Multimodal Magnetic Nanoclusters for Gene Delivery,Directed Migration,and Tracking of Stem Cells

This study develops multimodal magnetic nanoclusters (M‐MNCs) for gene transfer, directed migration, and tracking of human mesenchymal stem cells (hMSCs). The M‐MNCs are designed with 5 nm iron oxide nanoparticles and a fluorescent dye (i.e., Rhodamine B) in the matrix of the Food and Drug Administration approved polymer poly(lactide‐co‐glycolide) using a nanoemulsion method. The synthesized M‐MNCs have a hydrodynamic diameter of ≈150 nm, are internalized by stem cells via endocytosis, and deliver genes with high efficiency. The cellular internalization and gene expression efficiency of the clustered nanoparticles are significantly higher than that of single nanoparticles. The M‐MNC‐labeled hMSCs migrate upon application of a magnetic force and can be visualized by both optical and magnetic resonance (MR) imaging. In animal models, the M‐MNC‐labeled hMSCs are also successfully tracked using optical and MR imaging. Thus, the M‐MNCs not only allow the efficient delivery of genes to stem cells but also the tracking of cells in animal models. Taken together, the results show that this new type of nanocomposite can be of great help in future stem cell research and in the development of cell‐based therapeutic agents.     

Formation of Embryoid Bodies with Controlled Sizes and Maintained Pluripotency in Three‐Dimensional Inverse Opal Scaffolds

Embryoid bodies (EBs) are aggregates of cells derived from embryonic stem (ES) cells, which can serve as a good model system to investigate molecular and cellular interactions in the earliest stages of embryo development. Current methods for producing EBs mainly rely on the use of hanging drops or suspensions in non‐tissue culture treated plates, microwells, and spinner flasks. The capability of these methods is limited in terms of size uniformity and distribution as well as scalability. Here, a new platform based on three‐dimensional alginate inverse opal scaffolds with uniform pores is presented, where uniform EBs with controllable sizes could be produced in the pores and then recovered after disintegration of the scaffolds. The size of the EBs could be readily controlled by varying the culture time and/or by using scaffolds with different pore sizes. The EBs maintained their viability and undifferentiated state, and they were able to differentiate into specific lineages upon stimulation.     

3D‐Printed Microrobotic Transporters with Recapitulated Stem Cell Niche for Programmable and Active Cell Delivery

Poor retention rate, low targeting accuracy, and spontaneous transformation of stem cells present major clinical barriers to the success of therapies based on stem cell transplantation. To improve the clinical outcome, efforts should focus on the active delivery of stem cells to the target tissue site within a controlled environment, increasing survival, and fate for effective tissue regeneration. Here, a remotely steerable microrobotic cell transporter is presented with a biophysically and biochemically recapitulated stem cell niche for directing stem cells towards a pre‐destined cell lineage. The magnetically actuated double‐helical cell microtransporters of 76 µm length and 20 µm inner cavity diameter are 3D printed where biological and mechanical information regarding the stem cell niche are encoded at the single‐cell level. Cell‐loaded microtransporters are mobilized inside confined microchannels along computer‐controlled trajectories under rotating magnetic fields. The mesenchymal stem cells are shown retaining their differentiation capacities to commit to the osteogenic lineage when stimulated inside the microswimmers in vitro. Such a microrobotic approach has the potential to enable the development of active microcarriers with embedded functionalities for controlled and precisely localized therapeutic cell delivery.     

A Magneto-Responsive Hydrogel System for the Dynamic Mechano-Modulation of Stem Cell Niche

