Double-Fueled Janus Swimmers with ... - ACS Publications

Double-Fueled Janus Swimmers with Magnetotactic Behavior Philipp S. Schattling,† Miguel A. Ramos-Docampo,‡ Verónica Salgueiriño,‡ and Brigitte Stad̈ ler*,† †

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark Departamento de Física Aplicada, Universidade de Vigo, 36310 Vigo, Spain

‡

S Supporting Information *

ABSTRACT: Self-propelled particles attract a great deal of attention due to the auspicious range of applications for which nanobots can be used. In a biomedical context, self-propelled swimmers hold promise to autonomously navigate to a desired location in an attempt to counteract cell/tissue defects either by releasing drugs or by performing surgical tasks. The vast majority of prior reports deal with single engine assemblies, often utilizing fuel molecules which are considered to be highly cytotoxic. Herein, we introduce two engines: (1) a motor which couples enzymes (i.e., glucose oxidase) and inorganic nanoparticles (i.e., platinum nanoparticles) to gain power and (2) a peptide-fueled trypsin motor. We demonstrate that both engines can induce enhanced diffusion properties of (Janus) particles using bioavailable and completely harmless fuel molecules. By combining both engines on the same carrier, we show self-propelled particles employing two independent engines, using two different fuels. A collaborative enhancement of the swimmer’s diffusion properties upon powering-up both engines simultaneously is observed. Additionally, the incorporation of magnetic nanoparticles allows for the swimmer to move in a magnetic gradient upon applying an external magnetic field, yielding in directional motion of the doublefueled particles. These multiple-fueled biocompatible swimmers are a significant contribution to make them applicable in a biomedical context. KEYWORDS: Janus particle, self-propulsion, magnetic nanoparticle, directional motility, enzyme, magnetotaxis

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(substrates), which are readily available in biological systems and whose decomposition products are not harmful to the organism either. Enzymatic conversion can be used to induce propulsion due to a gradient of chemical species generated by the asymmetric distributed enzymes on the swimmer surface.15,16 Initially, the locomotion of particles, which was induced by the catalytic activity of enzymes, still relied on H2O2 as the fuel,17−21 and only recently, different alternatives have started to emerge. Successful examples in this context include the enzyme urease22−24 or glucose oxidase (GOx),22 which employ urea or glucose, respectively, to power microparticles. However, although GOx and urease-driven swimmers are fueled with biocompatible compounds, the combustion products of both (H2O2 and ammonia, respectively) are considered cytotoxic. In an effort to address this issue, our group25 and van Hest et al.26 have gone one step forward and employed the tandem reaction between GOx and catalase (Cat), during which the produced H2O2 was directly converted by Cat into oxygen and water. Despite the progress in the field,

eliberate transport of matter often relies on random diffusion and the subsequent statistical accumulation of the cargo at the target position, limiting the efficiency and specificity. A promising approach to challenge this shortcoming is to equip carriers with nanometer-sized engines for (directed) motility. Upon the combustion of specific fuel molecules, these carriers are able to autonomously navigate through fluids, with velocities that outperform Brownian motion multiple times. This young discipline has yielded impressive results as recently highlighted in four comprehensive reviews.1−4 Having applications in nanomedicine in mind, two major prerequisites of these selfpropelled nanomachines were identified: (i) the fuel molecules as well as their combustion products must be biocompatible, and (ii) the function of the nanorobots should be remotecontrollable by an operator. Many of the early5−9 and recent10−14 self-propelled assemblies utilized hydrogen peroxide (H2O2) as fuel and a platinum (Pt) surface for fuel conversion. However, due to the cytotoxic nature of H2O2, these swimmers are excluded from a straightforward translation into a biomedical context, and therefore, efforts were invested into the search for alternative biocompatible nanoengines. To this end, enzymes hold great promise because different types of enzymes can selectively employ a large variety of fuel molecules © 2017 American Chemical Society

Received: January 20, 2017 Accepted: March 22, 2017 Published: March 22, 2017 3973

