Magnetically-actuated hydrogel-based achiral planar microswimmers for SERS detection: In situ coprecipitation for continuous loading of iron oxide nanoparticles.

Ultraviolet lithography is a very promising technology used for the batch fabrication of biomedical microswimmers. However, creating microswimmers that can swim at low Reynolds number using biocompatible materials while retaining strong magnetic properties and excellent biomedical functionality is a great challenge. Most of the previously reported biomedical microswimmers possess either strong magnetic properties by using non-biocompatible nickel coating or good biocompatibility by using iron oxide particle-embedded hydrogel with weak magnetism, but not both. Alternatively, iron oxide nanoparticles can be coated on the surface of microswimmers to improve magnetic properties; however, this method limited the usability of the microswimmers' surfaces. To address these shortcomings, this work utilized an in situ synthesis technique to generate high magnetic content inside hydrogel-based achiral planar microswimmers while leaving their surfaces free to be functionalized for SERS detection. The hydrogel matrices of the magnetically actuated hydrogel-based microswimmers were first prepared by ultraviolet lithography. Then, the high concentration of iron oxide was achieved through multiple continuous in situ coprecipitation cycles. Finally, the SERS detection capability of magnetically actuated hydrogel-based microswimmers was enabled by uniformly growing silver nanoparticles on the surface of the microswimmers. In the motion control tests, the microswimmers showed a high swimming efficiency, high step-out frequency, and consistent synchronized motion. Furthermore, the magnetically actuated hydrogel-based microswimmers were able to improve the detection efficiency of analytes under magnetic guidance.

2D Magnetic Microswimmers for Targeted Cell Transport and 3D Cell Culture Structure Construction.

Cell delivery using magnetic microswimmers is a promising tool for targeted therapy. However, it remains challenging to rapidly and uniformly manufacture cell-loaded microswimmers that can be assembled into cell-supporting structures at diseased sites. Here, rapid and uniform manufacturable 2D magnetic achiral microswimmers with pores were fabricated to deliver bone marrow mesenchymal stem cells (BMSCs) to regenerate articular-damaged cartilage. Under actuation with magnetic fields, the BMSC-loaded microswimmers take advantage of the achiral structure to exhibit rolling or swimming motions to travel on smooth and rough surfaces, up inclined planes, or in the bulk fluid. Cell viability, proliferation, and differentiation tests performed days after cell seeding verified the microswimmers' biocompatibility. Long-distance targeting and in situ assemblies into 3D cell-supporting structures with BMSC-loaded microswimmers were demonstrated using a knee model and U-shaped wells. Overall, combining the advantages of preparing an achiral 2D structured microswimmer with magnetically driven motility results in a platform for cell transport and constructing 3D cell cultures that can improve cell delivery at lesion sites for biomedical applications.

Imaging-Guided Biomimetic M1 Macrophage Membrane-Camouflaged Magnetic Nanorobots for Photothermal Immunotargeting Cancer Therapy.

Biohybrid micro/nanorobots have demonstrated improved therapeutic outcomes for targeting and treating diseases in preclinical trials. However, in vivo applications remain challenging due to a lack of sufficient targeting. Based on evidence that immune cells play a role in the immune modulation in the tumor microenvironment, we developed M1 macrophage membrane-coated magnetic photothermal nanocomplexes (MPN) for photoacoustic (PA) imaging-guided tumor therapy. The MPN were able to inherit the protein from the original macrophage cells and exert a targeted immunosuppression role. Integrating black phosphorus quantum dots and DOX also greatly enhanced reactive oxygen species generation and chemo-phototherapy efficacy. The results suggest that the MPN can be employed as an excellent tumor immunotargeting nanorobotic platform for modulating the tumor microenvironment under PA imaging and magnetic guidance and, thus, exert synergistic therapeutic efficacies.

A rolled-up-based fabrication method of 3D helical microrobots.

