Antigen 43-mediated biotin display and fabrication of bacteria-driven microswimmers

Controlled attachment of bacterial cells to biotic and abiotic surfaces without affecting their fitness is of great interest in biotechnological applications, such as patterning surfaces with cell-based biosensors, cell-cell attachment in syntrophic communities and fabrication of bacteria-driven biohybrid microswimmers. For years genetically modified outer membrane proteins and autotransporters were used to functionalize the bacterial cell surface with peptides and small proteins used for peptide library screening, bioremediation and biocatalysis. In this study we modified Escherichia coli (E. coli) to autonomously display biotin on its cell surface via the engineered autotransporter antigen 43 (Ag43) and thus to bind to streptavidin modified surfaces. We could show that a biotin acceptor peptide (BAP) at the N-terminus of Ag43 is biotinylated in the cytoplasm, translocated to the cell surface and accessible to free or surface bound streptavidin. Flow cytometry measurements and fluorescence microscopy imaging of cells stained with fluorescently labelled streptavidin indicate that the biotinylation is strongly dependent on the intracellular levels of biotin and the biotin protein ligase BirA. Moreover, the staining pattern of Ag43 suggests that the majority of Ag43 is located at the cell poles. In addition, we modified Ag43 with the LOV2 domain of Arabidopsis thaliana, to control the accessibility of the displayed biotin through light controlled photocaging. To examine the effect of attachment on the fitness of E. coli, we used laser-assisted adsorption by photobleaching (LAPAP) to micro-pattern an abiotic surface with biotin. Such immobilized cells were able to grow for several generations and released their daughter cells into the medium. Aside from Ag43 alternative display mechanisms including OmpA (outer membrane protein A), INP (ice nucleating protein), AIDA-I (autotransporter) and FliC (flagellin), were investigated for biotin display, although only modified flagellin showed pronounced attachment to streptavidin. In a second part we used the Ag43 based biotin display system to fabricate bacteria-driven biohybrid microswimmers (bacteriabots). Bacteriabots combine synthetic cargo with motile bacteria that enable propulsion and steering. Although fabrication and potential use of such bacteriabots have attracted much attention, existing methods of fabrication require an extensive sample preparation that can drastically decrease the viability and motility of bacteria. Moreover, chemotactic behavior of bacteriabots in a liquid medium with chemical gradients has remained largely unclear. To overcome these shortcomings, we used our Ag43 based biotin display system to bind cells to streptavidin-coated cargo. We show that the cargo attachment to these bacteria is greatly enhanced by motility and occurs predominantly at the cell poles, which is greatly beneficial for the fabrication of motile bacteriabots. We further performed a systematic study to understand and optimize the ability of these bacteriabots to follow chemical gradients. We demonstrate that the chemotaxis of bacteriabots is primarily limited by the cargo-dependent reduction of swimming speed and show that the fabrication of bacteriabots using elongated E. coli cells can be used to overcome this limitation

Magnetically Driven Micro and Nanorobots

Manipulation and navigation of micro and nanoswimmers in different fluid environments can be achieved by chemicals, external fields, or even motile cells. Many researchers have selected magnetic fields as the active external actuation source based on the advantageous features of this actuation strategy such as remote and spatiotemporal control, fuel-free, high degree of reconfigurability, programmability, recyclability, and versatility. This review introduces fundamental concepts and advantages of magnetic micro/nanorobots (termed here as "MagRobots") as well as basic knowledge of magnetic fields and magnetic materials, setups for magnetic manipulation, magnetic field configurations, and symmetry-breaking strategies for effective movement. These concepts are discussed to describe the interactions between micro/nanorobots and magnetic fields. Actuation mechanisms of flagella-inspired MagRobots (i.e., corkscrew-like motion and traveling-wave locomotion/ciliary stroke motion) and surface walkers (i.e., surface-assisted motion), applications of magnetic fields in other propulsion approaches, and magnetic stimulation of micro/nanorobots beyond motion are provided followed by fabrication techniques for (quasi)spherical, helical, flexible, wire-like, and biohybrid MagRobots. Applications of MagRobots in targeted drug/gene delivery, cell manipulation, minimally invasive surgery, biopsy, biofilm disruption/eradication, imaging-guided delivery/therapy/surgery, pollution removal for environmental remediation, and (bio)sensing are also reviewed. Finally, current challenges and future perspectives for the development of magnetically powered miniaturized motors are discussed