DOI: 10.1021/acsnano.7b00441 ACS Nano 2017, 11, 3973−3983

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ACS Nano all of the self-propelled systems operate only under specific environmental conditions and employ one type of fuel. While this aspect is of minor importance in homogeneous systems, it becomes a limiting factor in a complex organism, where many different parameters (pH, salt concentration, presence of biomolecules, etc.) of the fluid can significantly and simultaneously vary. In this context, swimmers are required to adapt to these environmental changes in order to sustain their mobility. Although the rationale is clear, to the best of our knowledge, there is only one example of multi-fueled systems reported to date.27 However, this approach required H2O2 and strong acidic or alkaline conditions, limiting its applicability in a biomedical context. Further, the design did not allow the simultaneous operation of both engines. In the literature, selfinduced locomotion of fuel-based systems is discussed on the basis of three predominant mechanisms: self-electrophoresis, self-diffusiophoresis, and bubble propulsion. Whereas the former mechanism requires two conductive metals connected as the two hemispheres of a Janus particle,6,7,28 the latter mechanism involves micrometer tubular structures, which develop large gas bubbles in the fluid.12,18 In contrast, selfdiffusiophoresis is driven by a concentration gradient formed by a (bio)catalytic reaction occurring asymmetrically on the swimmers surface, making it the best fuel-based thrust in a biomedical context to date.22,25 From a different perspective, fuel-free systems also attract a great deal of attention. These systems induce mobility by external fields (ultrasound,29−33 magnetic fields, etc.) by environmental-triggered degradation34,35 or by exploiting flagellated bacteria or sperm.36−39 However, independent of the propulsion mode, ultrasound or enzyme-driven, the swimmers do not provide remotecontrolled locomotion per se and consequently undergo diffusive random motion. In this regard, the incorporation of magnetic nanoparticles in the final assembly of the swimmers gave access to swimmers which could be aligned,40,41 guided through microfluidic channels,42,43 or directed to approach, transport, and release cargo44,45 by applying/changing an external magnetic field. Herein, we report the assembly of biocompatible remotecontrolled sub-micrometer-sized self-propelled Janus particles powered by enzymes using two different biological fuels. Specifically, we (i) assembled Janus swimmers with a motor coupling inorganic−biological entities by employing the tandem reaction between GOx and Pt nanoparticles (Pt-NP) using glucose as fuel, (ii) engaged the digestive enzyme trypsin as an engine to fabricate a peptide-fueled Janus swimmer, (iii) combined both motor systems on the same Janus swimmers and illustrated the fuel-dependent motility, and (iv) integrated manganese ferrite nanoparticles (MF-NP) into the assembly, yielding double-powered swimmers with magnetic control over their direction.

Figure 1. GOx/Pt-NP swimmer. (a) Schematic representation of the Janus-shaped GOx/Pt-NP swimmers consisting of a PEG deactivated and a GOx/Pt-NP decorated hemispheres. (b) Representative CLSM (2.7 μm particles, left) and TEM (800 nm particles, right) images of the Janus swimmers showing the asymmetric distribution of fluorescently labeled GOx and Pt-NPs, respectively. Scale bars: CLSM 10 μm, TEM 200 nm (top), 20 nm (bottom). The dashed line indicates the border between the hemispheres with and without Pt-NPs.

lysine) (PLL) and subsequently equipped with the motor entities. The cationic PLL ensured the effective immobilization of the citrate-stabilized negatively charged Pt-NPs and has previously been used as a supporting layer for the immobilization of GOx.25 The successful spatial deposition of (fluorescein-labeled) GOx just on one hemisphere was confirmed on 2.7 μm sized assemblies by laser scanning confocal microscopy (CLSM), and the Pt-NP adsorption was confirmed by transmission electron microscopy (TEM) (Figure 1b and Supporting Information Figure S1). The mobility was assessed in PLL-coated microfluidic channels by recording and tracking the behavior of 800 nm swimmers in aqueous 20 wt % glycerol solutions with different glucose concentrations (Movie S1). (Please note that all mobility experiments were performed with 800 nm sized assemblies, whereas 2.7 μm sized particles were used for visualization purposes.) Besides translational diffusion, the particles also undergo rotational diffusion, which is quantified by the rotational diffusion coefficient and given by the equation Dr = kBT(8πηr3)−1, where kB is the Boltzmann constant, T the absolute temperature, η the viscosity of the liquid, and r the radius of the assembly.47