While the potential of using helical microrobots for biomedical applications, such as cargo transport, drug delivery, and micromanipulation, had been demonstrated, the viability to use them for practical applications is hindered by the cost, speed, and repeatability of current fabrication techniques. Hence, this paper introduces a simple, low-cost, high-throughput manufacturing process for single nickel layer helical microrobots with consistent dimensions. Photolithography and electron-beam (e-beam) evaporation were used to fabricate 2D parallelogram patterns that were sequentially rolled up into helical microstructures through the swelling effect of a photoresist sacrificial layer. Helical parameters were controlled by adjusting the geometric parameters of parallelogram patterns. To validate the fabrication process and characterize the microrobots' mobility, we characterized the structures and surface morphology of the microrobots using a scanning electron microscope and tested their steerability using feedback control, respectively. Finally, we conducted a benchmark comparison to demonstrate that the fabrication method can produce helical microrobots with swimming properties comparable to previously reported microrobots.

Fabrication of Bilayer Magnetically Actuated L-Shaped Microrobot Based on Chitosan via Photolithography.

Magnetically actuated microrobots showed increasing potential in various fields, especially in the biomedical area, such as invasive surgery, targeted cargo delivery, and treatment. However, it remains a challenge to incorporate biocompatible natural polymers that are favorable for practical biomedical applications. In this work, bilayer magnetic microrobots with an achiral planar design were fabricated using a biocompatible natural polymer and Fe3O4 nanoparticles through the photolithography by applying the layer-by-layer method. The microrobots consisted of a magnetic bottom layer and a photo-crosslinked chitosan top layer. The SEM results showed that the microrobot processed the L-shaped planar structure with the average width, length, and thickness of 99.18 ± 5.11 µm, 189.56 ± 11.37 µm, and 23.56 ± 4.08 µm, respectively. Moreover, microrobots actuated using a three-dimensional (3D) Helmholtz coil system was characterized and reached up to an average maximum velocity of 325.30 µm/s and a step-out frequency of 14 Hz. Furthermore, the microrobots exhibited excellent cell biocompatibility towards L929 cells in the CCK-8 assay. Therefore, the development of bi-layered chitosan-based microrobots offers a general solution for using magnetic microrobots in biomedical applications by providing an easy-to-fabricate, highly mobile microrobotic platform with the incorporation of biocompatible natural polymers for enhanced biocompatibility.

Improving Swimming Performance of Photolithography-Based Microswimmers Using Curvature Structures.

The emergence of robotic microswimmers and their huge potential in biomedical applications such as drug delivery, non-invasive surgery, and bio-sensing facilitates studies to improve their effectiveness. Recently, achiral microswimmers that have neither flexible nor helical structures have garnered attention because of their simple structures and fabrication process while preserving adequate swimming velocity and controllability. In this paper, the crescent shape was utilized to create photolithography-fabricated crescent-shaped achiral microswimmers. The microswimmers were actuated using rotating magnetic fields at low Reynolds numbers. Compared with the previously reported achiral microswimmers, the crescent-shaped microswimmers showed significant improvement in forward swimming speed. The effects of different curvatures, arm angles, and procession angles on the velocities of microswimmers were investigated. Moreover, the optimal swimming motion was defined by adjusting the field strength of the magnetic field. Finally, the effect of the thickness of the microswimmers on their swimming velocity was investigated.

High-performance and selective adsorption of ZIF-8/MIL-100 hybrids towards organic pollutants.

