnetic force. We conjugate a monolayer of RGD-bearing magnetic nanoparticles (MNPs) on a glass substrate via the flexible and coiled poly(ethylene glycol) linker of large molecular weight (PEG, average MW: 2000), and this increases the RGD tether mobility, which can be significantly reduced by applying magnetic attraction on MNPs. Our data show that high RGD tether mobility delays the early adhesion and spreading of human mesenchymal stem cells (hMSCs), leading to compromised osteogenic differentiation at later stage. In contrast, hMSCs cultured on substrate with restricted RGD tether mobility, achieved either via a shorter PEG linker (MW: 200) or magnetic force, show significantly better adhesion, spreading, and osteogenic differentiation. The control utilizing RGD-bearing nonmagnetic nanoparticles shows no such enhancing effect of magnetic field on cellular events, further supporting our conjecture of magnetic tuning of RGD tether mobility. We hypothesize that high tether mobility of RGD entails additional time and effort by the cells to fully develop traction force and mechanical feedback, thereby delaying the maturation of FAs and activation of subsequent mechanotransduction signaling. Our staining results of vinculin, a critical component of FAs, and Yes-associated protein (YAP), an important mechanosensitive transcriptional factor, support our hypothesis. We believe that our work not only sheds light on the impact of dynamic presentation of cell adhesive ligands on cellular behaviors, which should be taken into consideration for designing novel biomaterials, but also formulate an effective noncontact strategy that enables further investigation on the mechanobiological mechanisms underlying such cellular responses.

Immunotherapy has revolutionized oncology remarkably and gained great improvements in cancer therapy. However, tumor immunotherapy still encounters serious challenges, especially certain tumors barely respond to immunotherapy. The lack of immunogenicity and subsequent insufficient antitumor immune activation is a pivotal reason. Here, a general introduction and the strengthening strategies of immunogenicity of a tumor for enhanced immunotherapy are reviewed. Specifically, nanotechnology nowadays is playing important roles in increasing the antitumor efficacy of various treatments, including immunotherapy. This review highlights how nanomedicines integrating one or more anticancer therapeutic methods (e.g., cancer vaccines, chemotherapy, phototherapy, and radiotherapy) to increase the tumor immunogenicity for rousing T cell related immune responses and achieving inspiring antitumor efficacy. Given the sophisticated immune evasion mechanisms, rational designed nanodrugs with combinational formulations are summarized to improve therapeutic efficacy in synergistic ways. Nanoplatforms taking advantage of the distinct features of tumor tissue or tumor cell with stimuli-responsiveness and targeting functions are introduced to accelerate tumor accumulation of drugs successfully and greatly promote therapeutic efficacy with low-dose administration and programmed drug release. Finally, the related challenges and personal perspectives of nanomedicines for tumor immunotherapy are concluded.

Photoacoustic (PA) imaging and tracking of stem cells plays an important role in the real-time assessment of cell-based therapies. Nevertheless, the limitations of conventional inorganic PA contrast agents and the narrow range of the excitation wavelength in the first near-infrared (NIR-I) window hamper the applications of PA imaging in living subjects. Herein, we report the design and synthesis of a second near-infrared (NIR-II) absorptive organic semiconducting polymer (OSP)-based nanoprobe (OSPN+) for PA imaging and tracking of stem cells. Comparison studies in biological tissue show that NIR-II light excited PA imaging of the OSPN+ has significantly higher signal-to-noise ratio than NIR-I light excited PA imaging, thereby demonstrating the superiority of the OSPN+ for deep tissue imaging. With good biocompatibility, appropriate size, and optimized surface property, the OSPN+ shows enhanced cellular uptake for highly efficient PA labeling of stem cells. In vivo investigations reveal significant NIR-II PA contrast enhancement of the transplanted OSPN+-labeled human mesenchymal stem cells by 40.6- and 21.7-fold in subcutaneous and brain imaging, respectively, compared with unlabeled cases. Our work demonstrates a class of OSP-based nanomaterials for NIR-II PA stem cell imaging to facilitate a better understanding and evaluation of stem cell-based therapies.

Remote, noninvasive, and reversible control over the nanoscale presentation of bioactive ligands, such as Arg-Gly-Asp (RGD) peptide, is highly desirable for temporally regulating cellular functions in vivo. Herein, we present a novel strategy for physically uncaging RGD using a magnetic field that allows safe and deep tissue penetration. We developed a heterodimeric nanoswitch consisting of a magnetic nanocage (MNC) coupled to an underlying RGD-coated gold nanoparticle (AuNP) via a long flexible linker. Magnetically controlled movement of MNC relative to AuNP allowed reversible uncaging and caging of RGD that modulate physical accessibility of RGD for integrin binding, thereby regulating stem cell adhesion, both in vitro and in vivo. Reversible RGD uncaging by the magnetic nanoswitch allowed temporal regulation of stem cell adhesion, differentiation, and mechanosensing. This physical and reversible RGD uncaging utilizing heterodimeric magnetic nanoswitch is unprecedented and holds promise in the remote control of cellular behaviors in vivo.

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.