The biophysical microenvironment of cells dynamically evolves during embryonic development, leading to defined tissue specification. A versatile and highly adaptive magneto-responsive hydrogel system composed of magnetic nanorods (MNRs) and a stress-responsive polymeric matrix is developed to dynamically regulate the physical stem cell niche. The anisotropic magnetic/shape factor of nanorods is utilized to maximize the strains on the polymeric network, thus regulating the hydrogel modulus in a physiologically relevant range under a minimal magnitude of the applied magnetic fields below 4.5 mT. More significantly, the pre-alignment of MNRs induces greater collective strains on the polymeric network, resulting in a superior stiffening range, over a 500% increase as compared to that with randomly oriented nanorods. The pre-alignment of nanorods also enables a fast and reversible response under a magnetic field of the opposite polarity as well as spatially controlled heterogeneity of modulus within the hydrogel by applying anisotropic magnetic fields. The mechano-modulative capability of this system is validated by a mechanotransduction model with human-induced pluripotent stem cells where the locally controlled hydrogel modulus regulates the activation of mechano-sensitive signaling mediators and subsequent stem cell differentiation. Therefore, this magneto-responsive hydrogel system provides a platform to investigate various cellular behaviors under dynamic mechanical microenvironments.     

Multifunctional Upconversion Nanoparticles for Dual‐Modal Imaging‐Guided Stem Cell Therapy under Remote Magnetic Control

Stem cells have generated a great deal of excitement in cell‐based therapies. Here, a unique class of multifunctional nanoparticles (MFNPs) with both upconversion luminescence (UCL) and superparamagnetic properties is used for stem cell research. It is discovered that after being labeled with MFNPs, mouse mesenchymal stem cells (mMSCs) are able to maintain their viability and differentiation ability. In vivo UCL imaging of MFNP‐labeled mMSCs transplanted into animals is carried out, achieving ultrahigh tracking sensitivity with a detection limit as low as ≈10 cells in a mouse. Using both UCL optical and magnetic resonance (MR) imaging approaches, MFNP‐labeled mMSCs are tracked after being intraperitoneally injected into wound‐bearing mice under a magnetic field. The translocation of mMSCs from the injection site to the wound nearby the magnet is observed and, intriguingly, a remarkably improved tissue repair effect is observed as the result of magnetically induced accumulation of stem cells in the wound site. The results demonstrate the use MFNPs as novel multifunctional probes for labeling, in vivo tracking, and manipulation of stem cells, which is promising for imaging guided cell therapies and tissue engineering.     

MRI‐Visible siRNA Nanomedicine Directing Neuronal Differentiation of Neural Stem Cells in Stroke

A major challenge in stroke treatment is the restoration of neural circuit in which neuron function plays a central role. Although transplantation of exogenous neural stem cells (NSCs) is admittedly a promising therapeutical means, the treatment outcome is greatly affected due to the poor NSCs differentiation into neurons caused by myelin associated inhibitory factors binding to Nogo‐66 receptor (NgR). Herein, a nanoscale polymersome is developed to codeliver superparamagnetic iron oxide nanoparticles and siRNA targeting NgR gene (siNgR) into NSCs. This multifunctional nanomedicine directs neuronal differentiation of NSCs through silencing the NgR gene and meanwhile allows a noninvasive monitoring of NSC migration with magnetic resonance imaging. An improved recovery of neural function is achieved in rat ischemic stroke model. The results demonstrate the great potential of the multifunctional siRNA nanomedicine in stroke treatment based on stem cell transplantation.     

Generation of Monodisperse Inorganic–Organic Janus Microspheres in a Microfluidic Device

This study presents a simple synthetic approach for the in situ preparation of monodisperse hybrid Janus microspheres (HJM) having organic and inorganic parts in a PDMS‐based microfluidic device. Based on the mechanism of shear‐force‐driven break‐off, merged droplets of two photocurable oligomer solutions having distinctive properties are generated into an immiscible continuous phase. Functionalized perfluoropolyether (PFPE) as the organic phase and hydrolytic allylhydridopolycarbosilane (AHPCS) as the inorganic phase are used for the generation in aqueous medium of HJM with well‐defined morphology and high monodispersity (average diameter of 162 µm and a 3.5% coefficient of variation). The size and shape of the HJM is controlled by varying the flow rate of the disperse and continuous phases. The HJM have two distinctive regions: a hydrophobic hemisphere (PFPE) having a smooth surface and a relatively hydrophilic region (AHPCS) with a rough, porous surface. In addition, pyrolysis and subsequent oxidation of these HJM convert them into SiC‐based ceramic hemispheres through the removal of the organic portion and etching off the silica shell. The selective incorporation of magnetic nanoparticles into the inorganic part shows the feasibility of the forced assembly of HJM in an applied magnetic field.     