Environmental contamination by organic pollutants has become a pressing concern. In this study, metal-organic framework composites with a core-shell structure of MIL-100 wrapped around ZIF-8 (ZIF-MIL hybrids) were synthesized and characterized for their effectiveness to remove organic pollutants. First, a sequence of routine characterizations will examine the ZIF-MIL series samples' physicochemical properties and morphological characteristics. Then, the adsorption capacities of ZIF-MIL towards organic pollutants, including cationic dyes (methylene blue (MB), and rhodamine B (RHB)), anionic dyes (methyl orange (MO)), neutral pollutants (Sudan III (SD-III), tetracycline (TC) and amoxicillin (AMX)), were investigated. Among the ZIF-MIL series, ZIF-MIL-4 has an excellent specific surface area with high uptake of TC (1288 mg g-1) and RHB (1181 mg g-1). Based on the adsorption data from kinetic and dynamic studies, the adsorption process was closest to the pseudo-second-order kinetic model and Freundlich isotherm. In terms of thermodynamic parameter values, the adsorption of TC is an endothermic and spontaneous process, while the adsorption of RHB is an exothermic and spontaneous process. Furthermore, the reusability and selectivity studies of ZIF-MIL-4 towards TC and RHB exhibited significant regeneration ability and high selectivity. The effects of ionic strength and pH on pollutant removal efficiency were also tested. The experimental results showed that the main interactions between ZIF-MIL-4 and RHB or TC were weak coordination, electrostatic, hydrogen bonding, and π-π stacking interactions. Thus, the proposed MOF hybrid, by forming mixtures with other MOFs, can be a potential purifier with improved adsorption capacity and selectivity for organic pollutants as well as self-reusability.

Macrophage-compatible magnetic achiral nanorobots fabricated by electron beam lithography.

With the development and progress of nanotechnology, the prospect of using nanorobots to achieve targeted drug delivery is becoming possible. Although nanorobots can potentially improve nano-drug delivery systems, there remains a significant challenge to fabricating magnetically controllable nanorobots with a size suitable for drug delivery in complex in vivo environments. Most of the current research focused on the preparation and functionalization of microscale and milliscale robots due to the relative difficulties in fabricating nanoscale robots. To address this problem and move towards in vivo applications, this study uses electron beam lithography to fabricate achiral planar L-shaped nanorobots that are biocompatible with immune cells. Their minimal planar geometry enabled nanolithography to fabricate nanorobots with a minimum feature size down to 400 nm. Using an integrated imaging and control system, the locomotive behavior of the L-shaped nanorobots in a fluidic environment was studied by examining their velocity profiles and trajectories. Furthermore, the nanorobots exhibit excellent cell compatibility with various types of cells, including macrophage cells. Finally, the long-term cell culture medium immersion test demonstrated that the L-shaped nanorobots have robust stability. This work will demonstrate the potential to use these nanorobots to operate in vivo without triggering immune cell responses.

Immunomodulation and delivery of macrophages using nano-smooth drug-loaded magnetic microrobots for dual targeting cancer therapy.

To realize the potential to use micro/nanorobots for targeted cancer therapy, it is important to improve their biocompatibility and targeting ability. Here, we report on drug-loaded magnetic microrobots capable of polarizing macrophages into the antitumor phenotype to target and inhibit cancer cells. In vitro tests demonstrated that the microrobots have good biocompatibility with normal cells and immune cells. Positively charged DOX was loaded onto the surface of microrobots via electrostatic interactions and exhibited pH-responsive release behavior. The nano-smooth surfaces of the microrobots activated M1 polarization of macrophages, thus activating their intrinsic targeting and antitumor abilities toward cancer cells. Through dual targeting from magnetic guidance and M1 macrophages, the microrobots were able to target and kill cancer cells in a 3D tumor spheroid culture assay. These findings demonstrate a way to improve the tumor-targeting and antitumor abilities of microrobots through the combined use of magnetic control, macrophages, and pH-responsive drug release.

Stop-Flow Lithography for the Continuous Production of Degradable Hydrogel Achiral Crescent Microswimmers.

The small size of robotic microswimmers makes them suitable for performing biomedical tasks in tiny, enclosed spaces. Considering the effects of potentially long-term retention of microswimmers in biological tissues and the environment, the degradability of microswimmers has become one of the pressing issues in this field. While degradable hydrogel was successfully used to prepare microswimmers in previous reports, most hydrogel microswimmers could only be fabricated using two-photon polymerization (TPP) due to their 3D structures, resulting in costly robotic microswimmers solution. This limits the potential of hydrogel microswimmers to be used in applications where a large number of microswimmers are needed. Here, we proposed a new type of preparation method for degradable hydrogel achiral crescent microswimmers using a custom-built stop-flow lithography (SFL) setup. The degradability of the hydrogel crescent microswimmers was quantitatively analyzed, and the degradation rate in sodium hydroxide solution (NaOH) of different concentrations was investigated. Cytotoxicity assays showed the hydrogel crescent microswimmers had good biocompatibility. The hydrogel crescent microswimmers were magnetically actuated using a 3D Helmholtz coil system and were able to obtain a swimming efficiency on par with previously reported microswimmers. The results herein demonstrated the potential for the degradable hydrogel achiral microswimmers to become a candidate for microscale applications.