RESULTS AND DISCUSSION Bio-Inorganic Swimmer. In an effort to preserve the previously reported successful interplay between Pt-NP and H2O2, we aimed at employing GOx to provide the required H2O2 via the conversion of glucose. Therefore, GOx and Pt-NP were immobilized on the same hemisphere of Janus particles (Figure 1a). The Janus particles were assembled by adapting our previous reported protocol,46 employing a Pickering emulsion to initially break the symmetry of the particles. One hemisphere was deactivated with PEGylated poly(L-lysine) (PEG), and the second hemisphere was coated with poly(L3974


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ACS Nano The inverse of Dr describes the rotational diffusion time (tr), which can be understood as the reorientation time of the colloids.48 The value tr is important for the correct analysis of the diffusion properties. It was shown earlier that at delay times much smaller than tr the mean squared displacement (MSD) featured a nonlinear course, which changed into a linear increase at delay times longer than tr.47,49 This change is explained to occur due to the rotational diffusion of the propelled particles, which is changing their direction. As a consequence, the diffusion changes from ballistic to diffusive over longer time periods. The MSD analysis showed a linear increase of the MSD with increasing time intervals, clearly pointing toward enhanced (random) diffusion of the bioinorganic swimmers (Figure 2a and Supporting Information

increasing concentration of glucose led to their enhanced diffusion of the swimmers, with a maximum Deff of ∼0.40 μm2 s−1 (Figure 2b). On the other hand, glucose concentrations higher than 200 mM did not further increase Deff, likely due to the maximum conversion rate of the type and number of the employed enzymes. In this context, it is important to note that the found enhancement of the Deff did not imply a change of the Brownian diffusion coefficient. Instead, the increase was explained by an increase of the velocity due to the enzymatic conversion on the active hemisphere (Supporting Information Figure S2b). The instantaneous velocity of the bio-inorganic swimmers increased upon addition of fuel from 0.07 μm s−1 (no fuel) to 0.32 μm s−1 (400 mM of glucose). However, the instantaneous velocity only provides a rough estimate of the actual swimmer speed as it is a measure of the covered distance of the particle over a known time period and does not take into consideration the rapid reorientation of the particle.49 In the literature, the instantaneous velocity is commonly calculated for self-propelled swimmers with small feature sizes when the randomization of the swimmers’ trajectory complicated the MSD analysis.10,42,48 The diffusion properties were characterized by performing MSD analysis of two independent repeats. Between 25 and 150 swimmers per repeat were analyzed for each experimental condition. The statistical significance is expressed by both the average over the individual swimmers and the independent repeats. This approach differs from previous publications as it considers immobile particles, as well. In the context of this paper, immobility can be explained by a high amount of nonactive enzymes on the specific swimmer, a lack of Pt-NP or MF-NP, interactions with the surface of the microfluidic channel, the weight of the silica particles, or simply the absence of fuel molecules. Consequently, the obtained Deff is an overlay of particles, which are part of different “motility populations” and weighted with the occupation number of each population. In an effort to quantify the contribution of each motility population to Deff, the MSD analysis was performed by applying different displacement filters, which is a simple measure for the exploration ability of an individual swimmer. The maximum displacement which was found in the nonfueled situation was taken as a reference, and based on that, four populations were labeled as follows: (i) swimmers exhibiting up to 10%, (ii) between 10 and 50%, (iii) between 50 and 90%, and (iv) >90% of the maximum displacement. This statistical approach was exercised for the GOx/Pt-NP swimmers and allowed the change of the population of each motility group to be followed dependent on the different fuel concentrations. Additionally, that analysis is a suitable tool to distinguish between passive and accelerated diffusion. The pie charts in Figure 3 show that, with increasing concentration of glucose, the fraction of the least mobile swimmers (light gray) decreased from initially 81% (no fuel) to 30% (400 mM). In contrast, the amount of mobile particles increased in each population with an increasing amount of glucose. At 100 mM of glucose, almost 50% of the swimmers exhibited already a distinct mobility larger than 10% of the reference. This trend continued with increasing glucose concentration, yielding more than 70% of mobile particles at 200 and 400 mM glucose. This observation confirmed the impact of the enzymatic propulsion on the particles’ mobility upon addition of the fuel. In an effort to ensure that the increase of the mobility is solely explained by the enzymatic motors and not by interactions of the particles with glucose, enzyme-free swimmers were exposed to 400 mM

