Mahmood Mohammadi

Flagellated Magnetotactic Bacteria as Controlled MRI-trackable Propulsion and Steering Systems for Medical Nanorobots Operating in the Human Microvasculature

Although nanorobots may play critical roles for many applications in the human body, such as targeting tumoral lesions for therapeutic purposes, miniaturization of the power source with an effective onboard controllable propulsion and steering system have prevented the implementation of such mobile robots. Here, we show that the flagellated nanomotors combined with the nanometer-sized magnetosomes of a single magnetotactic bacterium can be used as an effective integrated propulsion and steering system for devices, such as nanorobots, designed for targeting locations only accessible through the smallest capillaries in humans while being visible for tracking and monitoring purposes using modern medical imaging modalities such as magnetic resonance imaging. Through directional and magnetic field intensities, the displacement speeds, directions, and behaviors of swarms of these bacterial actuators can be controlled from an external computer.

MRI-based Medical Nanorobotic Platform for the Control of Magnetic Nanoparticles and Flagellated Bacteria for Target Interventions in Human Capillaries

Medical nanorobotics exploits nanometer-scale components and phenomena with robotics to provide new medical diagnostic and interventional tools. Here, the architecture and main specifications of a novel medical interventional platform based on nanorobotics and nanomedicine, and suited to target regions inaccessible to catheterization, are described. The robotic platform uses magnetic resonance imaging (MRI) for feeding back information to a controller responsible for the real-time control and navigation along pre-planned paths in the blood vessels of untethered magnetic carriers, nanorobots, and/or magnetotactic bacteria (MTB) loaded with sensory or therapeutic agents acting like a wireless robotic arm, manipulator, or other extensions necessary to perform specific remote tasks. Unlike known magnetic targeting methods, the present platform allows us to reach locations deep in the human body while enhancing targeting efficacy using real-time navigational or trajectory control. We describe several versions of the platform upgraded through additional software and hardware modules allowing enhanced targeting efficacy and operations in very difficult locations such as tumoral lesions only accessible through complex microvasculature networks.

Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions

Oxygen depleted hypoxic regions in the tumour are generally resistant to therapies1. Although nanocarriers have been used to deliver drugs, the targeting ratios have been very low. Here, we show that the magneto-aerotactic migration behaviour2 of magnetotactic bacteria3, Magnetococcus marinus strain MC-14, can be used to transport drug-loaded nanoliposomes into hypoxic regions of the tumour. In their natural environment, MC-1 cells, each containing a chain of magnetic iron-oxide nanocrystals5, tend to swim along local magnetic field lines and towards low oxygen concentrations6 based on a two-state aerotactic sensing system2. We show that when MC-1 cells bearing covalently bound drug-containing nanoliposomes were injected near the tumour in SCID Beige mice and magnetically guided, up to 55% of MC-1 cells penetrated into hypoxic regions of HCT116 colorectal xenografts. Approximately 70 drug-loaded nanoliposomes were attached to each MC-1 cell. Our results suggest that harnessing swarms of microorganisms exhibiting magneto-aerotactic behaviour can significantly improve the therapeutic index of various nanocarriers in tumour hypoxic regions.

Using a swarm of self-propelled natural microrobots in the form of flagellated bacteria to perform complex micro-assembly tasks

Many science fiction novels have envisioned swarms of artificial microrobots capable of performing complex collective tasks. Unfortunately, todays technological constraints have prevented such powerful concept to be a reality when considering artificial microrobots. In this paper, we show that a swarm of computer-controlled flagellated Magnetotactic Bacteria (MTB) acting as natural microrobots of approximately 1 to 2 micrometers in diameter can perform many of the same complex collective tasks envisioned with these futuristic self-propelled artificial microrobots. To prove the concept, magnetotaxis-based control has been used to coordinate a swarm made of thousands of these self-propelled natural microrobots to build in a collective effort, a miniature version of an ancient Egyptian pyramid.

Covalent binding of nanoliposomes to the surface of magnetotactic bacteria for the synthesis of self-propelled therapeutic agents.