Bioactive Photodegradable Hydrogel for Cultivation and Retrieval of Embryonic Stem Cells

The development of a novel photodegradable heparin‐based hydrogel for cultivation and retrieval of embryonic stem cells is described. Mouse embryonic stem cells cultured atop the gel with encapsulated growth factors (GFs) express higher levels of differentiation markers compared to a standard protocol employing soluble GFs. Beyond improving differentiation of stem cells, the novel hydrogels can be used to release specific stem cell colonies without disturbing neighboring cells. This way, stem cell colonies can be retrieved at different time points and from different locations of the culture surface for polymerase chain reaction (PCR) analysis without the loss of the microenvironment context. The ability to retrieve some stem cell colonies without disturbing neighboring colonies will open possibilities for characterizing in‐dish heterogeneity of stem cell phenotype and will also allow to conserve cells/reagents. Overall, the bioactive photodegradable hydrogel developed in this study may offer new possibilities for cultivation and analysis of stem cells as well as other cell types.     

Labeling of Adipose‐Derived Stem Cells by Oleic‐Acid‐Modified Magnetic Nanoparticles

The in vivo tracking of adipose derived stem cells (ASCs) is of essential concern when they are used as seed cells in tissue engineering. This study explores the feasibility of using magnetic nanoparticles (MNs), a type of contrast agents in magnetic resonance imaging (MRI), to label ASCs such that the labeled ASCs could be tracked in vivo by MRI non‐invasively and repeatedly. To do this, MNs of <10 nm surface‐coated with oleic acid are synthesized via a high‐temperature solution‐phase reaction. Cytotoxicity of the as‐synthesized MNs at concentrations up to 0.1 mg mL^?1 on 10⁴ cells mL^?1 ASCs is evaluated by LDH release. Since only minor cytotoxicity is detected, the effects of the labeling technique on cellular behaviors and uptake by labeled cells are investigated. Cell proliferation and differentiation with and without MNs are compared. The results show that proliferation of ASCs (10⁴ cells mL^?1) labeled by MNs (0.05 mg mL^?1) is significantly enhanced and dependent on the labeling time. The MNs are located in the vesicles within cytoplasm of ASCs. The cellular uptake reaches as high as ～180 pg/cell. Nevertheless, the labeled ASCs still maintained adipogenic and osteogenic differentiation. Hence, the feasibility of labeling ASCs by oleic acid coated MNs is ascertained and it was better to label the cells during their quiescent stage. The labeled ASCs can also be in vivo detected by MRI in a subcutaneous model in vivo. Further MRI tracking of the labeled ASCs in long‐term follow‐up would thus follow this current study.     

Tissue Tapes—Phenolic Hyaluronic Acid Hydrogel Patches for Off‐the‐Shelf Therapy

Hydrogels have been applied to improve stem cell therapy and drug delivery, but current hydrogel‐based delivery methods are inefficient in clinical settings due to difficulty in handling and treatment processes, and low off‐the‐shelf availability. To overcome these limitations, an adhesive hyaluronic acid (HA) hydrogel patch is developed that acts as a ready‐to‐use tissue tape for therapeutic application. The HA hydrogel patches functionalized with phenolic moieties (e.g., catechol, pyrogallol) exhibit stronger tissue adhesiveness, greater elastic modulus, and increased off‐the‐shelf availability, compared with their bulk solution gel form. With this strategy, stem cells are efficiently engrafted onto beating ischemic hearts without injection, resulting in enhanced angiogenesis in ischemic regions and improving cardiac functions. HA hydrogel patches facilitate the in vivo engraftment of stem cell–derived organoids. The off‐the‐shelf availability of the hydrogel patch is also demonstrated as a drug‐loaded ready‐made tissue tape for topical drug delivery to promote wound healing. Importantly, the applicability of the cross‐linker‐free HA patch is validated for therapeutic cell and drug delivery. The study suggests that bioinspired phenolic adhesive hydrogel patches can provide an innovative method for simple but highly effective cell and drug delivery, increasing the off‐the‐shelf availability—a critically important component for translation to clinical settings.     

