This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. pubs.acs.org/NanoLett Letter Hybrid Biodegradable Nanomotors through Compartmentalized Synthesis Imke A. B. Pijpers, Shoupeng Cao, Antoni Llopis-Lorente, Jianzhi Zhu, Shidong Song, Rick R. M. Joosten, Fenghua Meng, Heiner Friedrich, David S. Williams, Samuel Sań chez, Jan C. M. van Hest,* and Loai K. E. A. Abdelmohsen* Cite This: Nano Lett. 2020, 20, 4472−4480 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Designer particles that are embued with nano- machinery for autonomous motion have great potential for biomedical applications; however, their development is highly demanding with respect to biodegradability/compatibility. Pre- viously, biodegradable propulsive machinery based on enzymes has been presented. However, enzymes are highly susceptible to proteolysis and deactivation in biological milieu. Biodegradable hybrid nanomotors powered by catalytic inorganic nanoparticles provide a proteolytically stable alternative to those based upon enzymes. Herein we describe the assembly of hybrid biodegradable nanomotors capable of transducing chemical energy into motion. Such nanomotors are constructed through a process of compartmentalized synthesis of inorganic MnO2 nanoparticles (MnPs) within the cavity of organic stomatocytes. We show that the nanomotors remain active in cellular environments and do not compromise cell viability. Effective tumor penetration of hybrid nanomotors is also demonstrated in proof-of-principle experiments. Overall, this work represents a new prospect for engineering of nanomotors that can retain their functionality within biological contexts. KEYWORDS: Biodegradable nanomotors, Compartmentalization, Stomatocyte, Hybrid nanosystems, Biomedical applications E ngineering of motile vehicles at the micro- and nanoscale with built-in systems of chemical propulsion has witnessed a surge of interest over the past decade.1−4 In such systems, Recently, the group of Sen showed the ability of liposome protocells to undergo positive and negative chemotaxis behavior, unlocking tremendous opportunities for biological autonomous motion is achieved by the conversion of chemical transport.25 Such systems not only demonstrate the advanta- energy into kinetic energy.5,6 Such chemical processes are geous aspects of non-Brownian motility within biological empowered by various forms of bio-organic and inorganic contexts but also highlight the challenges faced in engineering catalysts (e.g., enzymes and platinum nanoparticles).7,8 The of new features for such complex materials and emphasize the potential of motile materials in biomedical research (by, e.g., importance of molecular interactions between the motors and facilitating the active transport of biofunctional cargo) has their environmenta significant aspect for their effective been presented as a key opportunity for the exploitation of this function and application. technology.9−11 Indeed, developing robust chemistries that can Miniaturization of motile systems to the nanoscale is give rise to motility and diverse structural architectures that extremely challenging, but it is critical for their development form the molecular chassis for these vehicles drives fascinating toward biomedical applications by facilitating effective progress toward the actual translation of these systems.12−15 In interactions with cells and tissues while avoiding the particular, controlling physical properties such as particle unfavorable fate of microscopic particles (e.g., poor circulation morphology and composition is essential to expand the and distribution).26,27 Developing both the nanomotor chassis motors’ potential in applications that can benefit from non- and propulsive unit for biodegradability and compatibility Brownian motility.16−19 Developing micro- and nanomotors suitable for biomedical integration has been explored by various research groups, who have demonstrated motors’ Received: March 23, 2020 capability in undertaking complex tasks.20−23 Städler et al. Revised: May 19, 2020 showed the capacity of microswimmers for directed Published: May 19, 2020 penetration of three-dimensional (3D) cell spheroids, offering a wealth of opportunities for the application of micro- and nanomotors in tissue penetration and cargo/drug delivery.24 © 2020 American Chemical Society https://dx.doi.org/10.1021/acs.nanolett.0c01268 4472 Nano Lett. 2020, 20, 4472−4480 Nano Letters pubs.acs.org/NanoLett Letter Figure 1. (A) Schematic demonstrating the formation of spherical