Single-cell adhesive profiling in an optofluidic device elucidates CD8+ T lymphocyte phenotypes in inflamed vasculature-like microenvironments.

Tissue infiltration by circulating leukocytes occurs via adhesive interactions with the local vasculature, but how the adhesive quality of circulating cells guides the homing of specific phenotypes to different vascular microenvironments remains undefined. We developed an optofluidic system enabling fluorescent labeling of photoactivatable cells based on their adhesive rolling velocity in an inflamed vasculature-mimicking microfluidic device under physiological fluid flow. In so doing, single-cell level multidimensional profiling of cellular characteristics could be characterized and related to the associated adhesive phenotype. When applied to CD8+ T cells, ligand/receptor expression profiles and subtypes associated with adhesion were revealed, providing insight into inflamed tissue infiltration capabilities of specific CD8+ T lymphocyte subsets and how local vascular microenvironmental features may regulate the quality of cellular infiltration. This methodology facilitates rapid screening of cell populations for enhanced homing capabilities under defined biochemical and biophysical microenvironments, relevant to leukocyte homing modulation in multiple pathologies.

Microengineered In Vitro Assays for Screening and Sorting Manufactured Therapeutic T Cells.

Adoptively transferred T cells constitute a major class of current and emergent cellular immunotherapies for the treatment of disease, including but not limited to cancer. Although key advancements in molecular recognition, genetic engineering, and manufacturing have dramatically enhanced their translational potential, therapeutic potency remains limited by poor homing and infiltration of transferred cells within target host tissues. In vitro microengineered homing assays with precise control over micromechanical and biological cues can address these shortcomings by enabling interrogation, screening, sorting, and optimization of therapeutic T cells based on their homing capacity. In this article, the working principles, application, and integration of microengineered homing assays for the mechanistic study of biophysical and biomolecular cues relevant to homing of therapeutic T cells are reviewed. The potential for these platforms to enable scalable enrichment and screening of next-generation manufactured T cell therapies for cancer is also discussed.

Adhesion analysis via a tumor vasculature-like microfluidic device identifies CD8+ T cells with enhanced tumor homing to improve cell therapy.

CD8+ T cell recruitment to the tumor microenvironment is critical for the success of adoptive cell therapy (ACT). Unfortunately, only a small fraction of transferred cells home to solid tumors. Adhesive ligand-receptor interactions have been implicated in CD8+ T cell homing; however, there is a lack of understanding of how CD8+ T cells interact with tumor vasculature-expressed adhesive ligands under the influence of hemodynamic flow. Here, the capacity of CD8+ T cells to home to melanomas is modeled ex vivo using an engineered microfluidic device that recapitulates the hemodynamic microenvironment of the tumor vasculature. Adoptively transferred CD8+ T cells with enhanced adhesion in flow in vitro and tumor homing in vivo improve tumor control by ACT in combination with immune checkpoint blockade. These results show that engineered microfluidic devices can model the microenvironment of the tumor vasculature to identify subsets of T cells with enhanced tumor infiltrating capabilities, a key limitation in ACT.

Reduced rotational flows enable the translation of surface-rolling microrobots in confined spaces.

Biological microorganisms overcome the Brownian motion at low Reynolds numbers by utilizing symmetry-breaking mechanisms. Inspired by them, various microrobot locomotion methods have been developed at the microscale by breaking the hydrodynamic symmetry. Although the boundary effects have been extensively studied for microswimmers and employed for surface-rolling microrobots, the behavior of microrobots in the proximity of multiple wall-based "confinement" is yet to be elucidated. Here, we study the confinement effect on the motion of surface-rolling microrobots. Our experiments demonstrate that the locomotion efficiency of spherical microrollers drastically decreases in confined spaces due to out-of-plane rotational flows generated during locomotion. Hence, a slender microroller design, generating smaller rotational flows, is shown to outperform spherical microrollers in confined spaces. Our results elucidate the underlying physics of surface rolling-based locomotion in confined spaces and present a design strategy with optimal flow generation for efficient propulsion in such areas, including blood vessels and microchannels.

