Études des configurations spatio-temporelles du champ magnétique sur le contrôle des bactéries magnétotactiques

Depuis que les scientifiques se sont intéressés à travailler à l’échelle nano et micro, la création de véhicules qui puissent y travailler est devenue une nécessité. Ces véhicules sont des nano-micro robots qui doivent fonctionner dans ces milieux de manière autonome et contrôlée. L’une des plus grandes utilités de ces nano-micro robots, par exemple, est leur utilisation dans un système microvasculaire pour transporter des agents thérapeutiques vers les tumeurs cancéreuses de façon contrôlée. La technologie de fabrication des robots artificiels actuelle n’est pas en mesure de fournir ce nano-micro robot. Pour contourner cette limitation, nous avons choisi un micro robot déjà existant dans la nature. C’est la bactérie magnétotactique Magnetococcus Marinus souche MC-1, d’une taille de 2 µm de diamètre et ayant : 1) une autonomie de mouvement grâce à son propre système de propulsion fourni par deux moteurs moléculaires (flagelles), 2) une chaîne de particules nanométriques magnétiques (magnétosomes), qui permet à la bactérie de s’aligner avec le champ magnétique et de se propulser dans la direction du champ. En plus, les microrobots ont la capacité de réaliser des tâches dans l’environnement micrométrique comme : la microfabrication et le transport. L’équipe du laboratoire NanoRobotique de Polytechnique de Montréal a développé une plateforme de contrôle des bactéries magnétiques dans le but de contrôler leurs déplacements dans un système in vivo, et ainsi de transporter des agents thérapeutiques directement dans le cancer. Autrement dit, cette nouvelle plateforme permet de guider la bactérie magnétotactique vers une cible prédéfinie. L’objectif de ce mémoire de recherche est d’améliorer la modélisation du champ magnétique de cette plateforme. Cette nouvelle modélisation permettra de réduire les durées d’agrégation et de déplacement des bactéries magnétiques tout en augmentant la performance de la plateforme. D’abord, une méthode de contrôle basée sur la géométrie spatiale du champ magnétique a été développée et validée. Finalement, une étude de comportement des bactéries magnétiques exposées au champ magnétique alternatif a été effectuée afin de pouvoir développer une technique novatrice de contrôle.----------ABSTRACT Working at the nano and micro scale environment has provided scientists with an immense opportunity to explore within small and previously unreachable areas. Evidently, creation of vehicles that could facilitate such careful maneuver has gained a lot of interest. These vehicles are nanomicrorobots that perform autonomously under controlled environment. Among many research disciplines that could advance with such miniature system, drug delivery and navigation is one of the most beneficial uses for these controlled nanomicrorobots; acting as therapeutic agent carriers targeting cancerous tumors by traveling through complex microvascular structures. Current artificial robot technology lacks maturity in manufacturing mass scale nanomicrorobots. Therefore, inspired by nature, we chose special bacteria bona fide to serve as microrobots. Magnetotactic Magnetococcus Marinus strain MC-1 has: 1) an autonomy movement with its own propulsion system provided by two molecular motors (flagella) and 2) a chain of magnetic nanoparticles (magnetosomes) acting as a compass that aligns the moving bacteria in the direction of external magnetic field. These 2 µm diameter bacteria have the ability to perform as actuators, micro-fabricators and transporters. Polytechnique NanoRobotics Montreal laboratory team has developed a magnetic controller platform to control these bacteria in vivo and deliver therapeutic agents directly into the cancer tissue. In other words, this platform helps navigate the magnetotactic bacteria to the predefined target. The objective of this research thesis is to improve the magnetic field modeling of this platform. Our new proposed model will reduce the bacteria displacement and aggregation time while increasing the performance of the platform. At the beginning, a control method based on the spatial configuration of the magnetic field has been developed and validated. And at the end, a study on magnetic bacteria behavior exposed to alternating magnetic field is performed in order to develop an innovative control technique

Survey of Magneto-tactic Properties of Escherichia coli Under Static Magnetic Fields

