Janus microparticles-based targeted and spatially-controlled piezoelectric neural stimulation via low-intensity focused ultrasound.

Electrical stimulation is a fundamental tool in studying neural circuits, treating neurological diseases, and advancing regenerative medicine. Injectable, free-standing piezoelectric particle systems have emerged as non-genetic and wireless alternatives for electrode-based tethered stimulation systems. However, achieving cell-specific and high-frequency piezoelectric neural stimulation remains challenging due to high-intensity thresholds, non-specific diffusion, and internalization of particles. Here, we develop cell-sized 20 µm-diameter silica-based piezoelectric magnetic Janus microparticles (PEMPs), enabling clinically-relevant high-frequency neural stimulation of primary neurons under low-intensity focused ultrasound. Owing to its functionally anisotropic design, half of the PEMP acts as a piezoelectric electrode via conjugated barium titanate nanoparticles to induce electrical stimulation, while the nickel-gold nanofilm-coated magnetic half provides spatial and orientational control on neural stimulation via external uniform rotating magnetic fields. Furthermore, surface functionalization with targeting antibodies enables cell-specific binding/targeting and stimulation of dopaminergic neurons. Taking advantage of such functionalities, the PEMP design offers unique features towards wireless neural stimulation for minimally invasive treatment of neurological diseases.

Roadmap for Clinical Translation of Mobile Microrobotics.

Medical microrobotics is an emerging field to revolutionize clinical applications in diagnostics and therapeutics of various diseases. On the other hand, the mobile microrobotics field has important obstacles to pass before clinical translation. This article focuses on these challenges and provides a roadmap of medical microrobots to enable their clinical use. From the concept of a "magic bullet" to the physicochemical interactions of microrobots in complex biological environments in medical applications, there are several translational steps to consider. Clinical translation of mobile microrobots is only possible with a close collaboration between clinical experts and microrobotics researchers to address the technical challenges in microfabrication, safety, and imaging. The clinical application potential can be materialized by designing microrobots that can solve the current main challenges, such as actuation limitations, material stability, and imaging constraints. The strengths and weaknesses of the current progress in the microrobotics field are discussed and a roadmap for their clinical applications in the near future is outlined.

Acoustic Trapping and Manipulation of Hollow Microparticles under Fluid Flow Using a Single-Lens Focused Ultrasound Transducer.

Microparticle manipulation and trapping play pivotal roles in biotechnology. To achieve effective manipulation within fluidic flow conditions and confined spaces, it is necessary to consider the physical properties of microparticles and the types of trapping forces applied. While acoustic waves have shown potential for manipulating microparticles, the existing setups involve complex actuation mechanisms and unstable microbubbles. Consequently, the need persists for an easily deployable acoustic actuation setup with stable microparticles. Here, we propose the use of hollow borosilicate microparticles possessing a rigid thin shell, which can be efficiently trapped and manipulated using a single-lens focused ultrasound (FUS) transducer under physiologically relevant flow conditions. These hollow microparticles offer stability and advantageous acoustic properties. They can be scaled up and mass-produced, making them suitable for systemic delivery. Our research demonstrates the successful trapping dynamics of FUS within circular tubings of varying diameters, validating the effectiveness of the method under realistic flow rates and ultrasound amplitudes. We also showcase the ability to remove hollow microparticles by steering the FUS transducer against the flow. Furthermore, we present potential biomedical applications, such as active cell tagging and navigation in bifurcated channels as well as ultrasound imaging in mouse cadaver liver tissue.

Size-Dependent Locomotion Ability of Surface Microrollers on Physiologically Relevant Microtopographical Surfaces.