Magnetically steerable bacterial microrobots moving in 3D biological matrices for stimuli-responsive cargo delivery.

Bacterial biohybrids, composed of self-propelling bacteria carrying micro/nanoscale materials, can deliver their payload to specific regions under magnetic control, enabling additional frontiers in minimally invasive medicine. However, current bacterial biohybrid designs lack high-throughput and facile construction with favorable cargoes, thus underperforming in terms of propulsion, payload efficiency, tissue penetration, and spatiotemporal operation. Here, we report magnetically controlled bacterial biohybrids for targeted localization and multistimuli-responsive drug release in three-dimensional (3D) biological matrices. Magnetic nanoparticles and nanoliposomes loaded with photothermal agents and chemotherapeutic molecules were integrated onto Escherichia coli with ~90% efficiency. Bacterial biohybrids, outperforming previously reported E. coli-based microrobots, retained their original motility and were able to navigate through biological matrices and colonize tumor spheroids under magnetic fields for on-demand release of the drug molecules by near-infrared stimulus. Our work thus provides a multifunctional microrobotic platform for guided locomotion in 3D biological networks and stimuli-responsive delivery of therapeutics for diverse medical applications.

Light-driven carbon nitride microswimmers with propulsion in biological and ionic media and responsive on-demand drug delivery.

We propose two-dimensional poly(heptazine imide) (PHI) carbon nitride microparticles as light-driven microswimmers in various ionic and biological media. Their high-speed (15 to 23 micrometer per second; 9.5 ± 5.4 body lengths per second) swimming in multicomponent ionic solutions with concentrations up to 5 M and without dedicated fuels is demonstrated, overcoming one of the bottlenecks of previous light-driven microswimmers. Such high ion tolerance is attributed to a favorable interplay between the particle's textural and structural nanoporosity and optoionic properties, facilitating ionic interactions in solutions with high salinity. Biocompatibility of these microswimmers is validated by cell viability tests with three different cell lines and primary cells. The nanopores of the swimmers are loaded with a model cancer drug, doxorubicin (DOX), resulting in a high (185%) loading efficiency without passive release. Controlled drug release is reported under different pH conditions and can be triggered on-demand by illumination. Light-triggered, boosted release of DOX and its active degradation products are demonstrated under oxygen-poor conditions using the intrinsic, environmentally sensitive and light-induced charge storage properties of PHI, which could enable future theranostic applications in oxygen-deprived tumor regions. These organic PHI microswimmers simultaneously address the current light-driven microswimmer challenges of high ion tolerance, fuel-free high-speed propulsion in biological media, biocompatibility, and controlled on-demand cargo release toward their biomedical, environmental, and other potential applications.

Liquid Crystal Structure of Supercooled Liquid Gallium and Eutectic Gallium-Indium.

Understanding the origin of structural ordering in supercooled liquid gallium (Ga) has been a great scientific quest in the past decades. Here, reflective polarized optical microscopy on Ga sandwiched between glasses treated with rubbed polymers reveals the onset of an anisotropic reflection at 120 °C that increases on cooling and persists down to room temperature or below. The polymer rubbing usually aligns the director of thermotropic liquid crystals (LCs) parallel to the rubbing direction. On the other hand, when Ga is sandwiched between substrates that align conventional LC molecules normal to the surface, the reflection is isotropic, but mechanical shear force induces anisotropic reflection that relaxes in seconds. Such alignment effects and shear-induced realignment are typical to conventional thermotropic LCs and indicate a LC structure of liquid Ga. Specifically, Ga textures obtained by atomic force and scanning electron microscopy reveal the existence of a lamellar structure corresponding to a smectic LC phase, while the nanometer-thin lamellar structure is transparent under transmission polarized optical microscopy. Such spatial molecular arrangements may be attributed to dimer molecular entities in the supercooled liquid Ga. The LC structure observation of electrically conductive liquid Ga can provide new opportunities in materials science and LC applications.