Some of the microorganisms such as Escherichia coli have the ability to migrate to areas in which the intensity of magnetic fields (MFs) is higher, which is called magnetotactic properties. Magnetotaxis is a process implemented by a group of gram-negative bacteria that involves orienting and coordinating movement in response to magnetic fields. This study was conducted to investigate these properties of Escherichia coli in laboratory conditions. By means of coated wires (30 rounds) placed in two parts of the reactor (with five zones and a volume of 250 mL) and direct current (DC), an intensity of 0.18 mT for 42 minutes has been prepared. The most probable number of E. coli per 100 mL (MPN/100 mL) in each zone of the reactor, before and after exposure, was estimated. According to the results of this study, E. coli has magnetotactic properties, and the mean density of these bacteria in higher MFs (0.18 mT) is higher compared to the other zones in the reactor

MRI-Based Tumour Targeting Enhancement with Magnetotactic Bacterial Carriers

RÉSUMÉ Le cancer constitue la première cause de mortalité au Québec, avec 20,000 décès estimés par année. Parmi tous les patients atteints du cancer, une grande proportion pourrait profiter de l’avancement technologique en ce qui concerne le transport de médicaments. En effet, l’un des meilleurs moyens d’augmenter l’efficacité d’un médicament contre le cancer, tout en réduisant sa toxicité sur les cellules saines, est de le diriger vers la tumeur et de le maintenir à cet endroit jusqu’à ce qu’un effet thérapeutique se produise. Le transport ciblé de médicaments vers la tumeur peut considérablement améliorer l’efficacité thérapeutique, surtout si le transporteur est capable d’atteindre les zones nécrotiques et se répartir uniformément dans la zone à traiter. Les bactéries, de par leur motilité, sont d’excellents candidats pour une telle application, surtout qu’elles peuvent aussi être facilement fonctionnalisées. Ainsi, la recherche sur le traitement du cancer utilisant des bactéries s’est imposée comme une approche prometteuse surtout qu’elle pallie à une limitation majeure de la chimiothérapie et de la radiothérapie en permettant le traitement des zones anaérobies. Alors que des laboratoires à travers le monde tentent de fabriquer des systèmes miniatures en se basant sur le modèle bactérien, nous avons opté pour l’utilisation des bactéries qui existent dans la nature. Notre stratégie a été de trouver un système biologique ayant les caractéristiques essentielles (e.x. diamètre total de moins de deux micromètres, force de poussée de plus de 4 pN, etc.) et de concentrer nos efforts à identifier une interface et une méthode permettant son contrôle pour des fins de ciblages thérapeutiques dans les lésions tumorales. Nous avons identifié les bactéries magnétotactiques de type MC-1 comme le meilleur transporteur potentiel de médicaments pour le ciblage du cancer. Les MC-1 sont à la fois dirigeables par champs magnétiques et anaérobies, ce qui leur donne un grand avantage par rapport aux bactéries traditionnellement utilisées pour le ciblage du cancer. Le ciblage du cancer avec des bactéries exploite le plus souvent l’affinité des bactéries anaérobies à la région nécrotique faible en oxygène de la tumeur. Certes, ce ciblage manque de spécificité et un des problèmes le plus reconnu est la nécessité d’injecter une forte dose de bactéries pour assurer une croissance de celles-ci à l’intérieur de la tumeur. Ceci n’est pas le cas avec les MC-1 car elles sont à la fois anaérobies et magnétotactiques grâce à une chaîne de nanoparticules d’environ 70 nanomètres de diamètre, formant une sorte de « nano-boussole » magnétique à----------ABSTRACT Magnetotactic Bacteria (MTB) are being explored as potential drug transporters to solid tumours. The MTB’s active motility combined with magnetotaxism (their ability to swim following the direction of a magnetic field) offer new and potentially more accurate solutions in delivering drugs to tumours. In fact, the flagella bundles of the MC-1 bacteria (with an overall ideal cell diameter of approximately 50% the diameter of the tiniest human blood vessels) provide 4.0 to 4.7pN of thrust force for propulsion (roughly 10 times the value of many other well-known flagellated bacteria). Since there are no existing methods or technologies capable of inducing an equivalent force on a carrier of appropriate size for traveling inside a tumour’s microvasculature, live microorganisms are considered as a viable option. Many of the parameters in a tumour microenvironment, such as malformed angiogenesis capillaries, heterogeneous blood flow, and high interstitial pressure, hinder the delivery of blood-borne drugs to the affected area. Active motility might prove to be helpful in bypassing these limitations and may facilitate the uniform distribution of the drug in the targeted area. An MTB navigation technique that allows targeting without prior knowledge of the exact architecture of the vessels network has been developed. This navigation technique exploits both the ability of the MTB to swim following an imposed magnetic field and their random, continuous motion at low magnetic fields. Firstly, a focused magnetic field on the target sets the overall direction of the bacteria. Then, as the bacteria approach the targeted zone, the intensity of the magnetic field is decreased, which allows better bacteria repartition by exploiting their free motion. An additional approach that enhances MTB targeting relies on modulating the magnetic field direction in time, while keeping the magnetic field lines pointed toward the target. Navigation experiments in complex micro-channel networks highlight this process, where the successful targeting of bacteria is demonstrated when an appropriate magnetic field algorithm is applied, especially when it takes into account the nature of the channel network. Tridimensional control and navigation of MTB is also possible with the same technique through proper powering of the magnetic coils. In fact, by controlling their magnetic environment, it is possible to form a swarm of MTB, control its size and position within a given volume using a computer program

