Enzyme-powered nanomotors are an exciting technology for biomedical applications due to their ability to navigate within biological environments using endogenous fuels. However, limited studies into their collective behavior and demonstrations of tracking enzyme nanomotors in vivo have hindered progress toward their clinical translation. Here, we report the swarming behavior of urease-powered nanomotors and its tracking using positron emission tomography (PET), both in vitro and in vivo. For that, mesoporous silica nanoparticles containing urease enzymes and gold nanoparticles were used as nanomotors. To image them, nanomotors were radiolabeled with either 124I on gold nanoparticles or 18F-labeled prosthetic group to urease. In vitro experiments showed enhanced fluid mixing and collective migration of nanomotors, demonstrating higher capability to swim across complex paths inside microfabricated phantoms, compared with inactive nanomotors. In vivo intravenous administration in mice confirmed their biocompatibility at the administered dose and the suitability of PET to quantitatively track nanomotors in vivo. Furthermore, nanomotors were administered directly into the bladder of mice by intravesical injection. When injected with the fuel, urea, a homogeneous distribution was observed even after the entrance of fresh urine. By contrast, control experiments using nonmotile nanomotors (i.e., without fuel or without urease) resulted in sustained phase separation, indicating that the nanomotors’ self-propulsion promotes convection and mixing in living reservoirs. Active collective dynamics, together with the medical imaging tracking, constitute a key milestone and a step forward in the field of biomedical nanorobotics, paving the way toward their use in theranostic applications.

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)...

The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3′-Co­(1,2-C2B9H11)2]−), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.

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