Shape anisotropy-governed locomotion of surface microrollers on vessel-like microtopographies against physiological flows.

Surface microrollers are promising microrobotic systems for controlled navigation in the circulatory system thanks to their fast speeds and decreased flow velocities at the vessel walls. While surface propulsion on the vessel walls helps minimize the effect of strong fluidic forces, three-dimensional (3D) surface microtopography, comparable to the size scale of a microrobot, due to cellular morphology and organization emerges as a major challenge. Here, we show that microroller shape anisotropy determines the surface locomotion capability of microrollers on vessel-like 3D surface microtopographies against physiological flow conditions. The isotropic (single, 8.5 µm diameter spherical particle) and anisotropic (doublet, two 4 µm diameter spherical particle chain) magnetic microrollers generated similar translational velocities on flat surfaces, whereas the isotropic microrollers failed to translate on most of the 3D-printed vessel-like microtopographies. The computational fluid dynamics analyses revealed larger flow fields generated around isotropic microrollers causing larger resistive forces near the microtopographies, in comparison to anisotropic microrollers, and impairing their translation. The superior surface-rolling capability of the anisotropic doublet microrollers on microtopographical surfaces against the fluid flow was further validated in a vessel-on-a-chip system mimicking microvasculature. The findings reported here establish the design principles of surface microrollers for robust locomotion on vessel walls against physiological flows.

Multifunctional surface microrollers for targeted cargo delivery in physiological blood flow.

Mobile microrobots offer great promise for minimally invasive targeted medical theranostic applications at hard-to-access regions inside the human body. The circulatory system represents the ideal route for navigation; however, blood flow impairs propulsion of microrobots especially for the ones with overall sizes less than 10 micrometers. Moreover, cell- and tissue-specific targeting is required for efficient recognition of disease sites and long-term preservation of microrobots under dynamic flow conditions. Here, we report cell-sized multifunctional surface microrollers with ~3.0 and ~7.8-micrometer diameters, inspired by leukocytes in the circulatory system, for targeted drug delivery into specific cells and controlled navigation inside blood flow. The leukocyte-inspired spherical microrollers are composed of magnetically responsive Janus microparticles functionalized with targeting antibodies against cancer cells (anti-HER2) and light-cleavable cancer drug molecules (doxorubicin). Magnetic propulsion and steering of the microrollers resulted in translational motion speeds up to 600 micrometers per second, around 76 body lengths per second. Targeting cancer cells among a heterogeneous cell population was demonstrated by active propulsion and steering of the microrollers over the cell monolayers. The multifunctional microrollers were propelled against physiologically relevant blood flow (up to 2.5 dynes per square centimeter) on planar and endothelialized microchannels. Furthermore, the microrollers generated sufficient upstream propulsion to locomote on inclined three-dimensional surfaces in physiologically relevant blood flow. The multifunctional microroller platform described here presents a bioinspired approach toward in vivo controlled propulsion, navigation, and targeted active cargo delivery in the circulatory system.

Reprogrammable shape morphing of magnetic soft machines.

Shape-morphing magnetic soft machines are highly desirable for diverse applications in minimally invasive medicine, wearable devices, and soft robotics. Despite recent progress, current magnetic programming approaches are inherently coupled to sequential fabrication processes, preventing reprogrammability and high-throughput programming. Here, we report a high-throughput magnetic programming strategy based on heating magnetic soft materials above the Curie temperature of the embedded ferromagnetic particles and reorienting their magnetic domains by applying magnetic fields during cooling. We demonstrate discrete, three-dimensional, and reprogrammable magnetization with high spatial resolution (~38 µm). Using the reprogrammable magnetization capability, reconfigurable mechanical behavior of an auxetic metamaterial structure, tunable locomotion of a surface-walking soft robot, and adaptive grasping of a soft gripper are shown. Our approach further enables high-throughput magnetic programming (up to 10 samples/min) via contact transfer. Heat-assisted magnetic programming strategy described here establishes a rich design space and mass-manufacturing capability for development of multiscale and reprogrammable soft machines.

