Magnetotactic Sperm Cells for Assisted Reproduction.

Biohybrid micromotors are active microscopic agents consisting of biological and synthetic components that are being developed as novel tools for biomedical applications. By capturing motile sperm cells within engineered microstructures, they can be controlled remotely while being propelled forward by the flagellar beat. This makes them an interesting tool for reproductive medicine that can enable minimally invasive sperm cell delivery to the oocyte in vivo, as a treatment for infertility. The generation of sperm-based micromotors in sufficiently large numbers, as they are required in biomedical applications has been challenging, either due to the employed fabrication techniques or the stability of the microstructure-sperm coupling. Here, biohybrid micromotors, which can be assembled in a fast and simple process using magnetic microparticles, are presented. These magnetotactic sperm cells show a high motility and swimming speed and can be transferred between different environments without large detrimental effects on sperm motility and membrane integrity. Furthermore, clusters of micromotors are assembled magnetically and visualized using dual ultrasound (US)/photoacoustic (PA) imaging. Finally, a protocol for the scaled-up assembly of micromotors and their purification for use in in vitro fertilization (IVF) is presented, bringing them closer to their biomedical implementation.

Nanomaterial-decorated micromotors for enhanced photoacoustic imaging.

Micro-and nanorobots have the potential to perform non-invasive drug delivery, sensing, and surgery in living organisms, with the aid of diverse medical imaging techniques. To perform such actions, microrobots require high spatiotemporal resolution tracking with real-time closed-loop feedback. To that end,  photoacoustic imaging has appeared as a promising technique for imaging microrobots in deep tissue with higher molecular specificity and contrast. Here, we present different strategies to track magnetically-driven micromotors with improved contrast and specificity using dedicated contrast agents (Au nanorods and nanostars). Furthermore, we discuss the possibility of improving the light absorption properties of the employed nanomaterials considering possible light scattering and coupling to the underlying metal-oxide layers on the micromotor's surface. For that, 2D COMSOL simulation and experimental results were correlated, confirming that an increased spacing between the Au-nanostructures and the increase of thickness of the underlying oxide layer lead to enhanced light absorption and preservation of the characteristic absorption peak. These characteristics are important when visualizing the micromotors in a complex in vivo environment, to distinguish them from the light absorption properties of the surrounding natural chromophores. Supplementary Information: The online version contains supplementary material available at 10.1007/s12213-023-00156-7.

Multifunctional 4D-Printed Sperm-Hybrid Microcarriers for Assisted Reproduction.

Remotely controllable microrobots are appealing for various biomedical in vivo applications. In particular, in recent years, our group has focused on developing sperm-microcarriers to assist sperm cells with motion deficiencies or low sperm count (two of the most prominent male infertility problems) to reach the oocyte toward in-vivo-assisted fertilization. Different sperm carriers, considering their motion in realistic media and confined environments, have been optimized. However, the already-reported sperm carriers have been mainly designed to transport single sperm cell, with limited functionality. Thus, to take a step forward, here, the development of a 4D-printed multifunctional microcarrier containing soft and smart materials is reported. These microcarriers can not only transport and deliver multiple motile sperm cells, but also release heparin and mediate local enzymatic reactions by hyaluronidase-loaded polymersomes (HYAL-Psomes). These multifunctional facets enable in situ sperm capacitation/hyperactivation, and the local degradation of the cumulus complex that surrounds the oocyte, both to facilitate the sperm-oocyte interaction for the ultimate goal of in vivo assisted fertilization.

Electronically integrated microcatheters based on self-assembling polymer films.

Existing electronically integrated catheters rely on the manual assembly of separate components to integrate sensing and actuation capabilities. This strongly impedes their miniaturization and further integration. Here, we report an electronically integrated self-assembled microcatheter. Electronic components for sensing and actuation are embedded into the catheter wall through the self-assembly of photolithographically processed polymer thin films. With a diameter of only about 0.1 mm, the catheter integrates actuated digits for manipulation and a magnetic sensor for navigation and is capable of targeted delivery of liquids. Fundamental functionalities are demonstrated and evaluated with artificial model environments and ex vivo tissue. Using the integrated magnetic sensor, we develop a strategy for the magnetic tracking of medical tools that facilitates basic navigation with a high resolution below 0.1 mm. These highly flexible and microsized integrated catheters might expand the boundary of minimally invasive surgery and lead to new biomedical applications.

Self-sufficient self-oscillating microsystem driven by low power at low Reynolds numbers.