Enhanced Differentiation of Human Embryonic Stem Cells Toward Definitive Endoderm on Ultrahigh Aspect Ratio Nanopillars

Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have highlighted that the properties of the physical environment, such as substrate stiffness, affect cellular behavior. Here, mass‐produced, injection molded polycarbonate nanopillars are presented, where the surface mechanical properties, i.e., stiffness, can be controlled by the geometric design of the ultrahigh aspect ratio nanopillars (stiffness can be reduced by 25.0003). It is found that tall nanopillars, yielding softer surfaces, significantly enhance the induction of definitive endoderm cells from pluripotent human embryonic stem cells, resulting in more consistent differentiation of a pure population compared to planar control. By contrast, further differentiation toward the pancreatic ­endoderm is less successful on “soft” pillars when compared to “stiff” pillars or control, indicating differential cues during the different stages of differentiation. To accompany the mechanical properties of the nanopillars, the concept of surface shear modulus is introduced to describe the characteristics of engineered elastic surfaces through micro or nanopatterning. This provides a framework whereby comparisons can be drawn between such materials and bulk elastomeric materials.     

Protamine Functionalized Single‐Walled Carbon Nanotubes for Stem Cell Labeling and In Vivo Raman/Magnetic Resonance/Photoacoustic Triple‐Modal Imaging

Stem cells have shown great potential in regenerative medicine and attracted tremendous interests in recent years. Sensitive and reliable methods for stem cell labeling and in vivo tracking are thus urgently needed. Here, a novel approach to label human mesenchymal stem cells (hMSCs) with single‐walled carbon nanotubes (SWNTs) for in vivo tracking by triple‐modal imaging is presented. It is shown that polyethylene glycol (PEG) functionalized SWNTs conjugated with protamine (SWNT‐PEG‐PRO) exhibit extremely efficient cell entry into hMSCs, without affecting their proliferation and differentiation. The strong inherent resonance Raman scattering of SWNTs is used for in vitro and in vivo Raman imaging of SWNT‐PEG‐PRO‐labeled hMSCs, enabling ultrasensitive in vivo detection of as few as 500 stem cells administrated into mice. On the other hand, the metallic catalyst nanoparticles attached on nanotubes can be utilized as the T2‐contrast agent in magnetic resonance (MR) imaging of SWNT‐labeled hMSCs. Moreover, in vivo photoacoustic imaging of hMSCs in mice is also demonstrated. The work reveals that SWNTs with appropriate surface functionalization have the potential to serve as multifunctional nanoprobes for stem cell labeling and multi‐modal in vivo tracking.     

Active Regulation of On‐Demand Drug Delivery by Magnetically Triggerable Microspouters

Triggerable devices capable of on‐demand, controlled release of therapeutics are attractive options for the treatment of local diseases because of their potential to enhance therapeutic effectiveness with reduced systemic toxicity. Here, the design and fabrication of a miniaturized device, termed a microspouter, is described. This device is shown to provide active and precise control of localized delivery of drugs on demand. The microspouter is composed of a magnetic sponge to provide the force for drug release through magnetic field‐induced reversible deformation, a reservoir for the sponge installation and drug loading, and a soft membrane for sealing the device. Following application of a magnetic field to the microspouter, the shrinking of the sponge may trigger a spouting of drug through a membrane's microaperture. The efficiency of the device in controlling the dose and time course of drug release under different external magnetic fields has been demonstrated using methylene blue and docetaxel as model drugs. Additionally, the microspouter is found to have low background drug leakage that allows for tunable drug release in an ex vivo implantation experiment. All the results confirm the microspouter as a potential device for safe, long‐time, and controlled drug release in local disease treatment.     