Attachment of Therapeutic and Imaging Agents to Flagellated Magneto-Aerotactic Bacteria Cells for Tumor Treatment and Targeting Purposes

Malgré les progrès significatifs dans le domaine technologique et la compréhension du cancer (au niveau biologique), il y aura toujours des défis qui ralentiront le développement et l’implémentation de certaines options de traitements dans les essais cliniques. Les chercheurs dans les secteurs de l’administration du médicament et du génie tissulaire font face à des problèmes majeurs. Ceux-ci incluent notamment l’absence d’un système conventionnel et sélectif d’administration et de diffusion du médicament, les barrières physiologiques rencontrées par les agents antitumeur hématogènes avant de parvenir aux cellules cancéreuses dans les tumeurs solides et la séquestration des médicaments par le système immunitaire qui fait en sorte qu’une petite portion de la dose totale administrée atteint le site ciblé. Ainsi, un dosage fréquent est requis pour l’obtention de l’effet thérapeutique escompté, ce qui cause des effets adverses. Cela résulte ultimement par l’échec du traitement. De plus, l’imagerie médicale est essentielle dans le diagnostic et le traitement du cancer. Toutefois, dû à la complexité structurale des tumeurs et à la profondeur de pénétration limitée dans les tumeurs des agents de contraste disponibles, ceci était infaisable. Avec les développements récents, l’obtention d’images détaillées et à hautes résolutions a été facilitée. L’attachement et l’imagerie d’agents thérapeutiques nanométriques aux microorganismes magnéto-aerotactiques connus sous le nom de BN-1 magnetotactic bacteria (MTB) pour le ciblage des tumeurs ont été étudiés au cours de ce projet de maîtrise. Les microrobots MTB semblent être des agents de ciblage autopropulsés et navigable idéaux. Ils sont capables de voyager contre la pression du fluide interstitiel de la tumeur (TIFP) afin de cibler les régions profondes des tumeurs solides. Les complexes de MTB ont été formulés en attachant aux MTB (i) des liposomes encapsulés avec du SN38 (MTB-LP-SN38) et (ii) des nanoparticules fluidMAG-ARA superparamagnétique d’oxyde de fer magnétiques de 200 nm de diamètre (MTB-MNP). Puisque les nanoparticules magnétiques se comportent comme des agents d’imagerie par résonnance magnétique (IRM), les complexes MTB-MNP facilitent le monitoring de la structure de la tumeur et des zones hypoxiques, tout en agissant comme rétroaction dans les opérations de navigation des MTB. D'une part, les MTB-LP ont été développés par conjugaison covalente directe de liposomes fonctionnalisés à des groupements amine (–NH2) qui sont naturellement présents à la surface des bactéries MTB, via un couplage carbodiimide. D’autre part, le complexe MTB-MNP a été préparé vi selon une procédure en deux étapes. Tout d’abord, les nanoparticules magnétiques ont été fonctionnalisées avec l’anticorps BN-1 (AB) contre la protéine en surface des MTB en utilisant la chimie des carbodiimide. Par la suite, les MNP-AB ont été attachés aux MTB. Les échantillons de LP, LP-SN38 et MTB-LP-SN38 ont été analysés par chromatographie liquide/spectroscopie de mass (LC/MS), spectroscopie UV, diffusion dynamique de la lumière (DLS) et potentiel zeta (ZP). De plus, l’efficacité de l’attachement, l’alignement suivant le champ magnétique et la vitesse moyenne de natation d’échantillons de MTB-MNP soumis à un champ magnétique externe ont été examinés. Subséquemment, les résultats ont montré que les cellules de bactéries MTB sont capables de transporter des quantités thérapeutiques de médicaments et d’agents d’imagerie sans compromettre leur capacité naturelle de nager.