polymersomes and their subsequent osmotic-induced shape transformation into stomatocytes. (B) Schematic demonstrating the compartmentalized synthesis of MnPs within the stomatocytes’ lumen and subsequent formation of hybrid nanomotors that can convert fuel into mechanical motion. (C) Cryo-TEM image of an empty stomatocyte. (D) Cryo-TEM image of a MnP-loaded stomatocyte. (E) 3D rendering of a MnP-loaded stomatocyte obtained by TEM tomography. Scale bars = 200 nm. while preventing unwanted deactivation by inevitable environ- example, incorporation of photosensitizers and surface coating mental factors (e.g., the presence of proteolytic enzymes) is an with cell membrane components.34 On the basis of these important milestone toward the widespread adoption of such developments, biodegradable stomatocyte nanomotors with materials. Dual-compartmentalized stomatocytes, which are enzymes covalently tethered in their cavity have been bowl-shaped polymersomes,28,29 are uniquely suited for reported.35 However, as the neck size of these motors was nanomotor fabrication because of their asymmetric structure not controlled, the protection capacity was lost, leaving with an internal cavity (or stomach) that is in direct contact incorporated enzymes exposed to proteolytic deactivation with the external environment via a tunable neck. In our severely limiting, if not withholding, their application in previous work, we have showcased the potential of such biological contexts. stomatocytes to function as nanomotors by encapsulating Building on these recent developments and the considerable enzymatic cargo to drive their mechanical motion;30,31 capacity of stomatosomal architectures, exploiting the unique however, that work utilized nondegradable polystyrene block structure of PDLLA stomatocytes as nanomotors that can copolymers (BCPs) that, without doubt, lack any immediate function within biological contexts holds great future potential biomedical relevance. The motility of such systems using for biomedical applications. To this end, in the present work enzymatic cargo was achieved by modulating the size of the we employed inorganic nanoparticles as robust catalytic opening (or neck) during encapsulation so that the macro- nanomachinery that can drive the stomatocytes’ thrust in molecular cargo could be effectively sequestered, preventing harsh proteolytic media. Inorganic catalysts, such as manganese unwanted release or digestion by external proteases. From that dioxide (MnO2), have found application in, for example, work, the capacity of stomatocytes to confine and protect cancer therapy.36−38 In general, such nanoparticles are biocatalysts in their stomach, allowing enzymatic reactions to generated through surface stabilization or in situ formation take place that can drive motility in complex biological media, using protein or polymer nanostructures,39,40 making hybrid was apparent. Capitalizing on that research, our group has architectures. As MnO2 is capable of catalase-mimetic catalytic developed biodegradable stomatocytes by employing well- decomposition of hydrogen peroxide (H2O2) into water and defined poly(ethylene glycol)-block-poly(D,L-lactide) (PEG− oxygen (to drive motion through nanobubble formation), it is PDLLA) BCPs.32,33 Such PDLLA-based stomatocytes were a suitable candidate to replace enzymes as the propulsive units effectively integrated into biomedical research through, for for our biodegradable/compatible stomatocyte nanomotors. 4473 https://dx.doi.org/10.1021/acs.nanolett.0c01268 Nano Lett. 2020, 20, 4472−4480 Nano Letters pubs.acs.org/NanoLett Letter Furthermore, the utility of MnO2 particles as contrast agents structural features of stomatocytes by utilizing their inner has recently been shown through in vivo studies.41 In fact, nanocompartment as a 3D template for the synthesis of researchers have recently pointed out the broad potential of inorganic MnPs that would be more readily entrapped within enzyme-like inorganic particles for biomedical applications, as this domain and not escape through the 94 nm neck (Figures they hold interesting advantages: higher catalytic stability, ease 1E and S4 and Videos S1 and S2). This compartmentalized of modification/functionalization, lower manufacturing costs synthesis of MnPs was achieved by dissolving potassium compared with enzymes, utility as multimodal platforms, and permanganate (KMnO4) in the stomatocyte solution for 1 h remote control by external cues (magnetic fields, light, prior to reduction using sodium thiosulfate (Na2S2O3). BSA etc.).42,43 In this paper, we demonstrate the ability of was added to stabilize the resulting colloidal dispersion and to PDLLA stomatocytes to be used for the compartmentalized prevent uncontrolled aggregation. In this way, MnPs were synthesis of MnO2, a strategy that results in highly effective formed and compartmentalized within the stomatocyte, functional integration that yields fully biodegradable and whereas non-entrapped particles were removed readily using biocompatible nanomotors that are not vulnerable to centrifugation (Figures 1B,D, S5, and S6). It is noteworthy that enzymatic deactivation in complex biological media. Using diverse types of preformed nanoparticles, including MnPs, advanced characterization techniques, we explore the structure could not be physically encapsulated within the cavity of of hybrid nanomotors and demonstrate their enhanced motion, PDLLA stomatocytes, as observed in initial trials (Figure S7). stability against protease deactivation, degradability, and Therefore, the in situ synthesis/growth of MnO2 nanoparticles compatibility with cells. In this way, this work demonstrates within the stomatocyte cavity was necessary for the successful a novel concept for the design principles of a nanomotor realization of our hybrid nanomotor system. Cryo-TEM platform that is primed for further application in biomedical imaging and 3D tomographic reconstruction confirmed the research. In doing so, we also showcase a robust approach for well-defined nature of the resulting hybrid architectures the synthesis of inorganic catalysts using a compartmentaliza- (Figures 1D,E, S4, and S5 and Videos S1 and S2). tion approach to support the fabrication of complex, Interestingly, although the average size of individual MnPs multifunctional nanoarchitectures. was ca. 25 nm (Figure S8), fractionally smaller than the Stomatocyte nanomotors were assembled from the bottom- apparent size of the neck, the high density of particles inside up utilizing biodegradable copolymers comprising 1 and 2 kDa the stomach seemed to create a retaining force to prevent their PEG and PDLLA (n = 95). Such copolymers were synthesized escape, as observed from images from cryo-TEM and via organo-catalyzed ring-opening polymerization following tomographic analysis (Figures 1E, S4, and S5 and Videos S1 our previously published procedure (Supporting Information and S2). Analysis of tomography data indicated the formation (SI) section S2, Figures S1 and S2, and Table S1).33 Solvent- of nanoaggregates/agglomerates (i.e., particles attached to each induced supramolecular self-assembly of amphiphilic PEG− other) with sizes larger than the neck of the stomatocytes PDLLA into polymersomes was performed using equimolar (Figures 1C and S4 and Videos S1 and S2). Therefore, escape blends of both copolymers in organic solvent (10 wt % in of such nanoparticles from the inner cavity did not occur, even THF/dioxane, 1:4 v/v) followed by controlled addition of an after 3 months. Remarkably, the size of such nanoaggregates equal volume of Milli-Q water. The shape transformation of was significantly larger than that of the nanoparticles grown spherical polymersomes into bowl-shaped stomatocytes was outside the stomatocytes. This can be attributed to a then achieved by applying an osmotic pressure on the templating effect: the stomatocytes’ nanocavity templates the plasticized membrane through dialysis against 75 mM NaCl growth of the MnO2 nanoparticles. This same effect was at 4 °C (Figure 1A,C). Tailoring the salt concentration during previously observed in the case of either in situ-grown dialysis can, to a certain extent, tune the neck size of such nanoparticles or the assembly of enzymes in polystyrene- stomatocytes through increased deflation and consequent based stomatocytes.30,31,45 Such a difference in the sizes of volume reduction. Under the conditions employed, an average nanoparticles formed inside and outside of the stomatocytes neck size of 94 ± 50 nm was achieved; this is ca. 15 times can be due to the difference in nanoparticle growth rate. The larger than the size of a protein like bovine serum albumin low volume of the stomatocyte nanocavity compared with the (BSA). The formation of such stomatocytes was monitored outer solution could result in the formation of fewer nucleation and confirmed using dynamic light scattering (DLS), nano- centers and hence larger particles. This is in agreement with tracking analysis (NTA), and cryogenic transmission electron previous