Synthetic Micro/Nanomotors for Drug Delivery

Synthetic micro/nanomotors (MNMs) are human-made machines characterized by their capacity for undergoing self-propelled motion as a result of the consumption of chemical energy obtained from specific chemical or biochemical reactions, or as a response to an external actuation driven by a physical stimulus. This has fostered the exploitation of MNMs for facing different biomedical challenges, including drug delivery. In fact, MNMs are superior systems for an efficient delivery of drugs, offering several advantages in relation to conventional carriers. For instance, the self-propulsion ability of micro/nanomotors makes possible an easier transport of drugs to specific targets in comparison to the conventional distribution by passive carriers circulating within the blood, which enhances the drug bioavailability in tissues. Despite the promising avenues opened by the use of synthetic micro/nanomotors in drug delivery applications, the development of systems for in vivo uses requires further studies to ensure a suitable biocompatibility and biodegradability of the fabricated engines. This is essential for guaranteeing the safety of synthetic MNMs and patient convenience. This review provides an updated perspective to the potential applications of synthetic micro/nanomotors in drug delivery. Moreover, the most fundamental aspects related to the performance of synthetic MNMs and their biosafety are also discussed.This work was funded in part by MICINN under Grant PID2019-106557GB-C21 and by E.U. on the framework of the European Innovative Training Network—Marie Sklodowska-Curie Action Nano Paint (Grant Agreement 955612)

Enzyme Powered Nanomotors Towards Biomedical Applications

[eng] The advancements in nanotechnology enabled the development of new diagnostic tools and drug delivery systems based on nanosystems, which offer unique features such as large surface area to volume ratio, cargo loading capabilities, increased circulation times, as well as versatility and multifunctionality. Despite this, the majority of nanomedicines do not translate into clinics, in part due to the biological barriers present in the body. Synthetic nano- and micromotors could be an alternative tool in nanomedicine, as the continuous propulsion force and potential to modulate the medium may aid tissue penetration and drug diffusion across biological barriers. Enzyme-powered motors are especially interesting for biomedical applications, owing to their biocompatibility and use of bioavailable substrates as fuel for propulsion. This thesis aims at exploring the potential applications of urease-powered nanomotors in nanomedicine. In the first work, we evaluated these motors as drug delivery systems. We found that active urease- powered nanomotors showed active motion in phosphate buffer solutions, and enhanced in vitro drug release profiles in comparison to passive nanoparticles. In addition, we observed that the motors were more efficient in delivering drug to cancer cells and caused higher toxicity levels, due to the combination of boosted drug release and local increase of pH produced by urea breakdown into ammonia and carbon dioxide. One of the major goals in nanomedicine is to achieve localized drug action, thus reducing side-effects. A commonly strategy to attain this is the use moieties to target specific diseases. In our second work, we assessed the ability of urease-powered nanomotors to improve the targeting and penetration of spheroids, using an antibody with therapeutic potential. We showed that the combination of active propulsion with targeting led to a significant increase in spheroid penetration, and that this effect caused a decrease in cell proliferation due to the antibody’s therapeutic action. Considering that high concentrations of nanomedicines are required to achieve therapeutic efficiency; in the third work we investigated the collective behavior of urease-powered nanomotors. Apart from optical microscopy, we evaluated the tracked the swarming behavior of the nanomotors using positron emission tomography, which is a technique widely used in clinics, due to its noninvasiveness and ability to provide quantitative information. We showed that the nanomotors were able to overcome hurdles while swimming in confined geometries. We observed that the nanomotors swarming behavior led to enhanced fluid convection and mixing both in vitro, and in vivo within mice’s bladders. Aiming at conferring protecting abilities to the enzyme-powered nanomotors, in the fourth work, we investigated the use of liposomes as chassis for nanomotors, encapsulating urease within their inner compartment. We demonstrated that the lipidic bilayer provides the enzymatic engines with protection from harsh acidic environments, and that the motility of liposome-based motors can be activated with bile salts. Altogether, these results demonstrate the potential of enzyme-powered nanomotors as nanomedicine tools, with versatile chassis, as well as capability to enhance drug delivery and tumor penetration. Moreover, their collective dynamics in vivo, tracked using medical imaging techniques, represent a step-forward in the journey towards clinical translation.[spa] Recientes avances en nanotecnología han permitido el desarrollo de nuevas herramientas para el diagnóstico de enfermedades y el transporte dirigido de fármacos, ofreciendo propiedades únicas como encapsulación de fármacos, el control sobre la biodistribución de estos, versatilidad y multifuncionalidad. A pesar de estos avances, la mayoría de nanomedicinas no consiguen llegar a aplicaciones médicas reales, lo cual es en parte debido a la presencia de barreras biológicas en el organismo que limitan su transporte hacia los tejidos de interés. En este sentido, el desarrollo de nuevos micro- y nanomotores sintéticos, capaces de autopropulsarse y causar cambios locales en el ambiente, podrían ofrecer una alternativa para la nanomedicina, promoviendo una mayor penetración en tejidos de interés y un mejor transporte de fármacos a través de las barreras biológicas. En concreto, los nanomotores enzimáticos poseen un alto potencial para aplicaciones biomédicas gracias a su biocompatibilidad y a la posibilidad de usar sustancias presentes en el organismo como combustible. Los trabajos presentados en esta tesis exploran el potenical de nanomotores, autopropulsados mediante la enzima ureasa, para aplicaciones biomédicas, y investigan su uso como vehículos para transporte de fármacos, su capacidad para mejorar penetración de tejidos diana, su versatilidad y movimiento colectivo. En conjunto, los resultados presentados en esta tesis doctoral demuestran el potencial del uso de nanomotores autopropulsados mediante enzimas como herramientas biomédicas, ofreciendo versatilidad en su diseño y una alta capacidad para promover el transporte de fármacos y la penetración en tumores. Por último, su movimiento colectivo observado in vivo mediante técnicas de imagen médicas representan un significativo avance en el viaje hacia su aplicación en medicina