----------ABSTRACT Despite the significant progress in technology and in the biological understanding of cancer, there are still multiple challenges that slow down the development and implementation of certain treatment options in clinical trials. The researchers in the fields of drug delivery and tissue engineering are facing major problems. Some of these include the lack of a conventional and selective drug delivery and release system, the physiological barriers that the bloodborne antitumor agents encounter before reaching cancer cells in a solid tumor and sequestration of the drugs by the immune system that makes only a few percent of the total administered dose reaching the intended target site. Hence, there is a necessity for a frequent dosing to achieve the desired therapeutic effect, which can cause adverse side effects or sometimes even treatment failure. Furthermore, medical imaging is essential in cancer diagnosis and treatment. However, current medical imaging methods have limited use due to the structural complexity of the tumor and the limited penetration depth of the previously available contrast agents into tumor tissues. With recent developments, obtaining a high-resolution and detailed image of a tumor has been facilitated. The attachment of therapeutic and imaging nanosize agents to the magneto-aerotactic microorganisms known as BN-1 magnetotactic bacteria (MTB) for tumor targeting purposes has been studied during the accomplishment of this master’s project. MTB microbiorobots appear to be ideal self-propelling and navigable targeting agents. They are capable of traveling against the Tumor Interstitial Fluid Pressure (TIFP) to target deep regions in solid tumors. MTB complexes were formulated by attaching to MTB (i) SN38 anticancer drug encapsulated liposomes (MTB-LP-SN38) and (ii) 200 nm fluidMAG-ARA superparamagnetic iron oxide magnetic nanoparticles (MTB-MNP). As the magnetic nanoparticles act as magnetic resonance imaging (MRI) contrast agents, MTB-MNP complexes facilitate monitoring the tumor structure and hypoxic zones while acting as feedback control in the MTB navigation operations. On one hand, the MTB-LP was developed by direct covalent conjugation of functionalized liposomes to amine (–NH2) groups that are naturally present on the surface of MTB bacteria, via carbodiimide-mediated coupling. On the other hand, the MTB-MNP was prepared via a two-step procedure. First, the magnetic nanoparticles were functionalized with the BN-1 antibody (AB) against the MTB protein surface using carbodiimide chemistry, then the MNP-AB were attached to the MTB. The LP, LP-SN38 and MTB-LP-SN38 samples were analyzed with liquid chromatography/mass spectroscopy (LC/MS), UV-Spectroscopy, dynamic light scattering (DLS) and zeta potential (ZP). In addition, the attachment efficiency, alignment in the magnetic field and average swimming velocity of the MTB-MNP samples submitted to an external magnetic field were investigated. Subsequently, results showed that MTB bacteria cells are capable of carrying sufficient therapeutic and imaging agents without altering their natural swimming capability

Steering of magnetotactic bacterial microrobots by focusing magnetic field for targeted pathogen killing

International audienceTargeted steering of magnetotactic bacterial microrobots is a growing tendency for their various biomedical applications. However, real-time monitoring during their movements and targeted cell killing in specific locations remains challenging. Here, we steered bacterial microrobots to target and attach to Staphylococcus aureus that was subsequently killed in a magnetic target device, which can realize guiding, mixing, and killing for targeted therapy. The generated focusing magnetic field was applied to magnetotactic bacterial microrobots, and the realizability of control strategies was analyzed. We successfully guided magnetotactic bacterial microrobots in microfluidic chips without real-time monitoring of their location. After mixing with microrobots under a rotating magnetic field for their attachment, the pathogen was killed under a swinging magnetic field. These results suggest that targeted therapy with these microrobots by using a magnetic target device is a promising approach