reports that demonstrate the relationship between microscopy (cryo-TEM) (Figure S3). nanoparticle size and the concentration of both the inorganic Encapsulation of active components into the PDLLA-based salt and capping agent.45 This approach is general and would stomatocyte cavity is a challenging task. One example where be applicable to the compartmentalized synthesis of a range of this is most apparent is the covalent approach adopted by the metal/metal oxide nanoparticles, with the open neck group of Wilson to localize enzymes in the stomatocytes’ permitting the flux of molecular substrates, preventing cavity;35 however, controlling such complex, multistep, coalescence/aggregation while facilitating application in functionalization steps remains elusive and necessitates processing methods such as microfluidics because of the alternative strategies. Inspired by previous examples showing overall size of the compartment. the controlled growth of inorganic nanoparticles as an effective In order to study the ability of our hybrid stomatocytes to strategy for their inclusion in organic materials (e.g., protein undergo autonomous motion, NTA was used to record the and polymer cages),44 we developed a non-covalent approach nanomotors’ movement and track their trajectories as MnPs based on the compartmentalized synthesis of inorganic MnO2 catalyzed the formation of O2. In general, O2-driven propulsion nanoparticles (MnPs) within the organic stomatocytes as an is mediated by fuel decomposition and subsequent formation effective one-pot pathway for catalyst loading and subsequent of nanobubbles that propel the particle through their release construction of hybrid nanomotors. As evidenced by TEM from the motor’s chassis. The asymmetric structure of tomographic analysis, our approach exploited the unique stomatocytes, with a unidirectional neck, are particularly suited 4474 https://dx.doi.org/10.1021/acs.nanolett.0c01268 Nano Lett. 2020, 20, 4472−4480 Nano Letters pubs.acs.org/NanoLett Letter Figure 2. (A) Schematic demonstrating the spatial positioning of catalytic MnPs within stomatocytes, MnPs’ capacity to convert a range of fuel concentrations into O2 nanobubbles, and the (hypothesized) mechanism by which motion occurs. (B) MSD and velocity of hybrid nanomotors in the presence of a range of H2O2 concentrations. The velocities were theoretically calculated from MSD = (4D)Δt + (v2)(Δt2). NTA measurements were performed by diluting MnP-loaded stomatocytes (1:1000) in 1× PBS ([polymer] = 5 μg mL−1). Thereafter, aliquots of H2O2 were added (to reach a final concentration of 5, 25, or 50 mM). (C) Tracked Brownian and non-Brownian trajectories in the absence and presence of fuel, respectively. (D) Diffusion coefficients of MnP-loaded stomatocytes as a result of multiple cycles of fuel addition and depletion. In order to ensure complete depletion of fuel, the time interval between experiments (fuel addition) was 1 day. (E) MSDs of hybrid stomatocytes exposed to 50 μg mL−1 proteinase K (dotted line). to exploit this bubble-propelled motion. This is indeed H2O2 (100 mM) was added to unloaded stomatocytes. This reflected by their propulsion, which was found to be did not result in any autonomous motion, and all of the proportional to the concentration of H2O2 (Figure 2A and stomatocytes maintained their Brownian motion (Figure S11A Video S3). The mean square displacements (MSDs) and and Video S4). Further controls in which empty stomatocytes velocities of such hybrid stomatocyte nanomotors were were combined with free MnPs in solution yielded no motion, analyzed using Golestanian’s self-diffusiophoretic model (the indicating the importance of catalyst spatial positioning within detailed analysis is described in SI section S2).46 The range of the stomatocytes (Figure S11B and Video S5). observed MSD values for hybrid stomatocytes upon addition To highlight the robust nature of these nanomotors, we of an increasing concentration of fuel reflects the inherent studied their performance over time. To do so, nanomotors polydispersity in terms of MnO2 loading achieved in this were tested on their ability to be reactivated upon fuel system (Figure S9). Although one would expect linear fits of depletion (Figure 2D). After 3 days and over the course of the MSD curves at relatively high Δt (because of the short self- three cycles of fuel addition and depletion, no change in their rotation time τr of such small nanoparticles), nonlinear fits diffusion coefficient was recorded (as measured by NTA), were