Magnetic bio-hybrid micro actuators.

Over the past two decades, there has been a growing body of work on wireless devices that can operate on the length scales of biological cells and even smaller. A class of these devices receiving increasing attention are referred to as bio-hybrid actuators: tools that integrate biological cells or subcellular parts with synthetic or inorganic components. These devices are commonly controlled through magnetic manipulation as magnetic fields and gradients can be generated with a high level of control. Recent work has demonstrated that magnetic bio-hybrid actuators can address common challenges in small scale fabrication, control, and localization. Additionally, it is becoming apparent that these magnetically driven bio-hybrid devices can display high efficiency and, in many cases, have the potential for self-repair and even self-replication. Combining these properties with magnetically driven forces and torques, which can be transmitted over significant distances, can be highly controlled, and are biologically safe, gives magnetic bio-hybrid actuators significant advantages over other classes of small scale actuators. In this review, we describe the theory and mechanisms required for magnetic actuation, classify bio-hybrid actuators by their diverse organic components, and discuss their current limitations. Insights into the future of coupling cells and cell-derived components with magnetic materials to fabricate multi-functional actuators are also provided.

Propulsion of magnetically actuated achiral planar microswimmers in Newtonian and non-Newtonian fluids.

Magnetic achiral planar microswimmers can be massively fabricated at low cost and are envisioned to be useful for in vivo biomedical applications. To understand locomotion in representative in vivo environments, we investigated the swimming performance of achiral planar microswimmers in methylcellulose solutions. We observed that these microswimmers displayed very similar swimming characteristics in methylcellulose solutions as in water. Furthermore, this study indicated that the range of precession angles increased as the concentration of MC solution increased. Last, it was demonstrated that achiral planar microswimmers with similar precession angles exhibited nearly the same dimensionless speeds in different concentrations of the methylcellulose solutions. Upon understanding swimmer kinematics, more effective control over the achiral planar microswimmers can be achieved to perform multiple biomedical tasks in in vivo environments.

Magnetic tri-bead microrobot assisted near-infrared triggered combined photothermal and chemotherapy of cancer cells.

Magnetic micro/nanorobots attracted much attention in biomedical fields because of their precise movement, manipulation, and targeting abilities. However, there is a lack of research on intelligent micro/nanorobots with stimuli-responsive drug delivery mechanisms for cancer therapy. To address this issue, we developed a type of strong covalently bound tri-bead drug delivery microrobots with NIR photothermal response azobenzene molecules attached to their carboxylic surface groups. The tri-bead microrobots are magnetic and showed good cytocompatibility even when their concentration is up to 200 µg/mL. In vitro photothermal experiments demonstrated fast NIR-responsive photothermal property; the microrobots were heated to 50 °C in 4 min, which triggered a significant increase in drug release. Motion control of the microrobots inside a microchannel demonstrated the feasibility of targeted therapy on tumor cells. Finally, experiments with lung cancer cells demonstrated the effectiveness of targeted chemo-photothermal therapy and were validated by cell viability assays. These results indicated that tri-bead microrobots have excellent potential for targeted chemo-photothermal therapy for lung cancer cell treatment.

Nanorobots-Assisted Natural Computation for Multifocal Tumor Sensitization and Targeting.