Attachment of Therapeutic and Imaging Agents to Magnetotactic Bacteria Acting as Self-Propelled Bio-Carriers for Cancer Treatment

RÉSUMÉ Malgré les progrès de la médecine moderne, les traitements anticancéreux actuels n’arrivent toujours pas à vaincre le cancer. Seulement une fraction des doses de médicaments administrées parvient à la tumeur en raison d’un ciblage non spécifique, de barrières physiologiques au niveau du système vasculaire ainsi que de l’élimination immédiate de médicaments par le système immunitaire. Des dosages fréquents de médicaments deviennent nécessaires afin de surmonter ces obstacles, entraînant une toxicité systémique, des effets secondaires et un échec thérapeutique. De plus, les systèmes actuels d’imagerie médicale sont incapables de produire des images de haute qualité des structures tumorales pour les diagnostiques et les traitements. Ceci est dû aux restrictions de la résolution spatiale et de l’incapacité des agents de contraste à pénétrer dans les zones tumorales afin de générer un signal suffisamment intense. Le développement de nouveaux agents thérapeutiques ainsi que de nouvelles techniques de ciblage thérapeutique sont donc requis afin d’améliorer l’efficacité des traitements actuels. Pour ce projet de recherche doctorale, l'attachement de charges utiles à la surface de bactéries magnétotactiques flagellées Magnetococcus Marinus MC-1 (BMT) a été mise en place pour transporter de façon ciblée une quantité optimale de médicaments profondément dans les zones tumorales. Ces bio-robots autopropulsés de dimensions adéquates sont équipés d’un système de propulsion dirigeable, d’un système de navigation, et de capacités sensorielles. Divers types de complexes BMT ont été fabriquées en attachant aux BMT (i) des liposomes vides (BMT-LP), (ii) des liposomes contenant un agent anticancéreux SN38 (BMT-LSC), et (iii) des nanoparticules superparamagnétiques de magnétite (BMT-S200). L’efficacité de l'attachement des charges et du comportement des bactéries soumises à un champ magnétique directionnel ont été étudiés. Par la suite, la capacité des complexes BMT à naviguer le long d’une trajectoire prédéterminée, à infiltrer profondément l'espace interstitiel, et à cibler des zones tumorales inaccessibles, ont été étudiés dans un modèle animal soumis à un champ magnétique externe. Pour parvenir à des complexes BMT aptes à transporter suffisamment de produits pharmaceutiques et de s’accumuler préférentiellement dans les régions affectées, il faut assurer un attachement solide et stable qui ne compromet pas la motilité des BMT.----------ABSTRACT Despite the substantial achievements of modern medicine, current medical therapies cannot eradicate cancer. Due to nonspecific targeting, the multiple physiological barriers that blood-borne agents must encounter, and the rapid sequestration of drugs by the immune system, a suboptimal fraction of the total injected dose reaches the intended target. These obstacles necessitate frequent dosing to compensate therapeutic effects, resulting in systemic toxicity, undesirable side effects, and treatment failure. In addition, existing medical imaging modalities struggle to provide high quality clinical images of tumor structures for treatment purposes due to limitations in spatial resolution and lack of penetration of contrast agents into tumoral regions to induce sufficient signal intensity. To address these issues, the development of new therapeutic agents alongside improved strategies for targeting therapy with the ability to control their fate is required. The attachment of payloads to the flagellated Magnetococcus Marinus MC-1 magnetotactic bacteria (MTB) to directly transport optimal quantities of pharmaceutical agents to regions located deep in tumors is what has been proposed during the accomplishment of this PhD project. These engineered self-propelled bio-robots with an appropriate dimension are equipped with steerable propulsion, navigation system, and onboard sensory capabilities. MTB complexes were fabricated by attaching the MTB to (i) empty liposomes (MTB-LP), (ii) SN38 anticancer drug encapsulated in liposomes (MTB-LSC), and (iii) 200 nm superparamagnetic magnetite nanoparticles (MTB-S200). The attachment efficacy and magnetic response behavior from the influence of a directional magnetic field of loaded bacteria with therapeutic or imaging agents were studied. Subsequently, results showed that the attachment method was suitable to allow MC-1 MTB to transport therapeutic and imaging agents along a planned trajectory prior to penetrate deep through the interstitial space in order to reach the hypoxic regions of a tumor in an animal model. To achieve MTB complexes capable of carrying sufficient pharmaceutical agents and accumulating preferentially at disease sites, the attachment must be strong and stable without compromising the natural motility of MTB. The MTB-LP were prepared by direct covalent attachment of functionalized liposomes to the amine groups naturally presented on the surface of MTB using carbodiimide (EDC/NHS) chemistry