identified at high concentrations of fuel (Figure 2B). indicating the stability of this hybrid nanomotor system and Indeed, analysis of the anomalous diffusive exponent (α) value that the MnP platform is a highly suitable and robust catalytic after addition of fuel showed an increase, which suggests an nanomachine that is not degraded after a single use. Finally, enhanced superdiffusion (propulsive) behavior (Figure S10). the motility of our hybrid nanomotors was tested in a These results suggest that the autonomous movement of such proteolytic environment, namely, in the presence of proteinase nanomotors is a consequence of the bubble generation K at a concentration of 50 μg mL−1 (30 units mg−1). At first mechanism, which counteracts, to a certain extent, the effect sight, interestingly, it was clear that the Brownian motion of of the short τr. Nevertheless, effects of such a short τr on the the stomatocyte nanomotors in the absence of fuel was motors’ motion can be clearly seen in the shape of the impacted by the presence of proteinase K, with a ca. 2-fold nondirectional trajectories (Figure 2C). In order to confirm lower MSD (Figure 2E). This is likely due to normal protein that the autonomous motion is a result of the catalytic process interaction with the stomatocytes’ surface, which leads to an that takes place within the nanomotor cavity, control increase in the overall particle size and thus slower Brownian experiments were performed. First, a high concentration of motion (as evidenced by the ca. 150 nm increase in 4475 https://dx.doi.org/10.1021/acs.nanolett.0c01268 Nano Lett. 2020, 20, 4472−4480 Nano Letters pubs.acs.org/NanoLett Letter hydrodynamic size; Figure S12). This hypothesis was also confirmed by measurement of the MSD of empty carriers in the presence of proteinase K at the same concentration (50 μM), where a similar effect was observed (Figure S13). Despite this, the nanomotors’ autonomous motion was clearly maintained in the presence of fuel, and only a slight reduction in the MSD was observed (compared to measurements without free proteins), highlighting the stability of this system and its relevance for application in a biological context. Taken together, these studies demonstrate the robust properties of our hybrid nanomotor compared with the more sensitive catalysts generally used in nanoscale motile systems. To assess the potential of hybrid nanomotors for application in biomedical research, we first demonstrated the degradation of the MnP catalyst by glutathione (GSH), an intracellular antioxidant. In general, GSH is present at micromolar concentrations within the plasma, although this increases to millimolar levels within the cell. At high concentrations of GSH (10 mM), a significant color change was directly observed within 1 min (Figure 3A). The degradation of the MnP catalyst was also confirmed by the nanomotors’ deactivation and consequent recovery of their Brownian motion upon GSH addition (Figure 3B). Motor activity under less severe conditions (10 μM GSH) was not affected in the same time frame (Figure S15). These studies indicated that under certain intracellular conditions (e.g., elevated reductant concentration) nanomotors would be deactivated. In fact, developing glutathione-responsive systems has been regarded as an appealing strategy for targeted drug delivery in cancer cells.47,48 Taking into account the potential use of nanomotors as drug nanocarriers, this would be advantageous, as such systems would have autonomous motion in the extracellular environments but be degraded at the target. In our final set of experiments, we performed cellular testing using nanomotors to assess their biocompatibility and ability to function in a cellular environment. Particle toxicity was measured using HeLa cells treated with empty and loaded stomatocytes up to 250 μg mL−1 for 24 h (Figure 4A and Figure 3. (A) Visual of MnO2 catalyst (MnP) reduction by addition of high levels of glutathione (GSH) (10 mM) to a 10-fold dilution of Table S2). Relatively low toxicity was observed for hybrid MnPs in 1× PBS, where decoloration within 1 min indicates nanomotors at 250 μg mL−1 in comparison with other dissolution of the MnP catalyst. (B) Diffusion coefficients for fueled nanomotor systems that displayed similar (ca. 80%) viability nanomotors before and after GSH addition, showing motor at lower concentrations.49 When cells were treated with toxic deactivation at high levels of GSH even after repeated fuel addition. concentrations of H2O2 of either 