Controlled microrobotic navigation inside the body possesses significant potential for various biomedical engineering applications. Successful application requires considering imaging, control, and biocompatibility. Interaction with biological environments is also a crucial factor in ensuring safe application, but can also pose counterintuitive hydrodynamic barriers, limiting the use of microrobots. Surface rolling microrobots or surface microrollers is a robust microrobotic platform with significant potential for various applications; however, conventional spherical microrollers have limited locomotion ability over biological surfaces due to microtopography effects resulting from cell microtopography in the size range of 2-5 µm. Here, the impact of the microtopography effect on spherical microrollers of different sizes (5, 10, 25, and 50 µm) is investigated using computational fluid dynamics simulations and experiments. Simulations revealed that the microtopography effect becomes insignificant for increasing microroller sizes, such as 50 µm. Moreover, it is demonstrated that 50 µm microrollers exhibited smooth locomotion ability on in vitro cell layers and inside blood vessels of a chicken embryo model. These findings offer rational design principles for surface microrollers for their potential practical biomedical applications.

The mismatch between experimental and computational fluid dynamics analyses for magnetic surface microrollers.

Magnetically actuated Janus surface microrollers are promising microrobotic platform with numerous potential biomedical engineering applications. While the locomotion models based on a "rotating sphere on a nearby wall" can be adapted to surface microrollers, real-world dynamics may differ from the proposed theories/simulations. In this study, we examine the locomotion efficiency of surface microrollers with diameters of 5, 10, 25, and 50 µm and demonstrate that computational fluid dynamics simulations cannot accurately capture locomotion characteristics for different sizes of microrollers. Specifically, we observe a significant mismatch between lift forces predicted by simulations and opposite balancing forces, particularly for smaller microrollers. We propose the existence of an unaccounted force component in the direction of lift, which is not included in the computational fluid dynamics simulations. Overall, our findings provide a deeper understanding of the physical mechanisms underlying surface microroller locomotion and have important implications for future applications in biomedical engineering.

3D-printed micrometer-scale wireless magnetic cilia with metachronal programmability.

Biological cilia play essential roles in self-propulsion, food capture, and cell transportation by performing coordinated metachronal motions. Experimental studies to emulate the biological cilia metachronal coordination are challenging at the micrometer length scale because of current limitations in fabrication methods and materials. We report on the creation of wirelessly actuated magnetic artificial cilia with biocompatibility and metachronal programmability at the micrometer length scale. Each cilium is fabricated by direct laser printing a silk fibroin hydrogel beam affixed to a hard magnetic FePt Janus microparticle. The 3D-printed cilia show stable actuation performance, high temperature resistance, and high mechanical endurance. Programmable metachronal coordination can be achieved by programming the orientation of the identically magnetized FePt Janus microparticles, which enables the generation of versatile microfluidic patterns. Our platform offers an unprecedented solution to create bioinspired microcilia for programmable microfluidic systems, biomedical engineering, and biocompatible implants.

Multifunctional 3D-Printed Pollen Grain-Inspired Hydrogel Microrobots for On-Demand Anchoring and Cargo Delivery.

While a majority of wireless microrobots have shown multi-responsiveness to implement complex biomedical functions, their functional executions are strongly dependent on the range of stimulus inputs, which curtails their functional diversity. Furthermore, their responsive functions are coupled to each other, which results in the overlap of the task operations. Here, a 3D-printed multifunctional microrobot inspired by pollen grains with three hydrogel components is demonstrated: iron platinum (FePt) nanoparticle-embedded pentaerythritol triacrylate (PETA), poly N-isopropylacrylamide (pNIPAM), and poly N-isopropylacrylamide acrylic acid (pNIPAM-AAc) structures. Each of these structures exhibits their respective targeted functions: responding to magnetic fields for torque-driven surface rolling and steering, exhibiting temperature responsiveness for on-demand surface attachment (anchoring), and pH-responsive cargo release. The versatile multifunctional pollen grain-inspired robots conceptualized here pave the way for various future medical microrobots to improve their projected performance and functional diversity.

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.

On-demand anchoring of wireless soft miniature robots on soft surfaces.