Figure 2. (a) MSD analysis at 50, 100, and 400 mM glucose. Inset: Representative trajectories of individual particles at 50 and 400 mM glucose. Scale bar: 12 μm. (b) Effective diffusion coefficient (Deff) of the GOx/Pt-NP assemblies depending on the glucose concentration.

Figure S2a). This behavior was expected on the basis of the rapid reorientation of the mobile swimmers, due to the small rotational diffusion time (tr ∼ 0.69 s). The effective diffusion coefficient (Deff) was extracted from the slope of the MSD versus delay time plot, according to the equation MSD = (4D + v2tr)Δt = 4DeffΔt (Supporting Information Figure S2a).47−49 In the absence of fuel, Deff was found to be 0.05 (±0.01) μm2 s−1. Interestingly, this corresponds to approximately 15% of the theoretical diffusion coefficient (0.31 μm2 s−1) of 800 nm sized particles, which can be calculated using the Stokes−Einstein equation (D = kBT(6πηr)−1). The viscosity of the 20 wt % glycerol−water mixture was taken from Segur et al.50 This difference in Deff might be explained by additional contributions coming from the weight of the silica particles and their interactions with the bottom of the microfluidic channel. Further, other reports have also found differences between theoretical and experimental Deff values.11,48 As expected, an 3975


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into the fluorescent monoamide (MA-Rho-110) and the corresponding tripeptide.

Figure 3. Pie charts illustrating the population change of the different motility groups depending on the glucose concentration. The groups are categorized in relation to the maximum displacement of particles found in the nonfueled situation.

of glucose. The distribution of the population groups showed that over 85% of the particles exhibited no mobility at all, confirming that glucose was not able to induce mobility. In contrast to our earlier report on GOx/Cat Janus swimmers, the substitution of catalase with Pt-NP led to an increase in the diffusion properties of about 100%.25 This remarkable result can be explained by taking into account the fact that more GOx enzymes were immobilized in the current case, and the activity loss of enzymes upon immobilization, which did not occur for metallic nanoparticles, rendering this approach a promising concept for long-lasting swimmers. The glucose level of a healthy subject is below 7.7 mmol L−1.51 From this perspective, glucose concentrations above 100 mM are certainly rather high. However, the glucose level can temporarily be increased without server complications to the human organism, for example, as it is done in the standard diabetes test. The human organism is able to level high concentrations of glucose within the course of 2 h. That means, on the other hand, that the bio-inorganic swimmer would lose its mobility enhancement over time as the fuel runs low. In this context, a combination of differently fueled motors on the same assembly might overcome this challenge. Peptide-Fueled Swimmer. Proteins/peptides pose an attractive alternative to common fuels. They are abundant components in every living organism, and upon enzymatic cleavage, the decomposition products are typically harmless lower molecular weight molecules which are excreted through inherent pathways. Accordingly, from the natural occurring proteases, we chose the digestive enzyme trypsin (Trp), which cleaves peptide bonds at the carboxylic acid site of the basic amino acids arginine and lysine,52 to investigate its ability to serve as the complementary motor to the above-mentioned bio-inorganic swimmers, using peptides as fuel. Consequently, similar to the GOx/Pt-NP bio-inorganic swimmers, Trp was immobilized on the PLL-coated hemisphere of the Janus particles and the bis(benzyloxycarbonyl-L-arginine amide) derivative of rhodamine 110 (BA-Rho-110) was chosen as the model peptide fuel (Figure 4a). Upon exposure to trypsin, BA-Rho-110 is cleaved