125 or 250 μM in the Fuel was added at a final concentration of 100 mM. The diffusion presence of unloaded stomatocytes, no population of viable coefficient for passive particles is lower than those presented in Figure cells was visible (Figure S16). To assess the ability of 2 because of batch-to-batch differences in size distribution (Figure nanomotors to function under such conditions of low fuel S14). levels, we conducted additional motion studies at H2O2 concentrations equivalent to those employed in the cell studies H2O2 concentration of 250 μM, cell viability recovered up to (i.e., 125 and 250 μM). Under such conditions, enhanced ca. 45% because of the performance limits of the nanomotor. diffusion was observed, and the hybrid nanomotors retained To validate that the nanomotor-mediated increase in cellular their motile properties (Figure S17). H2O2 is a cytotoxic viability was commensurate with decreasing reactive oxygen species that is generated at increasing concentrations in the species (ROS) levels, additional experiments were performed tumor microenvironment and in cancerous cells; for instance, using a fluorescent ROS indicator (CM-H2DCFDA). Confocal Guo and co-workers determined H2O2 levels of around 715 microscopy (and quantitative analysis) confirmed that under μM in bladder cancer tissues.41 Because of their ability to the most extreme conditions (250 μM/250 μg mL−1), cellular scavenge H2O2, harnessing it for their enhanced diffusion, ROS was significantly reduced in the presence of hybrid nanomotors were utilized to rescue the viability of cells during nanomotors compared with unloaded stomatocytes, which did exposure to this harmful oxidative species. Indeed, around 50% not have a significant effect (Figures 4C−E, S18, and S19). viability was obtained after treatment of cells (exposed to 125 Although this effect was very pronounced, this only correlated μM H2O2) with nanomotors at 125 μg mL−1 (Figure 4B and to a partial recovery in viability, as shown in Figure 4B. Table S3). This significantly increased to ca. 80% when the As a proof-of-principle experiment, we compared the nanomotor concentration was elevated to 250 μg mL−1, near penetration behavior of fluorescently labeled hybrid nano- the background level as presented in Figure 4A. At an elevated motors and empty stomatocytes in 3D cell spheroids (the 4476 https://dx.doi.org/10.1021/acs.nanolett.0c01268 Nano Lett. 2020, 20, 4472−4480 Nano Letters pubs.acs.org/NanoLett Letter Figure 4. (A) Viability of HeLa cells after exposure to empty and MnP-loaded stomatocytes at a range of concentrations (15−250 μg mL−1) as determined by MTT assay. (B) Hybrid stomatocytes rescue the viability of HeLa cells after coincubation with H2O2. (C) ROS induction (as indicated by the green fluorescence of CM-H2DCFDA stain) in HeLa cells after treatment with H2O2 is significantly reduced by the presence of hybrid nanomotors (blue = Hoechst nuclear stain; scale bar = 50 μm). (F) Penetration of empty stomatocytes and (G) hybrid nanomotors into 3D cell spheroids (coincubated with 250 μM H2O2) with accompanying line profiles for comparison (experiments were replicated in triplicate, cf. Figures S21 and S22; scale bars = 1 mm). experiments were conducted in triplicate using 250 μM H2O2; to protect cells. This system has strong potential in biomedical Figures 4F,G and S20−S22). A nuclear cell stain (Hoechst) research and highlights the capacity of molecular engineering was used to provide an indication of overall spheroid for the creation of morphologically diverse, functional morphology, where treatment with 250 μM H2O2 did not nanosystems. To further showcase this application, we compromise cell stability (compared with those cultured in conducted proof-of-principle experiments to demonstrate two dimensions), in agreement with the literature that reports effective penetration of nanomotors into 3D cell spheroids. higher toxicity resistance of 3D cell models compared with 2D Future research will engage the utility of compartmentalized ones (Figure S20).50,51 We observed that nanomotors (fueled inorganic nanocatalysts (and assorted stabilizing molecules) to by H2O2) penetrated effectively into the spheroid, whereas drive autonomous (nano)motion and explore their biorelated control particles showed significantly reduced infiltration of the applications. We believe that our approach can offer a general dense cellular framework. These data highlight the applicability route toward assembling functional nanomotors and paves the of