Motility of Swimming Bacteria Hydrodynamics and Statistics

The present work contains original research on the field of biophysics, specifically the study of swimming bacteria. Swimming microorganisms can be modeled as active particles moving at low Reynolds number (Re ≪ 1) and subject to different sources of noise. The term “active” means that they are self-propelled, while Re ≪ 1 implies that their motion is dominated by viscous stresses, therefore relying on non-reciprocal deformations in time, in order to achieve movement. Noise arises from thermal fluctuations and the inherent stochasticity of their propelling machinery, as a result, bacteria follow random trajectories. Nevertheless, bacteria have evolved to display a number of strategies to overcome randomness and achieve directed locomotion, known as “taxis”. Here, we explore the mechanisms involved in the propulsion and navigation of swimming bacteria, using low Reynolds number flow techniques and random walks. First, we introduce the physical principles that govern the dynamics of a low Reynolds number swimmer. We pay special attention to the random walk model for the description of the swimming trajectories, since it allows to quantify motility in terms of statistical measures, such as diffusivity and drift velocity, which can be measured experimentally. After a general discussion of the model, we demonstrate its use by applying it to the dynamics of bacteria-driven microswimmers, which are active particles that use bacteria as a propulsion mechanism. We show in particular, that the diffusivity of such particles increases with the square of their size and that the microswimmers inherit the chemotactic capabilities from the bacteria that propel them. These results are in agreement with experiments and can be useful to improve the design of these artificial microswimmers. Next, we investigate the motility properties of Spiroplasma melliferum, which is special among bacteria, as it can swim without flagella. Instead, Spiroplasma can switch the handedness of its helical body and in the process, the helical domains rotate generating propulsion. Based on experimental observations, we develop a hydrodynamic model to describe Spiroplasma motility. We obtain expressions for the total linear and angular displacements of the cell body per swimming stroke. Observing that the cell body does not reorient at the end of one period, we define an effective swimming speed and a hydrodynamic efficiency. Then, we show that the helical shape that maximises speed and efficiency has a pitch angle close to that of Spiroplasma, φ ≃ 35◦, in agreement with experimental observations and with previous numerical simulations. Finally, we explore the dynamics of a low Reynolds number swimmer crossing a viscosity gradient. This is a work in collaboration with experimental groups in the National University of Mexico (UNAM) and Brown University. The experiments aim to shed light on the dynamics of the bacterium Helicobacter pylori, which inhabits the human gut and is capable of penetrating the mucus layer that protects the stomach. Experimentally, a magnetic swimmer is immersed in a stratified solution of miscible fluids with different viscosities. The swimmer consists of a helical tail and a cylindrical head that rotate at a fixed rate due to the action of an external magnetic field. As the swimmer advances, it accelerates or slows down, depending on its orientation with respect to the gradient. In general, the experimental results show that it is harder for a pusher-like swimmer to swim up the gradient, whereas for a puller-like swimmer it is the opposite. We rationalize this mathematically by assuming that the forces acting on the swimmer depend on the local viscosity that it experiences. This allows us to calculate the swimming speed as a function of the swimmer’s position along the gradient. The predictions of the model are in good agreement with the experimental observations. The results also suggest that viscotaxis is possible without viscoreceptors, and in fact governed solely by the motility pattern of the swimmer. Together, the results presented in this thesis contribute to the understanding of bacterial motility and low Reynolds number swimmers in general. Furthermore, these results may be useful for future developments in biophysics, including applications to targeted drug delivery and microrobotics.European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 682754 to Eric Lauga

A Magnetically and Electrically Powered Hybrid Micromotor in Conductive Solutions: Synergistic Propulsion Effects and Label-Free Cargo Transport and Sensing