Figure 4. Trp swimmer. (a) Schematic representation of the Janusshaped Trp swimmers consisting of PEG inactivated and a Trp decorated hemispheres. (b) Representative CLSM images of 2.7 μm assemblies using fluorescently labeled Trp. Scale bar: 10 μm. Inset: 3D stack of these Janus particles. Scale bar: 5 μm.

In order to preserve the activity of Trp after immobilization, different polymer precursor layers were tested (Supporting Information Discussion and Figure S3). PLL was found to be most suitable for this purpose, offering 10% remaining Trp activity. The Janus shape of the assemblies was again confirmed by CLSM using fluorescently labeled trypsin (Figure 4b). Similar to the GOx/Pt-NP swimmer, the peptide-fueled swimmers were expected to be propelled by self-diffusiophoresis. Due to the enzymatic reactions, a non-equilibrium density of product compounds on the active hemisphere was established, yielding in an osmotic flow in the microenvironment of the swimmer. As a consequence, the swimmers feature a motion in the direction opposite of the enzymatic hemisphere.1,47,53,54 The locomotion of the Trp swimmers was recorded and computer tracked in solutions containing different BA-Rho-110 concentrations (Movie S2). As expected, the Deff increased with increasing amount of fuel present, leveling off at the maximum of ∼0.45 μm2 s−1 for a fuel concentration between 50 and 100 μM (Figure 5a). The instantaneous velocity of the swimmers increased from 0.04 μm s−1 (no fuel) by approximately 8 times to 0.33 μm s−1 upon exposure to 200 μM of the peptide substrate (Supporting Information Figure S4). The motility groups of the Trp swimmers were analyzed, and as expected, it was found that, with increasing concentration of peptide fuel, the number of mobile particles considerably increased (Supporting Informa3976


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containing the MF-NPs spatially confined on one hemisphere of the assembly (Figure 6a). The successful integration of the

Figure 5. (a) Deff of 800 nm sized Trp swimmers depending on the BA-Rho-110 concentration. (b) MSD analysis using 15, 50, and 200 μM BA-Rho-110. Inset: Representative trajectories of individual particles at 15 and 200 μM BA-Rho-110. Scale bars: 12 μm.

tion Figure S5). Comparable to the bio-inorganic motor previously discussed, the MSD increased linearly with increasing delay times, pointing toward a diffusive movement (Figure 5b). Importantly, the Deff values of the Trp and the GOx/Pt-NP motors were rather similar. This is an important aspect when it comes to the interconnection of both engines. Since both motor systems performed comparably, there should be no compromises in terms of the motor power when switching from one to the other engine. It was shown in several papers that the concentration of salts had a significant effect on the propulsion, in particular, for bubble-propelled systems and metal-containing swimmers.55−57 Although the ionic strength might have an effect on the enhancement of the diffusion properties, within the scope of this paper, we limited the characterization of swimmers to environments containing the physiologically relevant pH in the absence of salt. Magnetic Swimmer. The next aim was to gain control over the direction of motion of the Janus swimmers. Ma et al. have shown that the combination of urease-driven propulsion coupled with magnetic guidance offers considerable higher velocities and distinct motion control.23 Instead of utilizing an e-beam technique for the magnetic layer deposition, we chose to equip the Janus swimmers with superparamagnetic manganese ferrite nanoparticles (MF-NPs). The synthetic method employed for the production of these MF-NPs offers two advantages: (1) MF-NPs were synthesized by a thermal decomposition in solution,58 which implies an important yield of sample of nanoparticles to work with (Supporting Information Figure S6); and (2) the fabricated MF-NPs can be wet-chemically manipulated,59 favoring different options for their incorporation in the final assembly in a controlled manner and without the need of advanced equipment. In a first step, Janus particles were prepared,

Figure 6. Magnetic swimmer. (a) Schematic illustration of the assembly. (b) Photograph of tubes containing the Janus particles with different number of deposited layers of MF-NP. (c) TEM image of the silica particles decorated with MF-NPs. Scale bar: 1 μm.