our technology and provide proof-of-principle results to way to overcome limitations associated with traditional guide future biomedical studies. physical encapsulation of active catalysts. Further studies will In summary, we have presented a new strategy for the expand the range of nanoparticles that can be integrated within fabrication of biodegradable hybrid stomatocyte nanomotors. stomatocyte motors and explore in more detail the mechanism of formation. ■ The unique structural features of such stomatocytes were utilized to template the in situ one-pot preparation of a ASSOCIATED CONTENT biodegradable MnO2 catalytic core through a process of compartmentalized synthesis. Such hybrid nanomotors show * Supporting Information sı autonomous motion in the presence of fuel and maintain their The Supporting Information is available free of charge at stability through extended periods of time. Moreover, such https://pubs.acs.org/doi/10.1021/acs.nanolett.0c01268. nanomotors maintained their activity in physiological medium Experimental procedures and supporting tables and and are highly biocompatibleable to reduce local ROS levels figures (PDF) 4477 https://dx.doi.org/10.1021/acs.nanolett.0c01268 Nano Lett. 2020, 20, 4472−4480 Nano Letters pubs.acs.org/NanoLett Letter Surface view from cryo-TEM tomography of MnP- Samuel Sánchez − Institute for Bioengineering of Catalonia loaded stomatocytes showing the overall structure of the (IBEC), The Barcelona Institute of Science and Technology, nanomotor (MP4) 08028 Barcelona, Spain; orcid.org/0000-0002-5845-8941 Cryo-TEM tomography of MnP-loaded stomatocytes Complete contact information is available at: showing the structure of the MnO2 nanoparticles inside https://pubs.acs.org/10.1021/acs.nanolett.0c01268 the cavity of the nanomotor (MP4) Author Contributions Compilation of NTA videos of motile MnP-loaded I.A.B.P., J.C.M.v.H., and L.K.E.A.A. designed the project. stomatocytes in the presence of fuel (MP4) I.A.B.P. conducted the synthesis, characterization, and motility NTA video of empty stomatocytes in the presence of experiments. S.C. performed the in vitro cell studies. R.R.M.J. fuel (MP4) and H.F. performed the tomography experiments and the 3D NTA video of empty stomatocytes and free MnPs in the rendering. J.C.M.v.H. and L.K.E.A.A. guided the research. presence of fuel(MP4) I.A.B.P. and L.K.E.A.A. wrote the manuscript. All of the ■ authors contributed to the critical discussion of the results and commented on the manuscript. AUTHOR INFORMATION Notes Corresponding Authors The authors declare no competing financial interest. ■ Loai K. E. A. Abdelmohsen − Department of Bio-Organic Chemistry, Institute of Complex Molecular Systems (ICMS), ACKNOWLEDGMENTS Eindhoven University of Technology, 5600 MB Eindhoven, The The authors acknowledge the support from the Dutch Ministry Netherlands; orcid.org/0000-0002-0094-1893; of Education, Culture and Science (Gravitation Program Email: l.k.e.a.abdelmohsen@tue.nl 024.001.035) and NWO-NSFC Advanced Materials (Project Jan C. M. van Hest − Department of Bio-Organic Chemistry, 792.001.015). The authors acknowledge the support by the Institute of Complex Molecular Systems (ICMS), Eindhoven National Natural Science Foundation of China University of Technology, 5600 MB Eindhoven, The (51561135010). We thank the Spanish MINECO (BOTSin- Netherlands; orcid.org/0000-0001-7973-2404; Fluids Project), the Foundation BBVA (MEDIROBOTS Email: j.c.m.v.hest@tue.nl Project), and the CERCA Program of the Generalitat de Authors Catalunya. We thank the Ser Cymru II Programme for support Imke A. B. Pijpers − Department of Bio-Organic Chemistry, of D.S.W.; this project received funding from the European Institute of Complex Molecular Systems (ICMS), Eindhoven Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie Grant Agreement 663830. ■ University of Technology, 5600 MB Eindhoven, The Netherlands Shoupeng Cao − Department of Bio-Organic Chemistry, REFERENCES Institute of Complex Molecular Systems (ICMS), Eindhoven (1) Wang, H.; Pumera, M. Fabrication of Micro/Nanoscale Motors. University of Technology, 5600 MB Eindhoven, The Chem. Rev. 2015, 115 (16), 8704−8735. Netherlands (2) Gao, W.; Wang, J. Synthetic Micro/Nanomotors in Drug Delivery. Nanoscale 2014, 6 (18), 10486−10494. Antoni Llopis-Lorente − Department of Bio-Organic (3) Lee, T. C.; Alarcón-Correa, M.; Miksch, C.; Hahn, K.; Gibbs, J. Chemistry, Institute of Complex Molecular Systems (ICMS), G.; Fischer, P. 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