The targeted and effective delivery of therapeutic agents remains an unmet goal in the field of controlled release systems. Magnetococcus marinus MC-1 magnetotactic bacteria (MTB) are investigated as potential therapeutic carriers. By combining directional magnetotaxis-microaerophilic control of these self-propelled agents, a larger amount of therapeutics can be delivered surpassing the diffusion limits of large drug molecules toward hard-to-treat hypoxic regions in solid tumors. The potential benefits of these carriers emphasize the need to develop an adequate method to attach therapeutic cargos, such as drug-loaded nanoliposomes, without substantially affecting the cells ability to act as delivery agents. In this study, we report on a strategy for the attachment of liposomes to MTB (MTB-LP) through carbodiimide chemistry. The attachment efficacy, motility, and magnetic response of the MTB-LP were investigated. Results confirm that a substantial number of nanoliposomes (∼70) are efficiently linked with MTB without compromising functionality and motility. Cytotoxicity assays using three different cell types (J774, NIH/3T3, and Colo205) reveal that liposomal attachments to MTB formulation improve the biocompatibility of MTB, whereas attachment does not interfere with liposomal uptake.

Three-dimensional remote aggregation and steering of magnetotactic bacteria microrobots for drug delivery applications

Magnetotactic bacteria (MTB) can be viewed as self-propelled natural microrobots. These bacterial microrobots can be remotely controlled using magnetic fields due to their internal chain of iron-oxide nanoparticles acting like a compass needle. This internal chain enables them to adopt a magnetotactic behavior that can be exploited to perform a variety of microscale tasks from microassembly and micro-manufacturing to the delivery through microvascular networks of therapeutic agents to tumors. To effectively support these applications, three-dimensional (3D) aggregations of MTB become essential in order to manipulate and guide the bacteria effectively in the human microvasculature to deliver a predefined dose of therapeutics. To achieve such aggregations in a 3D volume, time-varying magnetic field sequences were developed enabling us to simulate in time the existence of a magnetic monopole. This article presents and compares three different time-varying magnetic field sequences generated by three orthogonal pairs of electromagnets able to generate such 3D aggregations of MTB.

Structural Insight Into the Expanded Pcb-Degrading Abilities of a Biphenyl Dioxygenase Obtained by Directed Evolution.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzymes oxygenase component (BphAE(LB400)) are largely unknown. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzymes reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzymes substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.

Retuning Rieske-type Oxygenases to Expand Substrate Range

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAEp4, a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAELB400 by the double substitution T335A/F336M, and BphAERR41, obtained by changing Asn338, Ile341, and Leu409 of BphAEp4 to Gln338, Val341, and Phe409, metabolize dibenzofuran two and three times faster than BphAELB400, respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAELB400 showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

Flagellated bacterial nanorobots for medical interventions in the human body

We show that a combination of various types of nanorobots will prove to be more important as we attend to enhance targeting in the smallest blood vessels found in the human microvasculature. As such, various interdependent concepts for the implementation of these different types of medical bio-nanorobots including nanorobots propelled in the microvasculature by flagellated bacteria to target deep regions in the human body are presented. Through experimental results and theoretical formulations, we also showed the advantages of integrating biological components and more specifically Magnetotactic Bacteria (MTB) for the development of hybrid (made of synthetic and biological components) nanorobots adapted to operate in the human microvasculature. We also show a method capable to track using MRI as imaging modality, steerable microbeads and MTB that could be integrated in the implementation of future sophisticated bio-nanorobots operating inside the complex vascular network. As such, we show that these nanorobots including the ones propelled by a single flagellated bacterium could be guided or controlled directly towards specific locations deep inside the human body. We also show experimentally that flagellated bacterial nanorobots could be propelled and steered in vivo through the interstitial region of a tumor for enhanced therapeutic results.

Magnetic Resonance Imaging of Fe 3 O 4 Nanoparticles Embedded in Living Magnetotactic Bacteria for Potential Use as Carriers for In Vivo Applications

MC-1 Magnetotactic Bacteria (MTB) are studied for their potential use as bio-carriers for drug delivery. The exploitation of the flagella combined with nanoparticles magnetite or magnetosomes chain embedded in each bacterium and used to change the swimming direction of each MTB through magnetotaxis provide both propulsion and steering in small diameters blood vessels. But for guiding these MTB towards a target, being capable to image these living bacteria in vivo using an existing medical imaging modality is essential. Here, it is shown that the magnetosomes embedded in each MTB can be used to track the displacement of these bacteria using an MRI system. In fact, these magnetosomes disturb the local magnetic field affecting T1 and T2-relaxation times during MRI. MR T1- weighted and T2-weighted images as well as T2-relaxivity of MTB are studied in order to validate the possibility of monitoring MTB drug delivery operations using a clinical MR scanner. This study proves that MTB affect much more the T2-relaxation than T1-relaxation rate and can be though as a negative contrast agent. The signal decay in the T2-weighted images is found to change proportionally to the bacterial concentration. These results show that a bacterial concentration of 2.2times107 cells/mL can be detected using a T2-weighted image, which is very encouraging to further investigate the application of MTB for in vivo applications.