Shape Anisotropy and Magnetization Modulation in Hexagonal Cobalt Nanowires

Ferromagnetic cobalt nanowires with high‐crystalline quality are synthesized using a low‐voltage electrodeposition method. High‐resolution transmission electron microscopy (HRTEM) and X‐ray diffraction (XRD) results show that the nanowires are uniform in size, and consist of predominantly hexagonal close‐packed (hcp) structure with the magnetocrystalline easy axis (c‐axis) perpendicular to the wire axis. Superconducting quantum interference device (SQUID) measurements illustrate the dominance of shape anisotropy, manifested by the weak temperature dependence of the enhanced coercive field along the wire axis. Furthermore, the magnetic structures of individual, segmented, or intersected nanowires are studied using magnetic force microscopy. This reveals a strong dipole at the two ends of the wire, together with a spatial magnetization modulation along the wire. Based on theoretical modeling, such intrinsic modulation is attributed to magnetization frustration due to the competition between the magnetocrystalline polarization along the easy axis and the shape anisotropy along the wire axis.     

Wireless Manipulation of Magnetic/Piezoelectric Micromotors for Precise Neural Stem‐Like Cell Stimulation

Precise neural electrical stimulation, which is a means of promoting neuronal regeneration, is a promising solution for patients with neurotrauma and neurodegenerative diseases. In this study, wirelessly controllable targeted motion and precise stimulation at the single‐cell level using S.platensis@Fe₃O₄@tBaTiO₃ micromotors are successfully demonstrated for the first time. A highly versatile and multifunctional biohybrid soft micromotor is fabricated via the integration of S.platensis with magnetic Fe₃O₄ nanoparticles and piezoelectric BaTiO₃ nanoparticles. The results show that this micromotor system can achieve navigation in a highly controllable manner under a low‐strength rotating magnetic field. The as‐developed system can achieve single‐cell targeted motion and then precisely induce the differentiation of the targeted neural stem‐like cell by converting ultrasonic energy to an electrical signal in situ owing to the piezoelectric effect. This new approach toward the high‐precision stimulation of neural stem‐like cells opens up new applications for micromotors and has excellent potential for precise neuronal regenerative therapies.     

Controllable Particle Adhesion with a Magnetically Actuated Synthetic Gecko Adhesive

Particle capture and release using controllable adhesion is of growing interest in many fields including reusable adhesives, solar panel cleaning, micro‐manipulation and robot locomotion where current methods of particle management are not sufficient. Here controllable adhesion to glass spheres with a new magnetically actuated synthetic gecko adhesive made from a magnetoelastomer composite is demonstrated. Adhesion is controlled by changing the effective elastic modulus of the surface through actuation of micro surface features with an external magnetic field. A compliant mechanics and magnetic torque analysis explains this general principle and generalizes the results for various geometries. Results show sphere pull‐off forces can be increased 10‐fold by changing the ridge orientation via the external magnetic field, and that the effective elastic modulus can be changed from 65 kPa to 1.5 MPa. Particle transport and release of 500‐ and 1000‐micrometer glass spheres is also demonstrated.     

A Smart Hyperthermia Nanofiber with Switchable Drug Release for Inducing Cancer Apoptosis

A smart hyperthermia nanofiber is described with simultaneous heat generation and drug release in response to ‘on‐off’ switching of alternating magnetic field (AMF) for induction of skin cancer apoptosis. The nanofiber is composed of a chemically‐crosslinkable temperature‐responsive polymer with an anticancer drug (doxorubicin; DOX) and magnetic nanoparticles (MNPs), which serve as a trigger of drug release and a source of heat, respectively. By chemical crosslinking, the nanofiber mesh shows switchable changes in the swelling ratio in response to alternating ‘on‐off’ switches of AMF because the self‐generated heat from the incorporated MNPs induces the deswelling of polymer networks in the nanofiber. Correspondingly, the ‘on‐off’ release of DOX from the nanofibers is observed in response to AMF. The 70% of human melanoma cells died in only 5 min application of AMF in the presence of the MNPs and DOX incorporated nanofibers by double effects of heat and drug. Taken together these advantages on both the nano‐ and macroscopic scale of nanofibers demonstrate that the dynamically and reversibly tunable structures have the potential to be utilized as a manipulative hyperthermia material as well as a switchable drug release platform by simple switching an AMF ‘on’ and ‘off’.     