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

Hybrid bio-robotics: from the nanoscale to the macroscale

[eng] Hybrid bio-robotics is a discipline that aims at integrating biological entities with synthetic materials to incorporate features from biological systems that have been optimized through millions of years of evolution and are difficult to replicate in current robotic systems. We can find examples of this integration at the nanoscale, in the field of catalytic nano- and micromotors, which are particles able to self-propel due to catalytic reactions happening in their surface. By using enzymes, these nanomotors can achieve motion in a biocompatible manner, finding their main applications in active drug delivery. At the microscale, we can find single-cell bio-swimmers that use the motion capabilities of organisms like bacteria or spermatozoa to transport microparticles or microtubes for targeted therapeutics or bio-film removal. At the macroscale, cardiac or skeletal muscle tissue are used to power small robotic devices that can perform simple actions like crawling, swimming, or gripping, due to the contractions of the muscle cells. This dissertation covers several aspects of these kinds of devices from the nanoscale to the macro-scale, focusing on enzymatically propelled nano- and micromotors and skeletal muscle tissue bio-actuators and bio-robots. On the field of enzymatic nanomotors, there is a need for a better description of their dynamics that, consequently, might help understand their motion mechanisms. Here, we focus on several examples of nano- and micromotors that show complex dynamics and we propose different strategies to analyze their motion. We develop a theoretical framework for the particular case of enzymatic motors with exponentially decreasing speed, which break the assumptions of constant speed of current methods of analysis and need different strategies to characterize their motion. Finally, we consider the case of enzymatic nanomotors moving in complex biological matrices, such as hyaluronic acid, and we study their interactions and the effects of the catalytic reaction using dynamic light scattering, showing that nanomotors with negative surface charge and urease-powered motion present enhanced parameters of diffusion in hyaluronic acid. Moving towards muscle-based robotics, we investigate the application of 3D bioprinting for the bioengineering of skeletal muscle tissue. We demonstrate that this technique can yield well-aligned and functional muscle fibers that can be stimulated with electric pulses. Moreover, we develop and apply a novel co-axial approach to obtain thin and individual muscle fibers that resemble the bundle-like organization of native skeletal muscle tissue. We further exploit the versatility of this technique to print several types of materials in the same process and we fabricate bio-actuators based on skeletal muscle tissue with two soft posts. Due to the deflection of these cantilevers when the tissue contracts upon stimulation, we can measure the generated forces, therefore obtaining a force measurement platform that could be useful for muscle development studies or drug testing. With these applications in mind, we study the adaptability of muscle tissue after applying various exercise protocols based on different stimulation frequencies and different post stiffness, finding an increase of the force generation, especially at medium frequencies, that resembles the response of native tissue. Moreover, we adapt the force measurement platform to be used with human-derived myoblasts and we bioengineer two models of young and aged muscle tissue that could be used for drug testing purposes. As a proof of concept, we analyze the effects of a cosmetic peptide ingredient under development, focusing on the kinematics of high stimulation contractions. Finally, we present the fabrication of a muscle-based bio-robot able to swim by inertial strokes in a liquid interface and a nanocomposite-laden bio-robot that can crawl on a surface. The first bio-robot is thoroughly characterized through mechanical simulations, allowing us to optimize the skeleton, based on a serpentine or spring-like structure. Moreover, we compare the motion of symmetric and asymmetric designs, demonstrating that, although symmetric bio-robots can achieve some motion due to spontaneous symmetry breaking during its self-assembly, asymmetric bio-robots are faster and more consistent in their directionality. The nanocomposite-laden crawling bio-robot consisted of embedded piezoelectric boron nitride nanotubes that improved the differentiation of the muscle tissue due to a feedback loop of piezoelectric effect activated by the same spontaneous contractions of the tissue. We find that bio-robots with those nanocomposites achieve faster motion and stronger force