Remote Control of Heterodimeric Magnetic Nanoswitch Regulates the Adhesion and Differentiation of Stem Cells.

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.

Remote Manipulation of Ligand Nano-Oscillations Regulates Adhesion and Polarization of Macrophages 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.

Smart Design of Nanostructures for Boosting Tumor Immunogenicity in Cancer Immunotherapy.

Although tumor immunotherapy has emerged as a promising therapeutic method for oncology, it encounters several limitations, especially concerning low response rates and potential off-targets that elicit side effects. Furthermore, tumor immunogenicity is the critical factor that predicts the success rate of immunotherapy, which can be boosted by the application of nanotechnology. Herein, we introduce the current approach of cancer immunotherapy and its challenges and the general methods to enhance tumor immunogenicity. Importantly, this review highlights the integration of anticancer chemo/immuno-based drugs with multifunctional nanomedicines that possess imaging modality to determine tumor location and can respond to stimuli, such as light, pH, magnetic field, or metabolic changes, to trigger chemotherapy, phototherapy, radiotherapy, or catalytic therapy to upregulate tumor immunogenicity. This promotion rouses immunological memory, such as enhanced immunogenic cell death, promoted maturation of dendritic cells, and activation of tumor-specific T cells against cancer. Finally, we express the related challenges and personal perspectives of bioengineered nanomaterials for future cancer immunotherapy.

Photocontrolled SiRNA Delivery and Biomarker-Triggered Luminogens of Aggregation-Induced Emission by Up-Conversion NaYF4:Yb3+Tm3+@SiO2 Nanoparticles for Inducing and Monitoring Stem-Cell Differentiation.

Controlling the differentiation of stem cells and monitoring cell differentiation has attracted much research interest since the discovery of stem cells. In this regard, a novel near-infrared (NIR) light-activated nanoplatform is obtained by encapsulating the photoactivatable caged compound (DMNPE/siRNA) and combining a MMP13 cleaved imaging peptide-tetrapheny-lethene (TPE) unit conjugated with the mesoporous silica-coated up-conversion nanoparticles (UCNPs) for the remote control of cell differentiation and, simultaneously, for the real-time monitoring of differentiation. Upon NIR light illumination, the photoactivated caged compound is activated, and the siRNA is released from UCNPs, allowing controlled differentiation of stem cells by light. More importantly, MMP13 enzyme triggered by osteogenic differentiation would effectively cleave the TPE probe peptide, thereby allowing the real-time monitoring of differentiation in living stem cells by aggregation-induced emission (AIE).

Near-infrared light-controlled regulation of intracellular calcium to modulate macrophage polarization.

Macrophages are multifunctional immune cells with diverse physiological functions such as fighting against infection, influencing progression of pathologies, maintaining homeostasis, and regenerating tissues. Macrophages can be induced to adopt distinct polarized phenotypes, such as classically activated pro-inflammatory (M1) phenotypes or alternatively activated anti-inflammatory and pro-healing (M2), to execute diverse and dynamic immune functions. However, unbalanced polarizations of macrophage can lead to various pathologies, such as atherosclerosis, obesity, tumor, and asthma. Thus, the capability to remotely control macrophage phenotypes is important to the success of treating many pathological conditions involving macrophages. In this study, we developed an upconversion nanoparticle (UCNP)-based photoresponsive nanocarrier for near-infrared (NIR) light-mediated control of intracellular calcium levels to regulate macrophage polarization. UCNP was coated with mesoporous silica (UCNP@mSiO2), into which loaded calcium regulators that can either supply or deplete calcium ions. UCNP@mSiO2 was chemically modified through serial coupling of photocleavable linker and Arg-Gly-Asp (RGD) peptide-bearing molecular cap via cyclodextrin-adamantine host-guest complexation. The RGD-bearing cap functioned as the photolabile gating structure to control the release of calcium regulators and facilitated the cellular uptake of UCNP@mSiO2 nanocarrier. The upconverted UV light emission from the UCNP@mSiO2 under NIR light excitation triggered the cleavage of cap and intracellular release of calcium regulators, thereby allowing temporal regulation on the intracellular calcium levels. Application of NIR light through skin tissue promoted M1 or M2 polarization of macrophages, by elevating or depleting intracellular calcium levels, respectively. To the best of our knowledge, this is the first demonstration of NIR light-mediated remote control on macrophage polarization. This photoresponsive nanocarrier offers the potential to remotely manipulate in vivo immune functions, such as inflammation or tissue regeneration, via NIR light-controlled macrophage polarization.