Photoacoustic Imaging of Embryonic Stem Cell‐Derived Cardiomyocytes in Living Hearts with Ultrasensitive Semiconducting Polymer Nanoparticles

Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) have become promising tools to repair injured hearts. To achieve optimal outcomes, advanced molecular imaging methods are essential to accurately track these transplanted cells in the heart. In this study, it is demonstrated for the first time that a class of photoacoustic nanoparticles (PANPs) incorporating semiconducting polymers (SPs) as contrast agents can be used in the photoacoustic imaging (PAI) of transplanted hESC‐CMs in living mouse hearts. This is achieved by virtue of two benefits of PANPs. First, strong photoacoustic (PA) signals and specific spectral features of SPs allow PAI to sensitively detect and distinguish a small number of PANP‐labeled cells (2000) from background tissues. Second, the PANPs show a high efficiency for hESC‐CM labeling without adverse effects on cell structure, function, and gene expression. Assisted by ultrasound imaging, the delivery and engraftment of hESC‐CMs in living mouse hearts can be assessed by PANP‐based PAI with high spatial resolution (≈100 µm). In summary, this study explores and validates a novel application of SPs as a PA contrast agent to track labeled cells with high sensitivity and accuracy in vivo, highlighting the advantages of integrating PAI and PANPs to advance cardiac regenerative therapies.     

Soft Iron/Silicon Composite Tubes for Magnetic Peristaltic Pumping: Frequency‐Dependent Pressure and Volume Flow

The combination of force and flexibility enables controlled and soft movements. In sharp contrast, presently used machines are solid and mostly based on stiff driveshafts or cog wheels. Magnetic elastomers are realized through dispersion of small particles in polymer matrices and have attracted significant interest as soft actuators for controlled movement or conveying and are particularly attractive candidates for magnetic pump applications. At present, low magnetic particle loading and thus limited actuator strength have restricted the application of such materials. Here, the direct incorporation of metal microparticles into a very soft and flexible silicone and its application as an ultra‐flexible, yet strong magnetic tube, is described. Because metals have a far higher saturation magnetization and higher density than oxides, the resulting increased force/volume ratio afforded significantly stronger magnetic actuators with high mechanical stability, flexibility, and shape memory. Elliptical inner diameter shape of the tubing allowed a very efficient contraction of the tube by applying an external magnetic field. The combination of magnetic silicone tubes and a magnetic field generating device results in a magnetic peristaltic pump.     

An NIR‐II Fluorescence/Dual Bioluminescence Multiplexed Imaging for In Vivo Visualizing the Location,Survival, and Differentiation of Transplanted Stem Cells

The in vivo distribution, viability, and differentiation capability of transplanted stem cells are vital for the therapeutic efficacy of stem cell–based therapy. Herein, an NIR‐II fluorescence/dual bioluminescence multiplexed imaging method covering the visible and the second near‐infrared window from 400 to 1700 nm is successfully developed for in vivo monitoring the location, survival, and osteogenic differentiation of transplanted human mesenchymal stem cells (hMSCs) in a calvarial defect mouse model. The exogenous Ag₂S quantum dot–based fluorescence imaging in the second near‐infrared window is applied for visualizing the long‐term biodistribution of transplanted hMSCs. Endogenous red firefly luciferase (RFLuc)‐based bioluminescence imaging (BLI) and the collagen type 1 promoter–driven Gaussia luciferase (GLuc)‐based BLI are employed to report the survival and osteogenic differentiation statuses of the transplanted hMSCs. Meanwhile, by integrating the three imaging channels, multiple dynamic biological behaviors of transplanted hMSCs and the promotion effects of immunosuppression and the bone morphogenetic protein 2 on the survival and osteogenic differentiation of transplanted hMSCs are directly observed. The novel multiplexed imaging method can greatly expand the capability for multifunctional analysis of the fates and therapeutic capabilities of the transplanted stem cells, and aid in the improvement of stem cell–based regeneration therapies and their clinical translation.