We have proposed a new tumor sensitization and targeting (TST) framework, named in vivo computation, in our previous investigations. The problem of TST for an early and microscopic tumor is interpreted from the computational perspective with nanorobots being the "natural" computing agents, the high-risk tissue being the search space, the tumor targeted being the global optimal solution, and the tumor-triggered biological gradient field (BGF) providing the aided knowledge for fitness evaluation of nanorobots. This natural computation process can be seen as on-the-fly path planning for nanorobot swarms with an unknown target position, which is different from the traditional path planning methods. Our previous works are focusing on the TST for a solitary lesion, where we proposed the weak priority evolution strategy (WP-ES) to adapt to the actuating mode of the homogeneous magnetic field used in the state-of-the-art nanorobotic platforms, and some in vitro validations were performed. In this paper, we focus on the problem of TST for multifocal tumors, which can be seen as a multimodal optimization problem for the "natural" computation. To overcome this issue, we propose a sequential targeting strategy (Se-TS) to complete TST for the multiple lesions with the assistance of nanorobot swarms, which are maneuvered by the external actuating and tracking devices according to the WP-ES. The Se-TS is used to modify the BGF landscape after a tumor is detected by a nanorobot swarm with the gathered BGF information around the detected tumor. Next, another nanorobot swarm will be employed to find the second tumor according to the modified BGF landscape without being misguided to the previous one. In this way, all the tumor lesions will be detected one by one. In other words, the paths of nanorobots to find the targets can be generated successively with the sequential modification of the BGF landscape. To demonstrate the effectiveness of the proposed Se-TS, we perform comprehensive simulation studies by enhancing the WP-ES based swarm intelligence algorithms using this strategy considering the realistic in-body constraints. The performance is compared against that of the "brute-force" search, which corresponds to the traditional systemic tumor targeting, and also against that of the standard swarm intelligence algorithms from the algorithmic perspective. Furthermore, some in vitro experiments are performed by using Janus microparticles as magnetic nanorobots, a two-dimensional microchannel network as the human vasculature, and a magnetic nanorobotic control system as the external actuating and tracking system. Results from the in silico simulations and in vitro experiments verify the effectiveness of the proposed Se-TS for two representative BGF landscapes.

Perspective: Computational Nanobiosensing.

With the development of nanotechnology, externally manipulable or self-regulatable smart nanosystems can be utilized as effective tools for computational nanobiosensing, where natural computing strategies are exploited for knowledge-aided nanobiosensing.

µ-PIV Measurements of Flows Generated by Photolithography-Fabricated Achiral Microswimmers.

Robotic micro/nanoswimmers can potentially be used as tools for medical applications, such as drug delivery and noninvasive surgery. Recently, achiral microswimmers have gained significant attention because of their simple structures, which enables high-throughput fabrication and size scalability. Here, microparticle image velocimetry (µ-PIV) was used to study the hydrodynamics of achiral microswimmers near a boundary. The structures of these microswimmers resemble the letter L and were fabricated using photolithography and thin-film deposition. Through µ-PIV measurements, the velocity flow fields of the microswimmers rotating at different frequencies were observed. The results herein yield an understanding of the hydrodynamics of the L-shaped microswimmers, which will be useful in applications such as fluidic manipulation.

Biosensing-by-Learning Direct Targeting Strategy for Enhanced Tumor Sensitization.

We propose a novel iterative-optimization-inspired direct targeting strategy (DTS) for smart nanosystems, which harness swarms of externally manipulable nanoswimmers assembled by magnetic nanoparticles (MNPs) for knowledge-aided tumor sensitization and targeting. We aim to demonstrate through computational experiments that the proposed DTS can significantly enhance the accumulation of MNPs in the tumor site, which serve as a contrast agent in various medical imaging modalities, by using the shortest possible physiological routes and with minimal systemic exposure. The epicenter of a tumor corresponds to the global maximum of an externally measurable objective function associated with an in vivo tumor-triggered biological gradient; the domain of the objective function is the tissue region at a high risk of malignancy; swarms of externally controllable magnetic nanoswimmers for tumor sensitization are modeled as the guess inputs. The objective function may be resulted from a passive phenomenon such as reduced blood flow or increased kurtosis of microvasculature due to tumor angiogenesis; otherwise, the objective function may involve an active phenomenon such as the fibrin formed during the coagulation cascade activated by tumor-targeted "activator" nanoparticles. Subsequently, the DTS can be interpreted from the iterative optimization perspective: guess inputs (i.e., swarms of nanoswimmers) are continuously updated according to the gradient of the objective function in order to find the optimum (i.e., tumor) by moving through the domain (i.e., tissue under screening). Along this line of thought, we propose the computational model based on the gradient descent (GD) iterative method to describe the GD-inspired DTS, which takes into account the realistic in vivo propagation scenario of nanoswimmers. By means of computational experiments, we show that the GD-inspired DTS yields higher probabilities of tumor sensitization and more significant dose accumulation compared to the "brute-force" search, which corresponds to the systemic targeting scenario where drug nanoparticles attempt to target a tumor by enumerating all possible pathways in the complex vascular network. The knowledge-aided DTS has potential to enhance the tumor sensitization and targeting performance remarkably by exploiting the externally measurable, tumor-triggered biological gradients. We believe that this work motivates a novel biosensing-by-learning framework facilitated by externally manipulable, smart nanosystems.