Untethered soft miniature robots capable of accessing hard-to-reach regions can enable new, disruptive, and minimally invasive medical procedures. However, once the control input is removed, these robots easily move from their target location because of the dynamic motion of body tissues or fluids, thereby restricting their use in many long-term medical applications. To overcome this, we propose a wireless spring-preloaded barbed needle release mechanism, which can provide up to 1.6 N of force to drive a barbed needle into soft tissues to allow robust on-demand anchoring on three-dimensional (3D) surfaces. The mechanism is wirelessly triggered using radio-frequency remote heating and can be easily integrated into existing untethered soft robotic platforms without sacrificing their mobility. Design guidelines aimed at maximizing anchoring over the range of the most biological tissues (kPa range) and extending the operating depth of the device inside the body (up to 75%) are also presented. Enabled by these advances, we achieve robust anchoring on a variety of ex vivo tissues and demonstrate the usage of such a device when integrated with existing soft robotic platforms and medical imaging. Moreover, by simply changing the needle, we demonstrate additional functionalities such as controlled detachment and subsurface drug delivery into 3D cancer spheroids. Given these capabilities, our proposed mechanism could enable the development of a new class of biomedical-related functionalities, such as local drug delivery, disease monitoring, and hyperthermia for future untethered soft medical robots.

Real-time 3D optoacoustic tracking of cell-sized magnetic microrobots circulating in the mouse brain vasculature.

Mobile microrobots hold remarkable potential to revolutionize health care by enabling unprecedented active medical interventions and theranostics, such as active cargo delivery and microsurgical manipulations in hard-to-reach body sites. High-resolution imaging and control of cell-sized microrobots in the in vivo vascular system remains an unsolved challenge toward their clinical use. To overcome this limitation, we propose noninvasive real-time detection and tracking of circulating microrobots using optoacoustic imaging. We devised cell-sized nickel-based spherical Janus magnetic microrobots whose near-infrared optoacoustic signature is enhanced via gold conjugation. The 5-, 10-, and 20-µm-diameter microrobots are detected volumetrically both in bloodless ex vivo tissues and under real-life conditions with a strongly light-absorbing blood background. We further demonstrate real-time three-dimensional tracking and magnetic manipulation of the microrobots circulating in murine cerebral vasculature, thus paving the way toward effective and safe operation of cell-sized microrobots in challenging and clinically relevant intravascular environments.

High shear rate propulsion of acoustic microrobots in complex biological fluids.

Untethered microrobots offer a great promise for localized targeted therapy in hard-to-access spaces in our body. Despite recent advancements, most microrobot propulsion capabilities have been limited to homogenous Newtonian fluids. However, the biological fluids present in our body are heterogeneous and have shear rate-dependent rheological properties, which limit the propulsion of microrobots using conventional designs and actuation methods. We propose an acoustically powered microrobotic system, consisting of a three-dimensionally printed 30-micrometer-diameter hollow body with an oscillatory microbubble, to generate high shear rate fluidic flow for propulsion in complex biofluids. The acoustically induced microstreaming flow leads to distinct surface-slipping and puller-type propulsion modes in Newtonian and non-Newtonian fluids, respectively. We demonstrate efficient propulsion of the microrobots in diverse biological fluids, including in vitro navigation through mucus layers on biologically relevant three-dimensional surfaces. The microrobot design and high shear rate propulsion mechanism discussed herein could open new possibilities to deploy microrobots in complex biofluids toward minimally invasive targeted therapy.

3D Microprinting of Iron Platinum Nanoparticle-Based Magnetic Mobile Microrobots.

Wireless magnetic microrobots are envisioned to revolutionize minimally invasive medicine. While many promising medical magnetic microrobots are proposed, the ones using hard magnetic materials are not mostly biocompatible, and the ones using biocompatible soft magnetic nanoparticles are magnetically very weak and, therefore, difficult to actuate. Thus, biocompatible hard magnetic micro/nanomaterials are essential toward easy-to-actuate and clinically viable 3D medical microrobots. To fill such crucial gap, this study proposes ferromagnetic and biocompatible iron platinum (FePt) nanoparticle-based 3D microprinting of microrobots using the two-photon polymerization technique. A modified one-pot synthesis method is presented for producing FePt nanoparticles in large volumes and 3D printing of helical microswimmers made from biocompatible trimethy- lolpropane ethoxylate triacrylate (PETA) polymer with embedded FePt nanoparticles. The 30 µm long helical magnetic microswimmers are able to swim at speeds of over five body lengths per second at 200 Hz, making them the fastest helical swimmer in the tens of micrometer length scale at the corresponding low- magnitude actuation fields of 5-10 mT. It is also experimentally in vitro verified that the synthesized FePt nanoparticles are biocompatible. Thus, such 3D-printed microrobots are biocompatible and easy to actuate toward creating clinically viable future medical microrobots.