Magnetic Manipulation of Reversible Nanocaging Controls In Vivo Adhesion and Polarization of Macrophages.

Macrophages are key immune cells that perform various physiological functions, such as the maintenance of homeostasis, host defense, disease progression, and tissue regeneration. Macrophages adopt distinctly polarized phenotypes, such as pro-inflammatory M1 phenotype or anti-inflammatory (pro-healing) M2 phenotype, to execute disparate functions. The remotely controlled reversible uncaging of bioactive ligands, such as Arg-Gly-Asp (RGD) peptide, is an appealing approach for temporally regulating the adhesion and resultant polarization of macrophages on implants in vivo. Here, we utilize physical and reversible uncaging of RGD by a magnetic field that allows facile tissue penetration. We first conjugated a RGD-bearing gold nanoparticle (GNP) to the substrate and then a magnetic nanocage (MNC) to the GNP via a flexible linker to form the heterodimeric nanostructure. We magnetically manipulated nanoscale displacement of MNC and thus its proximity to the GNP to reversibly uncage and cage RGD. The uncaging of RGD temporally promoted the adhesion and subsequent M2 polarization of macrophages while inhibiting their M1 polarization both in vitro and in vivo. The RGD uncaging-mediated adhesion and M2 polarization of macrophages involved rho-associated protein kinase signaling. This study demonstrates physical and reversible uncaging of RGD to regulate the adhesion and polarization of host macrophages in vivo. This approach of magnetically regulating the heterodimer conformation for physical and reversible uncaging of RGD offers the promising potential to manipulate inflammatory or tissue-regenerative immune responses to the implants in vivo.

Self-assembled N-cadherin mimetic peptide hydrogels promote the chondrogenesis of mesenchymal stem cells through inhibition of canonical Wnt/ß-catenin signaling.

N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37 °C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3ß (GSK-3ß), which phosphorylates ß-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of ß-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of ß-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing ß-catenin nuclear translocation and the associated transcriptional activity of ß-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.

Optical µ-Printing of Cellular-Scale Microscaffold Arrays for 3D Cell Culture.

Guiding cell culture via engineering extracellular microenvironment has attracted tremendous attention due to its appealing potentials in the repair, maintenance, and development of tissues or even whole organs. However, conventional biofabrication technologies are usually less productive in fabricating microscale three-dimensional (3D) constructs because of the strident requirements in processing precision and complexity. Here we present an optical µ-printing technology to rapidly fabricate 3D microscaffold arrays for 3D cell culture and cell-scaffold interaction studies on a single chip. Arrays of 3D cubic microscaffolds with cubical sizes matching the single-cell size were fabricated to facilitate cell spreading on suspended microbeams so as to expose both apical and basal cell membranes. We further showed that the increasing of the cubical size of the microscaffolds led to enhanced spreading of the seeded human mesenchymal stem cells and activation of mechanosensing signaling, thereby promoting osteogenesis. Moreover, we demonstrated that the spatially selective modification of the surfaces of suspended beams with a bioactive coating (gelatin methacrylate) via an in-situ printing process allowed tailorable cell adhesion and spreading on the 3D microscaffolds.

Remote Control of Multimodal Nanoscale Ligand Oscillations Regulates Stem Cell Adhesion and Differentiation.

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.

Mussel-mimetic hydrogels with defined cross-linkers achieved via controlled catechol dimerization exhibiting tough adhesion for wet biological tissues.

The engineering of catechol-based hydrogels to bind to wet biological tissues with high adhesion energy remains a challenge. Herein, fast quinone-nucleophile coupling and dynamic boronate ester bond were used to enhance the interfacial adhesion and bulk cohesion of our dual-crosslinked hydrogels, respectively, for fabricating mussel-mimetic tough adhesives.