Electrically powered micro- and nanomotors are promising tools for in-vitro single-cell analysis. In particular, single cells can be trapped, transported and electroporated by a Janus particle (JP) using an externally applied electric field. However, while dielectrophoretic (DEP)-based cargo manipulation can be achieved at high-solution conductivity, electrical propulsion of these micromotors becomes ineffective at solution conductivities exceeding 0.3mS/cm. Here, we successfully extended JP cargo manipulation and transport capabilities to conductive near-physiological (<6mS/cm) solutions by combining magnetic field-based micromotor propulsion and navigation with DEP-based manipulation of various synthetic and biological cargos. Combination of a rotating magnetic field and electric field resulted in enhanced micromotor mobility and steering control through tuning of the electric field frequency. conditions are necessary. In addition, we demonstrated the micromotors ability of identifying apoptotic cell among viable and necrotic cells based their dielectrophoretic difference, thus, enabling to analyze the apoptotic status in the single cell samples for drug discovery, cell therapeutics and immunotherapy. We also demonstrated the ability to trap and transport live cells towards regions containing doxorubicin-loaded liposomes. This hybrid micromotor approach for label-free trapping, transporting and sensing of selected cells within conductive solutions, opens new opportunities in drug delivery and single cell analysis, where close-to-physiological medi

Innovative designs and applications of Janus micromotors with (photo)-catalytic and magnetic motion

El objetivo principal de esta Tesis Doctoral es el diseño y desarrollo de micromotores Janus biocompatibles y su aplicación en ámbitos relevantes de la salud y de la protección medioambiental. Los micromotores Janus son dispositivos en la microescala autopropulsados que tienen al menos dos regiones en su superficie con diferentes propiedades físicas y químicas, lo que les convierte en una clase distintiva de materiales que pueden combinar características ópticas, magnéticas y eléctricas en una sola entidad. Como la naturaleza del micromotor Janus -el dios romano de las dos caras- los objetivos de esta Tesis Doctoral presentan naturaleza dual y comprenden desarrollos de química fundamental y de química aplicada. En efecto, por una parte, el objetivo central aborda el diseño, síntesis y ensamblaje, así como la caracterización de micromotores Janus poliméricos propulsados por mecanismos (foto)-catalíticos y/o accionados por campos magnéticos. Por otra parte, el objetivo central implica la aplicación de los micromotores desarrollados para resolver desafíos sociales relevantes en los ámbitos químico-analítico, biomédico y ambiental. Partiendo de estas premisas, en la primera parte de la Tesis Doctoral, se sintetizaron micromotores Janus de policaprolactona propulsados químicamente integrando nanomateriales para el diseño de sensores móviles para la detección selectiva de endotoxinas bacterianas. De esta forma, el movimiento autónomo del micromotor mejora la mezcla de fluidos y la eficacia de las reacciones implicadas permitiendo detectar el analito en pocos minutos, incluso en muestras viscosas y medios donde la agitación no es posible. Además, esta autopropulsión es altamente compatible con su empleo en formatos ultra-miniaturizados para el desarrollo de futuros dispositivos portátiles en el marco de la tecnología point of care para aplicaciones clínicas y agroalimentarias. Con el fin de incrementar su biocompatibilidad para aplicaciones in vivo, en una segunda etapa de la Tesis Doctoral, se diseñaron micromotores Janus con propulsión autónoma utilizando luz visible para la eliminación de toxinas relevantes en procesos inflamatorios. El fenómeno autopropulsivo del micromotor y su capacidad de interacción con agentes tóxicos condujo a metodologías más rápidas y eficaces infiriéndose un futuro prometedor de estos micromotores para el tratamiento del shock séptico o intoxicación. En una tercera etapa, se sintetizaron micromotores propulsados por campos magnéticos. Estos micromotores utilizan una aproximación elegante de propulsión, exenta del empleo de combustibles químicos tóxicos como sucede en la propulsión catalítica y, en consecuencia, biocompatible. Asimismo, este mecanismo propulsivo permite controlar e incluso programar su trayectoria para aplicaciones que requieran de un guiado y de un control preciso de esta. De manera específica, estos micromotores han sido aplicados en esta Tesis Doctoral para la liberación controlada de fármacos en el tratamiento de cáncer pancreático y como elementos de remediación ambiental en la eliminación de agentes nerviosos en aguas contaminadas

Sperm Micromotors for Cargo Delivery through Flowing Blood

Micromotors are recognized as promising candidates for untethered micromanipulation and targeted cargo delivery in complex biological environments. However, their feasibility in the circulatory system has been limited due to the low thrust force exhibited by many of the reported synthetic micromotors, which is not sufficient to overcome the high flow and complex composition of blood. Here we present a hybrid sperm micromotor that can actively swim against flowing blood (continuous and pulsatile) and perform the function of heparin cargo delivery. In this biohybrid system, the sperm flagellum provides a high propulsion force while the synthetic microstructure serves for magnetic guidance and cargo transport. Moreover, single sperm micromotors can assemble into a train-like carrier after magnetization, allowing the transport of multiple sperm or medical cargoes to the area of interest, serving as potential anticoagulant agents to treat blood clots or other diseases in the circulatory system