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.

Recent advances in the design of implantable insulin secreting heterocellular islet organoids.

Islet transplantation has proved one of the most remarkable transmissions from an experimental curiosity into a routine clinical application for the treatment of type I diabetes (T1D). Current efforts for taking this technology one-step further are now focusing on overcoming islet donor shortage, engraftment, prolonged islet availability, post-transplant vascularization, and coming up with new strategies to eliminate lifelong immunosuppression. To this end, insulin secreting 3D cell clusters composed of different types of cells, also referred as heterocellular islet organoids, spheroids, or pseudoislets, have been engineered to overcome the challenges encountered by the current islet transplantation protocols. ß-cells or native islets are accompanied by helper cells, also referred to as accessory cells, to generate a cell cluster that is not only able to accurately secrete insulin in response to glucose, but also superior in terms of other key features (e.g. maintaining a vasculature, longer durability in vivo and not necessitating immunosuppression after transplantation). Over the past decade, numerous 3D cell culture techniques have been integrated to create an engineered heterocellular islet organoid that addresses current obstacles. Here, we first discuss the different cell types used to prepare heterocellular organoids for islet transplantation and their contribution to the organoids design. We then introduce various cell culture techniques that are incorporated to prepare a fully functional and insulin secreting organoids with select features. Finally, we discuss the challenges and present a future outlook for improving clinical outcomes of islet transplantation.

Elucidating the interaction dynamics between microswimmer body and immune system for medical microrobots.

The structural design parameters of a medical microrobot, such as the morphology and surface chemistry, should aim to minimize any physical interactions with the cells of the immune system. However, the same surface-borne design parameters are also critical for the locomotion performance of the microrobots. Understanding the interplay of such parameters targeting high locomotion performance and low immunogenicity at the same time is of paramount importance yet has so far been overlooked. Here, we investigated the interactions of magnetically steerable double-helical microswimmers with mouse macrophage cell lines and splenocytes, freshly harvested from mouse spleens, by systematically changing their helical morphology. We found that the macrophages and splenocytes can recognize and differentially elicit an immune response to helix turn numbers of the microswimmers that otherwise have the same size, bulk physical properties, and surface chemistries. Our findings suggest that the structural optimization of medical microrobots for the locomotion performance and interactions with the immune cells should be considered simultaneously because they are highly entangled and can demand a substantial design compromise from one another. Furthermore, we show that morphology-dependent interactions between macrophages and microswimmers can further present engineering opportunities for biohybrid microrobot designs. We demonstrate immunobots that can combine the steerable mobility of synthetic microswimmers and the immunoregulatory capability of macrophages for potential targeted immunotherapeutic applications.

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.

Zwitterionic 3D-Printed Non-Immunogenic Stealth Microrobots.

Microrobots offer transformative solutions for non-invasive medical interventions due to their small size and untethered operation inside the human body. However, they must face the immune system as a natural protection mechanism against foreign threats. Here, non-immunogenic stealth zwitterionic microrobots that avoid recognition from immune cells are introduced. Fully zwitterionic photoresists are developed for two-photon polymerization 3D microprinting of hydrogel microrobots with ample functionalization: tunable mechanical properties, anti-biofouling and non-immunogenic properties, functionalization for magnetic actuation, encapsulation of biomolecules, and surface functionalization for drug delivery. Stealth microrobots avoid detection by macrophage cells of the innate immune system after exhaustive inspection (>90 hours), which has not been achieved in any microrobotic platform to date. These versatile zwitterionic materials eliminate a major roadblock in the development of biocompatible microrobots, and will serve as a toolbox of non-immunogenic materials for medical microrobot and other device technologies for bioengineering and biomedical applications.