Bacteria-inspired nanorobots with flagellar polymorphic transformations and bundling.

Wirelessly controlled nanoscale robots have the potential to be used for both in vitro and in vivo biomedical applications. So far, the vast majority of reported micro- and nanoscale swimmers have taken the approach of mimicking the rotary motion of helical bacterial flagella for propulsion, and are often composed of monolithic inorganic materials or photoactive polymers. However, currently no man-made soft nanohelix has the ability to rapidly reconfigure its geometry in response to multiple forms of environmental stimuli, which has the potential to enhance motility in tortuous heterogeneous biological environments. Here, we report magnetic actuation of self-assembled bacterial flagellar nanorobotic swimmers. Bacterial flagella change their helical form in response to environmental stimuli, leading to a difference in propulsion before and after the change in flagellar form. We experimentally and numerically characterize this response by studying the swimming of three flagellar forms. Also, we demonstrate the ability to steer these devices and induce flagellar bundling in multi-flagellated nanoswimmers.

Autonomous dynamic obstacle avoidance for bacteria-powered microrobots (BPMs) with modified vector field histogram.

In order to broaden the use of microrobots in practical fields, autonomous control algorithms such as obstacle avoidance must be further developed. However, most previous studies of microrobots used manual motion control to navigate past tight spaces and obstacles while very few studies demonstrated the use of autonomous motion. In this paper, we demonstrated a dynamic obstacle avoidance algorithm for bacteria-powered microrobots (BPMs) using electric field in fluidic environments. A BPM consists of an artificial body, which is made of SU-8, and a high dense layer of harnessed bacteria. BPMs can be controlled using externally applied electric fields due to the electrokinetic property of bacteria. For developing dynamic obstacle avoidance for BPMs, a kinematic model of BPMs was utilized to prevent collision and a finite element model was used to characteristic the deformation of an electric field near the obstacle walls. In order to avoid fast moving obstacles, we modified our previously static obstacle avoidance approach using a modified vector field histogram (VFH) method. To validate the advanced algorithm in experiments, magnetically controlled moving obstacles were used to intercept the BPMs as the BPMs move from the initial position to final position. The algorithm was able to successfully guide the BPMs to reach their respective goal positions while avoiding the dynamic obstacles.

Versatile microrobotics using simple modular subunits.

The realization of reconfigurable modular microrobots could aid drug delivery and microsurgery by allowing a single system to navigate diverse environments and perform multiple tasks. So far, microrobotic systems are limited by insufficient versatility; for instance, helical shapes commonly used for magnetic swimmers cannot effectively assemble and disassemble into different size and shapes. Here by using microswimmers with simple geometries constructed of spherical particles, we show how magnetohydrodynamics can be used to assemble and disassemble modular microrobots with different physical characteristics. We develop a mechanistic physical model that we use to improve assembly strategies. Furthermore, we experimentally demonstrate the feasibility of dynamically changing the physical properties of microswimmers through assembly and disassembly in a controlled fluidic environment. Finally, we show that different configurations have different swimming properties by examining swimming speed dependence on configuration size.