Nanoerythrosome-functionalized biohybrid microswimmers.

Biohybrid microswimmers, which are realized through the integration of motile microscopic organisms with artificial cargo carriers, have a significant potential to revolutionize autonomous targeted cargo delivery applications in medicine. Nonetheless, there are many open challenges, such as motility performance and immunogenicity of the biological segment of the microswimmers, which should be overcome before their successful transition to the clinic. Here, we present the design and characterization of a biohybrid microswimmer, which is composed of a genetically engineered peritrichously flagellated Escherichia coli species integrated with red blood cell-derived nanoliposomes, also known as nanoerythrosomes. Initially, we demonstrated nanoerythrosome fabrication using the cell extrusion technique and characterization of their size and functional cell membrane proteins with dynamic light scattering and flow cytometry analyses, respectively. Then, we showed the construction of biohybrid microswimmers through the conjugation of streptavidin-modified bacteria with biotin-modified nanoerythrosomes by using non-covalent streptavidin interaction. Finally, we investigated the motility performance of the nanoerythrosome-functionalized biohybrid microswimmers and compared it with the free-swimming bacteria. The microswimmer design approach presented here could lead to the fabrication of personalized biohybrid microswimmers from patients' own cells with high fabrication efficiencies and motility performances.

Paper-based microchip electrophoresis for point-of-care hemoglobin testing.

Nearly 7% of the world's population live with a hemoglobin variant. Hemoglobins S, C, and E are the most common and significant hemoglobin variants worldwide. Sickle cell disease, caused by hemoglobin S, is highly prevalent in sub-Saharan Africa and in tribal populations of Central India. Hemoglobin C is common in West Africa, and hemoglobin E is common in Southeast Asia. Screening for significant hemoglobin disorders is not currently feasible in many low-income countries with the high disease burden. Lack of early diagnosis leads to preventable high morbidity and mortality in children born with hemoglobin variants in low-resource settings. Here, we describe HemeChip, the first miniaturized, paper-based, microchip electrophoresis platform for identifying the most common hemoglobin variants easily and affordably at the point-of-care in low-resource settings. HemeChip test works with a drop of blood. HemeChip system guides the user step-by-step through the test procedure with animated on-screen instructions. Hemoglobin identification and quantification is automatically performed, and hemoglobin types and percentages are displayed in an easily understandable, objective way. We show the feasibility and high accuracy of HemeChip via testing 768 subjects by clinical sites in the United States, Central India, sub-Saharan Africa, and Southeast Asia. Validation studies include hemoglobin E testing in Bangkok, Thailand, and hemoglobin S testing in Chhattisgarh, India, and in Kano, Nigeria, where the sickle cell disease burden is the highest in the world. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. Study design, methods, and results are presented according to the Standards for Reporting Diagnostic Accuracy (STARD). HemeChip correctly identified all subjects with hemoglobin S, C, and E variants with 100% sensitivity, and displayed an overall diagnostic accuracy of 98.4% in comparison to reference standard methods. HemeChip is a versatile, mass-producible microchip electrophoresis platform that addresses a major unmet need of decentralized hemoglobin analysis in resource-limited settings.

Cohesive self-organization of mobile microrobotic swarms.