Oscillations at several hertz are a key feature of dynamic behavior of various biological entities, such as the pulsating heart, firing neurons, or the sperm-beating flagellum. Inspired by nature's fundamental self-oscillations, we use electroactive polymer microactuators and three-dimensional microswitches to create a synthetic electromechanical parametric relaxation oscillator (EMPRO) that relies on the shape change of micropatterned polypyrrole and generates a rhythmic motion at biologically relevant stroke frequencies of up to ~95 Hz. We incorporate an Ag-Mg electrochemical battery into the EMPRO for autonomous operation in a nontoxic environment. Such a self-sufficient self-oscillating microsystem offers new opportunities for artificial life at low Reynolds numbers by, for instance, mimicking and replacing nature's propulsion and pumping units.

Dual Ultrasound and Photoacoustic Tracking of Magnetically Driven Micromotors: From In Vitro to In Vivo.

The fast evolution of medical micro- and nanorobots in the endeavor to perform non-invasive medical operations in living organisms has boosted the use of diverse medical imaging techniques in the last years. Among those techniques, photoacoustic imaging (PAI), considered a functional technique, has shown to be promising for the visualization of micromotors in deep tissue with high spatiotemporal resolution as it possesses the molecular specificity of optical methods and the penetration depth of ultrasound. However, the precise maneuvering and function's control of medical micromotors, in particular in living organisms, require both anatomical and functional imaging feedback. Therefore, herein, the use of high-frequency ultrasound and PAI is reported to obtain anatomical and molecular information, respectively, of magnetically-driven micromotors in vitro and under ex vivo tissues. Furthermore, the steerability of the micromotors is demonstrated by the action of an external magnetic field into the uterus and bladder of living mice in real-time, being able to discriminate the micromotors' signal from one of the endogenous chromophores by multispectral analysis. Finally, the successful loading and release of a model cargo by the micromotors toward non-invasive in vivo medical interventions is demonstrated.

Medical Imaging of Microrobots: Toward In Vivo Applications.

Medical microrobots (MRs) have been demonstrated for a variety of non-invasive biomedical applications, such as tissue engineering, drug delivery, and assisted fertilization, among others. However, most of these demonstrations have been carried out in in vitro settings and under optical microscopy, being significantly different from the clinical practice. Thus, medical imaging techniques are required for localizing and tracking such tiny therapeutic machines when used in medical-relevant applications. This review aims at analyzing the state of the art of microrobots imaging by critically discussing the potentialities and limitations of the techniques employed in this field. Moreover, the physics and the working principle behind each analyzed imaging strategy, the spatiotemporal resolution, and the penetration depth are thoroughly discussed. The paper deals with the suitability of each imaging technique for tracking single or swarms of MRs and discusses the scenarios where contrast or imaging agent's inclusion is required, either to absorb, emit, or reflect a determined physical signal detected by an external system. Finally, the review highlights the existing challenges and perspective solutions which could be promising for future in vivo applications.

IRONSperm: Sperm-templated soft magnetic microrobots.

We develop biohybrid magnetic microrobots by electrostatic self-assembly of nonmotile sperm cells and magnetic nanoparticles. Incorporating a biological entity into microrobots entails many functional advantages beyond shape templating, such as the facile uptake of chemotherapeutic agents to achieve targeted drug delivery. We present a single-step electrostatic self-assembly technique to fabricate IRONSperms, soft magnetic microswimmers that emulate the motion of motile sperm cells. Our experiments and theoretical predictions show that the swimming speed of IRONSperms exceeds 0.2 body length/s (6.8 ± 4.1 µm/s) at an actuation frequency of 8 Hz and precision angle of 45°. We demonstrate that the nanoparticle coating increases the acoustic impedance of the sperm cells and enables localization of clusters of IRONSperm using ultrasound feedback. We also confirm the biocompatibility and drug loading ability of these microrobots, and their promise as biocompatible, controllable, and detectable biohybrid tools for in vivo targeted therapy.

Real-Time Optoacoustic Tracking of Single Moving Micro-objects in Deep Phantom and Ex Vivo Tissues.

Medical imaging plays an important role in diagnosis and treatment of multiple diseases. It is a field which seeks for improved sensitivity and spatiotemporal resolution to allow the dynamic monitoring of diverse biological processes that occur at the micro- and nanoscale. Emerging technologies for targeted diagnosis and therapy such as nanotherapeutics, microimplants, catheters, and small medical tools also need to be precisely located and monitored while performing their function inside the human body. In this work, we show for the first time the real-time tracking of moving single micro-objects below centimeter thick phantom tissue and ex vivo chicken breast, using multispectral optoacoustic tomography (MSOT). This technique combines the advantages of ultrasound imaging regarding depth and resolution with the molecular specificity of optical methods, thereby facilitating the discrimination between the spectral signatures of the micro-objects from those of intrinsic tissue molecules. The resulting MSOT signal is further improved in terms of contrast and specificity by coating the micro-objects' surface with gold nanorods, possessing a unique absorption spectrum, which facilitate their discrimination from surrounding biological tissues when translated to future in vivo settings.