Engineering microrobots for targeted cancer therapies from a medical perspective

Systemic chemotherapy remains the backbone of many cancer treatments. Due to its untargeted nature and the severe side effects it can cause, numerous nanomedicine approaches have been developed to overcome these issues. However, targeted delivery of therapeutics remains challenging. Engineering microrobots is increasingly receiving attention in this regard. Their functionalities, particularly their motility, allow microrobots to penetrate tissues and reach cancers more efficiently. Here, we highlight how different microrobots, ranging from tailor-made motile bacteria and tiny bubble-propelled microengines to hybrid spermbots, can be engineered to integrate sophisticated features optimised for precision-targeting of a wide range of cancers. Towards this, we highlight the importance of integrating clinicians, the public and cancer patients early on in the development of these novel technologies

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

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

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

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

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

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