Correction: Enzyme-powered micro- and nano-motors: key parameters for an application-oriented design.

[This corrects the article DOI: 10.1039/D2SC01806C.].

Enzyme-powered micro- and nano-motors: key parameters for an application-oriented design.

Nature has inspired the creation of artificial micro- and nanomotors that self-propel converting chemical energy into mechanical action. These tiny machines have appeared as promising biomedical tools for treatment and diagnosis and have also been used for environmental, antimicrobial or sensing applications. Among the possible catalytic engines, enzymes have emerged as an alternative to inorganic catalysts due to their biocompatibility and the variety and bioavailability of fuels. Although the field of enzyme-powered micro- and nano-motors has a trajectory of more than a decade, a comprehensive framework on how to rationally design, control and optimize their motion is still missing. With this purpose, herein we performed a thorough bibliographic study on the key parameters governing the propulsion of these enzyme-powered devices, namely the chassis shape, the material composition, the motor size, the enzyme type, the method used to incorporate enzymes, the distribution of the product released, the motion mechanism, the motion media and the technique used for motion detection. In conclusion, from the library of options that each parameter offers there needs to be a rational selection and intelligent design of enzymatic motors based on the specific application envisioned.

Autonomous Treatment of Bacterial Infections in Vivo Using Antimicrobial Micro- and Nanomotors.

The increasing resistance of bacteria to existing antibiotics constitutes a major public health threat globally. Most current antibiotic treatments are hindered by poor delivery to the infection site, leading to undesired off-target effects and drug resistance development and spread. Here, we describe micro- and nanomotors that effectively and autonomously deliver antibiotic payloads to the target area. The active motion and antimicrobial activity of the silica-based robots are driven by catalysis of the enzyme urease and antimicrobial peptides, respectively. These antimicrobial motors show micromolar bactericidal activity in vitro against different Gram-positive and Gram-negative pathogenic bacterial strains and act by rapidly depolarizing their membrane. Finally, they demonstrated autonomous anti-infective efficacy in vivo in a clinically relevant abscess infection mouse model. In summary, our motors combine navigation, catalytic conversion, and bactericidal capacity to deliver antimicrobial payloads to specific infection sites. This technology represents a much-needed tool to direct therapeutics to their target to help combat drug-resistant infections.

Enzyme-Powered Porous Micromotors Built from a Hierarchical Micro- and Mesoporous UiO-Type Metal-Organic Framework.

Here, we report the design, synthesis, and functional testing of enzyme-powered porous micromotors built from a metal-organic framework (MOF). We began by subjecting a presynthesized microporous UiO-type MOF to ozonolysis, to confer it with mesopores sufficiently large to adsorb and host the enzyme catalase (size: 6-10 nm). We then encapsulated catalase inside the mesopores, observing that they are hosted in those mesopores located at the subsurface of the MOF crystals. In the presence of H2O2 fuel, MOF motors (or MOFtors) exhibit jet-like propulsion enabled by enzymatic generation of oxygen bubbles. Moreover, thanks to their hierarchical pore system, the MOFtors retain sufficient free space for adsorption of additional targeted species, which we validated by testing a MOFtor for removal of rhodamine B during self-propulsion.

Ionic Species Affect the Self-Propulsion of Urease-Powered Micromotors.

Enzyme-powered motors self-propel through the catalysis of in situ bioavailable fuels, which makes them excellent candidates for biomedical applications. However, fundamental issues like their motion in biological fluids and the understanding of the propulsion mechanism are critical aspects to be tackled before a future application in biomedicine. Herein, we investigated the physicochemical effects of ionic species on the self-propulsion of urease-powered micromotors. Results showed that the presence of PBS, NaOH, NaCl, and HEPES reduced self-propulsion of urease-powered micromotors pointing towards ion-dependent mechanisms of motion. We studied the 3D motion of urease micromotors using digital holographic microscopy to rule out any motor-surface interaction as the cause of motion decay when salts are present in the media. In order to protect and minimize the negative effect of ionic species on micromotors' performance, we coated the motors with methoxypolyethylene glycol amine (mPEG) showing higher speed compared to noncoated motors at intermediate ionic concentrations. These results provide new insights into the mechanism of urease-powered micromotors, study the effect of ionic media, and contribute with potential solutions to mitigate the reduction of mobility of enzyme-powered micromotors.

Self-Propulsion of Active Colloids via Ion Release: Theory and Experiments.

We study the self-propulsion of a charged colloidal particle that releases ionic species using theory and experiments. We relax the assumptions of thin Debye length and weak nonequilibrium effects assumed in classical phoretic models. This leads to a number of unexpected features that cannot be rationalized considering the classic phoretic framework: an active particle can reverse the direction of motion by increasing the rate of ion release and can propel even with zero surface charge. Our theory predicts that there are optimal conditions for self-propulsion and a novel regime in which the velocity is insensitive to the background electrolyte concentration. The theoretical results quantitatively capture the salt-dependent velocity measured in our experiments using active colloids that propel by decomposing urea via a surface enzymatic reaction.

