Monitoring the collective behavior of enzymatic nanomotors in vitro and in vivo by PET-CT

Abstract

Enzyme powered nanomotors hold great potential for biomedical applications, as they show improved diffusion and navigation within biological environments using endogenous fuels. Yet, understanding their collective behavior and tracking them in vivo is paramount for their clinical translation. Here, we report on the in vitro and in vivo study of swarms of selfpropelled enzyme-nanomotors and the effect of collective behavior on the nanomotors distribution within the bladder. For that purpose, mesoporous silica nanomotors were functionalized with urease enzymes and gold nanoparticles. Two radiolabeling strategies, i.e. absorption of 124 I on gold nanoparticles and covalent attachment of an 18 F-labeled prosthetic group to urease, were assayed. In vitro experiments using optical microscopy and positron emission tomography (PET) showed enhanced fluid mixing and collective migration of nanomotors in phantoms containing complex paths. Biodistribution studies after intravenous administration in mice confirmed the biocompatibility of the nanomotors at the administered dose, the suitability of PET to quantitatively track nanomotors in vivo, and the convenience of the 18 F-labeling strategy. Furthermore, intravesical instillation of nanomotors within the bladder in the presence of urea resulted in a homogenous distribution after the entrance of fresh urine. Control experiments using BSA-coated nanoparticles or nanomotors in water resulted in sustained phase separation inside the bladder, demonstrating that the catalytic decomposition of urea can provide urease-nanomotors with active motion, convection and mixing capabilities in living reservoirs. This active collective dynamics, together with the medical imaging tracking, constitutes a key milestone and a step forward in the field of biomedical nanorobotics, paving the way towards their use in theranostic applications.to the use of endogenous fuels, which enables nanomotors' on-site activation and the design of fully biocompatible motor-fuel complexes. Moreover, the library of enzyme/substrate combinations permits the design of application-tailored enzymatic nanomotors, as is the case of urease-powered nanomotors for bladder cancer therapy. (4) Yet, the large number of nanoparticles required to treat tumors (18) demands for a better understanding, control and visualization of nanoparticle swarms to aid in the evaluation of motile nanomedicines and facilitate the eventual translation into clinics. Indeed, collective phenomena commonly observed in nature (active filaments,(19) bacteria quorum sensing,(20) cell migration,(21) swarms of fish, ants, and birds)(22) also occurs in micro-/nanomotors, which demonstrated collective migration,(23-26) assembly,(27-30) or aggregation/diffusion behaviors in vitro.(31-38) Ex vivo, swarms of magnetic micropropellers demonstrated longrange propulsion through porcine eyes to the retina, suggesting potential as active ocular delivery devices.(39, 40) In vivo, controlled swimming of micromotor swarms was shown in mouse peritoneal cavities,(41)...

Biohybrid robotics: From the nanoscale to the macroscale

“…More investigations into these fundamental aspects will be necessary to optimize the fabrication and performance of these devices, but also reduce their side effects. Future research should focus more on the fundamental interactions with biological matrices (Palagi et al, 2017; Walker et al, 2015; Z. Wu et al, 2018) and also in the interaction between several of these nanomotors, which would also offer an interesting system to study in active matter physics (Hortelao, Simó, et al, 2020; Illien et al, 2017). The effects elements like ionic species or pH on the motion efficiency still need to be further investigated, as seemingly contradictory results arise (Arqué et al, 2020; De Corato et al, 2020; Tang et al, 2020), hinting at a much more complex interaction between all the actors involved in the motion.…”

“…Enhanced diffusion of urease‐powered nanomotors can improve the efficiency of anticancer drugs in 2D and 3D cultures of bladder cancer cells by showing an increased internalization when the nanomotors are targeted to a transmembrane protein (Figure 3a) (Hortelao et al, 2019; Hortelão et al, 2018). The same system was recently used in combination with the isotopes Iodine‐124 and Fluorine‐18 to study their suitability for in vitro and in vivo imaging via positron emission tomography (PET) coupled with computed tomography (PET‐CT) (Hortelao, Simó, et al, 2020), following a previous study that showed the successful imaging of motors of micrometer scale by chemisorption of Iodine into their gold surface (Vilela et al, 2018). Upon addition of urease, the nanomotors presented complex swarming behavior that could be tracked by PET‐CT (Figure 3b) and were homogeneously distributed in the bladder cavity of mice after intravesical administration (Figure 3c) (Hortelao, Simó, et al, 2020).…”

“…Bottom images show swarming behavior of these nanomotors after the addition of urea via PET in vitro. Adapted with permission from Hortelao, Simó, et al (2020). (c) in vivo PET‐CT images of urease nanomotors radiolabeled with Fluodine‐18 after intravesicular administration, with and without urea, showing their distribution in the bladder of mice.…”

“…(c) in vivo PET‐CT images of urease nanomotors radiolabeled with Fluodine‐18 after intravesicular administration, with and without urea, showing their distribution in the bladder of mice. Adapted with permission from Hortelao, Simó, et al (2020). (d) Schematic representation of an asymmetric polymerosome nanomotor propelled by the tandem reaction of GOx and catalase.…”

Spontaneous polarization and locomotion of an active particle with surface-mobile enzymes

“…Mimicking living cells and given the availability of chemical energy in the environment, artificial colloidal particles can be designed to self-propel through surface chemical reactions [1,2]. Besides serving as a model system to explore collective nonequilibrium phenomena [3], several technological applications have been envisaged for these active particles: from biomedical [4][5][6] to environmental remediation [7]. To achieve self-propulsion, different mechanism have been proposed such as diffusiophoresis [8], thermophoresis [9,10], momentum exchange [11], release of ions [12], and liquid-liquid phase separation [13].…”