outputs, demonstrating the beneficial effects in their differentiation. This research presented in this thesis contributes to the development of the field of bio-hybrid robotic devices. On enzymatically propelled nano- and micromotors, the novel theoretical framework and the results regarding the interaction of nanomotors with complex media might offer useful fundamental knowledge for future biomedical applications of these systems. The bioengineering approaches developed to fabricate murine- or human-based bio-actuators might find applications in drug screening or to model heterogeneous muscle diseases in biomedicine using the patient’s own cells. Finally, the fabrication of bio-hybrid swimmers and nanocomposite crawlers will help understand and improve the swimming motion of these devices, as well as pave the way towards the use of nanocomposite to enhance the performance of future actuators.[spa] La bio-robótica híbrida es una disciplina cuyo objetivo es la integración de entidades biológicas con materiales sintéticos para superar los desafíos existentes en el campo de la robótica blanda, incorporando características de los sistemas biológicos que han sido optimizadas durante millones de años de evolución natural y no son fáciles de reproducir artificialmente. Esta tesis cubre varios aspectos de este tipo de dispositivos desde la nanoescala a la macroescala, enfocándose en nano- y micromotores propulsados enzimáticamente y bio-actuadores y bio-robots basados en tejido muscular esquelético. En el campo de nanomotores enzimáticos, existe la necesidad de encontrar mejores modelos que puedan describir la dinámica de su movimiento para llegar a entender sus mecanismos de propulsión subyacentes. Aquí, nos enfocamos en diversos ejemplos de nano- y micromotores que muestran dinámicas de movimiento complejas y proponemos diferentes estrategias que se pueden utilizar para analizar y caracterizar este movimiento. Moviéndonos hacia robots basados en células musculares, investigamos la aplicación de la técnica de bioimpresión en 3D para la biofabricación de músculo esquelético. Demostramos que esta técnica puede producir fibras musculares funcionales y bien alineadas que puede ser estimuladas y contraerse con pulsos eléctricos. Investigamos la versatilidad de esta técnica para imprimir varios tipos de materiales en el mismo proceso y fabricamos bio-actuadores basados en músculo esquelético. Debido a los movimientos de unos postes gracias a las contracciones musculares, podemos obtener medidas de la fuerza ejercida, obteniendo una plataforma de medición de fuerzas que podría ser de utilidad para estudios sobre el desarrollo del músculo o para testeo de fármacos. Finalmente, presentamos la fabricación de un bio-robot basado en músculo esquelético capaz de nadar en la superficie de un líquido y un bio-robot con nanocompuestos incrustados que puede arrastrarse por una superficie sólida. El primer de ellos es minuciosamente caracterizado a través de simulaciones mecánicas, permitiéndonos optimizar su esqueleto, basado en una estructura tipo serpentina o muelle. El segundo bio-robot contiene nanotubos piezoeléctricos incrustados en su tejido, los cuales ayudan en la diferenciación del músculo debido a una retroalimentación basada en su efecto piezoeléctrico y activada por las contracciones espontáneas del tejido. Mostramos que estos bio-robots pueden generar un movimiento más rápido y una mayor generación de fuerza, demostrando los efectos beneficiales en la diferenciación del tejido

Engineering derivatives from biological systems for advanced aerospace applications

The present study consisted of a literature survey, a survey of researchers, and a workshop on bionics. These tasks produced an extensive annotated bibliography of bionics research (282 citations), a directory of bionics researchers, and a workshop report on specific bionics research topics applicable to space technology. These deliverables are included as Appendix A, Appendix B, and Section 5.0, respectively. To provide organization to this highly interdisciplinary field and to serve as a guide for interested researchers, we have also prepared a taxonomy or classification of the various subelements of natural engineering systems. Finally, we have synthesized the results of the various components of this study into a discussion of the most promising opportunities for accelerated research, seeking solutions which apply engineering principles from natural systems to advanced aerospace problems. A discussion of opportunities within the areas of materials, structures, sensors, information processing, robotics, autonomous systems, life support systems, and aeronautics is given. Following the conclusions are six discipline summaries that highlight the potential benefits of research in these areas for NASA's space technology programs