Mobile microrobots are envisioned to be useful in a wide range of high-impact applications, many of which require cohesive group formation to maintain self-bounded swarms in the absence of confining boundaries. Cohesive group formation relies on a balance between attractive and repulsive interactions between agents. We found that a balance of magnetic dipolar attraction and multipolar repulsion between self-assembled particle chain microrobots enables their self-organization into cohesive clusters. Self-organized microrobotic clusters move above a solid substrate via a hydrodynamic self-propulsion mechanism. Cluster velocity increases with cluster size, resulting from collective hydrodynamic effects. Clustering is promoted by the strength of cohesive interactions and is hindered by the heterogeneities of individual microrobots. The scalability of cohesive interactions allows the formation of larger groups, whose internal spatiotemporal organization undergoes a transition from solid-like ordering to a liquid-like behavior with increasing cluster size. Our work elucidates the dynamics of clustering under cohesive interactions, and presents an approach for addressing the operation of microrobots as localized collectives.

Shape-encoded dynamic assembly of mobile micromachines.

Field-directed and self-propelled colloidal assembly have been used to build micromachines capable of performing complex motions and functions. However, integrating heterogeneous components into micromachines with specified structure, dynamics and function is still challenging. Here, we describe the dynamic self-assembly of mobile micromachines with desired configurations through pre-programmed physical interactions between structural and motor units. The assembly is driven by dielectrophoretic interactions, encoded in the three-dimensional shape of the individual parts. Micromachines assembled from magnetic and self-propelled motor parts exhibit reconfigurable locomotion modes and additional rotational degrees of freedom that are not available to conventional monolithic microrobots. The versatility of this site-selective assembly strategy is demonstrated on different reconfigurable, hierarchical and three-dimensional micromachine assemblies. Our results demonstrate how shape-encoded assembly pathways enable programmable, reconfigurable mobile micromachines. We anticipate that the presented design principle will advance and inspire the development of more sophisticated, modular micromachines and their integration into multiscale hierarchical systems.

Programmable Collective Behavior in Dynamically Self-Assembled Mobile Microrobotic Swarms.

Collective control of mobile microrobotic swarms is indispensable for their potential high-impact applications in targeted drug delivery, medical diagnostics, parallel micromanipulation, and environmental sensing and remediation. Without integrated electronics for sensing and actuation, current microrobotic systems should rely on physical interactions among individual microrobots for local communication and cooperation. Here, it is shown that mobile microrobotic swarms with well-defined collective behavior can be designed by engineering magnetic interactions among individual units. Microrobots, dynamically self-assembled from magnetic microparticles into linear chains, locomote on surfaces in response to a precessing magnetic field. Control over precessing magnetic field allows engineering attractive and repulsive interactions among microrobots and, thus, collective order with well-defined spatial organization and stable parallel operation over macroscale distances (≈1 cm) and through confining obstacles. The design approach described here addresses programmable assembly, propulsion, and collective behavior of dense mobile microrobot swarms simultaneously by engineering magnetic interactions and dynamic actuation of microrobots. The presented approach will advance swarm microrobotics by enabling facile and rapid formation of self-organized and reconfigurable microrobotic swarms with programmable collective order and stability.

Temperature Gradients Drive Bulk Flow Within Microchannel Lined by Fluid-Fluid Interfaces.

Surface tension gradients induce Marangoni flow, which may be exploited for fluid transport. At the micrometer scale, these surface-driven flows can be quite significant. By introducing fluid-fluid interfaces along the walls of microfluidic channels, bulk fluid flows driven by temperature gradients are observed. The temperature dependence of the fluid-fluid interfacial tension appears responsible for these flows. In this report, the design concept for a biocompatible microchannel capable of being powered by solar irradiation is provided. Using microscale particle image velocimetry, a bulk flow generated by apparent surface tension gradients along the walls is observed. The direction of flow relative to the imposed temperature gradient agrees with the expected surface tension gradient. The phenomenon's ability to replace bulky peripherals, like traditional syringe pumps, on a diagnostic microfluidic device that captures and detects leukocyte subpopulations within blood is demonstrated. Such microfluidic devices may be implemented for clinical assays at the point of care without the use of electricity.

Microalga-Powered Microswimmers toward Active Cargo Delivery.