Intrinsic enzymatic properties modulate the self-propulsion of micromotors.

Bio-catalytic micro- and nanomotors self-propel by the enzymatic conversion of substrates into products. Despite the advances in the field, the fundamental aspects underlying enzyme-powered self-propulsion have rarely been studied. In this work, we select four enzymes (urease, acetylcholinesterase, glucose oxidase, and aldolase) to be attached on silica microcapsules and study how their turnover number and conformational dynamics affect the self-propulsion, combining both an experimental and molecular dynamics simulations approach. Urease and acetylcholinesterase, the enzymes with higher catalytic rates, are the only enzymes capable of producing active motion. Molecular dynamics simulations reveal that urease and acetylcholinesterase display the highest degree of flexibility near the active site, which could play a role on the catalytic process. We experimentally assess this hypothesis for urease micromotors through competitive inhibition (acetohydroxamic acid) and increasing enzyme rigidity (ß-mercaptoethanol). We conclude that the conformational changes are a precondition of urease catalysis, which is essential to generate self-propulsion.

Fundamental Aspects of Enzyme-Powered Micro- and Nanoswimmers.

Self-propulsion at the nanoscale constitutes a challenge due to the need for overcoming viscous forces and Brownian motion. Inspired by nature, artificial micro- and nanomachines powered by catalytic reactions have been developed. Due to the toxicity of the most commonly used fuels, enzyme catalysis has emerged as a versatile and biocompatible alternative to generate self-propulsion. Different swimmer sizes, ranging from the nanoscale to the microscale, and geometries, including tubular and spherical shapes, have been explored. However, there is still a lack of understanding of the mechanisms underlying enzyme-mediated propulsion. Size, shape, enzyme quantity and distribution, as well as the intrinsic enzymatic properties, may play crucial roles in motion dynamics. In this Account, we present the efforts carried out by our group and others by the community on the use of enzymes to power micro- and nanoswimmers. We examine the different structures, materials, and enzymes reported so far to fabricate biocatalytic micro- and nanoswimmers with special emphasis on their effect in motion dynamics. We discuss the development of tubular micro- and nanojets, focusing on the different fabrication methods and the effect of length and enzyme localization on their motion behavior. In the case of spherical swimmers, we highlight the role of asymmetry in enzyme coverage and how it can affect their motion dynamics. Different approaches have been described to generate asymmetric distribution of enzymes, namely, Janus particles, polymeric vesicles, and non-Janus particles with patch-like enzyme distribution that we recently reported. We also examine the correlation between enzyme kinetics and active motion. Enzyme activity, and consequently speed, can be modulated by modifying substrate concentration or adding specific inhibitors. Finally, we review the theory of active Brownian motion and how the size of the particles can influence the analysis of the results. Fundamentally, nanoscaled swimmers are more affected by Brownian fluctuations than microsized swimmers, and therefore, their motion is presented as an enhanced diffusion with respect to the passive case. Microswimmers, however, can overcome these fluctuations and show propulsive or ballistic trajectories. We provide some considerations on how to analyze the motion of these swimmers from an experimental point of view. Despite the rapid progress in enzyme-based micro- and nanoswimmers, deeper understanding of the mechanisms of motion is needed, and further efforts should be aimed to study their lifetime, long-term stability, and ability to navigate in complex media.

Influence of Enzyme Quantity and Distribution on the Self-Propulsion of Non-Janus Urease-Powered Micromotors.

The use of enzyme catalysis to power micro- and nanomachines offers unique features such as biocompatibility, versatility, and fuel bioavailability. Yet, the key parameters underlying the motion behavior of enzyme-powered motors are not completely understood. Here, we investigate the role of enzyme distribution and quantity on the generation of active motion. Two different micromotor architectures based on either polystyrene (PS) or polystyrene coated with a rough silicon dioxide shell (PS@SiO2) were explored. A directional propulsion with higher speed was observed for PS@SiO2 motors when compared to their PS counterparts. We made use of stochastically optical reconstruction microscopy (STORM) to precisely detect single urease molecules conjugated to the micromotors surface with a high spatial resolution. An asymmetric distribution of enzymes around the micromotor surface was observed for both PS and PS@SiO2 architectures, indicating that the enzyme distribution was not the only parameter affecting the motion behavior. We quantified the number of enzymes present on the micromotor surface and observed a 10-fold increase in the number of urease molecules for PS@SiO2 motors compared to PS-based micromotors. To further investigate the number of enzymes required to generate a self-propulsion, PS@SiO2 particles were functionalized with varying amounts of urease molecules and the resulting speed and propulsive force were measured by optical tracking and optical tweezers, respectively. Surprisingly, both speed and force depended in a nonlinear fashion on the enzyme coverage. To break symmetry for active propulsion, we found that a certain threshold number of enzymes molecules per micromotor was necessary, indicating that activity may be due to a critical phenomenon. Taken together, these results provide new insights into the design features of micro/nanomotors to ensure an efficient development.