High-Yield Production of Biohybrid Microalgae for On-Demand Cargo Delivery.

Biohybrid microswimmers exploit the swimming and navigation of a motile microorganism to target and deliver cargo molecules in a wide range of biomedical applications. Medical biohybrid microswimmers suffer from low manufacturing yields, which would significantly limit their potential applications. In the present study, a biohybrid design strategy is reported, where a thin and soft uniform coating layer is noncovalently assembled around a motile microorganism. Chlamydomonas reinhardtii (a single-cell green alga) is used in the design as a biological model microorganism along with polymer-nanoparticle matrix as the synthetic component, reaching a manufacturing efficiency of ≈90%. Natural biopolymer chitosan is used as a binder to efficiently coat the cell wall of the microalgae with nanoparticles. The soft surface coating does not impair the viability and phototactic ability of the microalgae, and allows further engineering to accommodate biomedical cargo molecules. Furthermore, by conjugating the nanoparticles embedded in the thin coating with chemotherapeutic doxorubicin by a photocleavable linker, on-demand delivery of drugs to tumor cells is reported as a proof-of-concept biomedical demonstration. The high-throughput strategy can pave the way for the next-generation generation microrobotic swarms for future medical active cargo delivery tasks.

A novel method for PEGylation of chitosan nanoparticles through photopolymerization.

An ultrafast and convenient method for PEGylation of chitosan nanoparticles has been established through a photopolymerization reaction between the acrylate groups of PEG and methacrylated-chitosan nanoparticles. The nanoparticle characteristics under physiological pH conditions were optimized through altered PEG chain length, concentration and duration of UV exposure. The method developed here has potential for clinical translation of chitosan nanoparticles. It also allows for the scalable and fast synthesis of nanoparticles with colloidal stability.

Deep Insight into PEGylation of Bioadhesive Chitosan Nanoparticles: Sensitivity Study for the Key Parameters Through Artificial Neural Network Model.

Ionically cross-linked chitosan nanoparticles have great potential in nanomedicine due to their tunable properties and cationic nature. However, low solubility of chitosan severely limits their potential clinical translation. PEGylation is a well-known method to increase solubility of chitosan and chitosan nanoparticles in neutral media; however, effect of PEG chain length and chitosan/PEG ratio on particle size and zeta potential of nanoparticles are not known. This study presents a systematic analysis of the effect of PEG chain length and chitosan/PEG ratio on size and zeta potential of nanoparticles. We prepared PEGylated chitosan chains prior to the nanoparticle synthesis with different PEG chain lengths and chitosan/PEG ratios. To precisely estimate the influence of critical parameters on size and zeta potential of nanoparticles, we both developed an artificial neural network (ANN) model and performed experimental characterization using the three independent input variables: (i) PEG chain length, (ii) chitosan/PEG ratio, and (iii) pH of solution. We studied the influence of PEG chain lengths of 2, 5, and 10 kDa and three different chitosan/PEG ratios (25 mg chitosan to 4, 12, and 20 µmoles of PEG) for the synthesis of chitosan nanoparticles within the pH range of 6.0-7.4. Artificial neural networks is a modeling tool used in nanomedicine to optimize and estimate inherent properties of the system. Inherent properties of a nanoparticle system such as size and zeta potential can be estimated based on previous experiment results, thus, nanoparticles with desired properties can be obtained using an ANN. With the ANN model, we were able to predict the size and zeta potential of nanoparticles under different experimental conditions and further confirmed the cell-nanoparticle adhesion behavior through experiments. Nanoparticle groups that had higher zeta potentials promoted adhesion of HEK293-T cells to nanoparticle-coated surfaces in cell culture medium, which was predicted through ANN model prior to experiments. Overall, this study comprehensively presents the PEGylation of chitosan, synthesis of PEGylated chitosan nanoparticles, utilizes ANN model as a tool to predict important properties such as size and zeta potential, and further captures the adhesion behavior of cells on surfaces prepared with these engineered nanoparticles.