Nature presents intriguing biological swimmers with innate energy harvesting abilities from their local environments. Use of natural swimmers as cargo delivery agents presents an alternative strategy to transport therapeutics inside the body to locations otherwise difficult to access by traditional delivery strategies. Herein, a biocompatible biohybrid microswimmer powered by a unicellular freshwater green microalga, Chlamydomonas reinhardtii, is reported. Polyelectrolyte-functionalized magnetic spherical cargoes (1 µm in diameter) are attached to surface of the microalgae via noncovalent interactions without the requirement for any chemical reaction. The 3D swimming motility of the constructed biohybrid algal microswimmers is characterized in the presence and absence of a uniform magnetic fields. In addition, motility of both microalgae and biohybrid algal microswimmers is investigated in various physiologically relevant conditions, including cell culture medium, human tubal fluid, plasma, and blood. Furthermore, it is demonstrated that the algal microswimmers are cytocompatible when co-cultured with healthy and cancerous cells. Finally, fluorescent isothiocyanate-dextran (a water-soluble polysaccharide) molecules are effectively delivered to mammalian cells using the biohybrid algal microswimmers as a proof-of-concept active cargo delivery demonstration. The microswimmer design described here presents a new class of biohybrid microswimmers with greater biocompatibility and motility for targeted delivery applications in medicine.

Seed-mediated synthesis of plasmonic gold nanoribbons using cancer cells for hyperthermia applications.

A surfactant-less, seed mediated, biological synthesis of two dimensional (2-D) nanoribbons in the presence of breast cancer cells (MCF7) is demonstrated. The diameter and yield of nanoribbons are tunable via seeds and gold precursor concentration. Such crystalline nanoribbons serve to enhance the Raman signals over MCF7 cells. The side and slopes of the triangular nanoplatelets fused as nanoribbons exhibit plasmon excitement in quadrupole resonance modes in the infrared region. Consequently, when irradiated with an infrared laser they show an excellent photothermal effect and rapid rise in temperature. The experimental results verified by finite-difference time-domain (FTDT) calculations reveal the presence of wedge-plasmon polaritons propagating along the edges of the nanoribbons. These simulations confirm that long aspect ratio nanoribbon's edges and vertices act as an active optical waveguide, allowing for heat propagation along the long axis, killing cancer cells in the process at lower power doses.

Soft erythrocyte-based bacterial microswimmers for cargo delivery.

Bacteria-propelled biohybrid microswimmers have recently shown to be able to actively transport and deliver cargos encapsulated into their synthetic constructs to specific regions locally. However, usage of synthetic materials as cargo carriers can result in inferior performance in load-carrying efficiency, biocompatibility, and biodegradability, impeding clinical translation of biohybrid microswimmers. Here, we report construction and external guidance of bacteria-driven microswimmers using red blood cells (RBCs; erythrocytes) as autologous cargo carriers for active and guided drug delivery. Multifunctional biohybrid microswimmers were fabricated by attachment of RBCs [loaded with anticancer doxorubicin drug molecules and superparamagnetic iron oxide nanoparticles (SPIONs)] to bioengineered motile bacteria, Escherichia coli MG1655, via biotin-avidin-biotin binding complex. Autonomous and on-board propulsion of biohybrid microswimmers was provided by bacteria, and their external magnetic guidance was enabled by SPIONs loaded into the RBCs. Furthermore, bacteria-driven RBC microswimmers displayed preserved deformability and attachment stability even after squeezing in microchannels smaller than their sizes, as in the case of bare RBCs. In addition, an on-demand light-activated hyperthermia termination switch was engineered for RBC microswimmers to control bacteria population after operations. RBCs, as biological and autologous cargo carriers in the biohybrid microswimmers, offer notable advantages in stability, deformability, biocompatibility, and biodegradability over synthetic cargo-carrier materials. The biohybrid microswimmer design presented here transforms RBCs from passive cargo carriers into active and guidable cargo carriers toward targeted drug and other cargo delivery applications in medicine.