MF-NPs during the layer-by-layer deposition was monitored visually by the gradual color change of the silica particles from white to dark brown with increasing MF-NP layer number (Figure 6b) and was further confirmed by QCM-D measurements (Supporting Information Figure S7). Up to three layers of MF-NPs were successfully deposited onto the Janus swimmers, yielding assemblies with a controllable magnetic response (Supporting Information Figure S8). The diffusion properties of these assemblies asymmetrically coated with MFNPs were assessed in a microfluidic channel. A rectangularshaped NdFeB magnet was placed at one side of the microfluidic channel in order to establish a spatial gradient of a time-independent magnetic field (Figure 7a). When a magnetic field was applied, the assemblies experienced a ballistic drift along the magnetic field gradient, which was quantified by the velocity of the swimmers. The velocities were extracted in the parabolic regime of the MSD at short delay times, according to equation MSD = 4DΔt + v2Δt2, where v is the velocity (Supporting Information Figure S9).48,49 The MSD results are summarized in Figure 7b, comparing three different static magnetic fields (60, 180, and 224 mT) and the number of 3977


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Figure 7. Magnetic swimmer. (a) Photograph of the experimental setup to magnetotactic swimmers. (b) Velocity of particles asymmetrically coated with different number of layers of MF-NPs depending on the strength of the magnetic field. MSD analysis (c) and representative trajectories (d) of swimmers decorated with three layers of MF-NPs upon exposure to a magnetic field of 224 mT. Scale bar: 250 μm.

are summarized in Figure 9a and in Supporting Information Table S1. The x and y axes display the glucose and BA-Rho-110 concentrations, respectively, the z axis represents the determined Deff. First, as shown earlier, the Deff of the Janus swimmers increased with increasing concentration of the individual fuel species. Further, when adding the second fuel (i.e., activating the second engine), an additional enhancement of the diffusion properties of the particles was observed. The additional gain of the propulsion increased with increasing feed of the second fuel at a constant concentration of the first fuel species, yielding a maximum Deff of 0.78 μm2 s−1 for the highest tested fuel concentrations (400 mM glucose, 100 μM BA-Rho100). The mean instantaneous velocity of the swimmers at the highest fuel combination was estimated to be 0.36 μm s−1 (Supporting Information Table S2 and Figure S12). Interestingly, this Deff was higher than the maximum observed for the Janus swimmers with only one type of immobilized enzymes. This finding suggests that the powering-up of the second type of enzymes did not just simply increase the diffusion properties but collaboratively enhanced the mobility of the Janus swimmers. Deff of the dual-powered swimmers increased by 19 times compared to the nonfueled situation. This increase was quite remarkable, in particular, when compared to previously published reports on enzyme-driven swimmers, in which a 1.3- to 1.5-fold enhancement of the Deff was found in comparison to their nonfueled situation.22 In addition, swimmers which did not experience a magnetic field gradient exhibited random motion illustrated by the trajectories of the mobile particles and confirmed by the linear increase of the MSD with increasing time intervals (Figure 9b,c). This finding was in agreement with earlier reports on enzymatic swimmers.22,23,25 Double-Fueled Swimmer with Magnetotactic Behavior. Finally, with the aim of gaining control over the direction of the assembled Janus swimmers, we took advantage of the responsiveness of the MF-NPs to an external magnetic field. As

MF-NP layers. As expected, the magnetically induced mobility of the swimmers increased with increasing number of MF-NP layers and increasing strength of the magnetic field, reaching velocities up to ∼9 μm s−1. However, just one layer of MF-NP showed no mobility orin the case of the strongest magnetic fielda rather low mobility as indicated by the low velocity. The MSD analysis of silica particles containing three layers of MF-NPs exposed to an external magnetic field of 224 mT showed a parabolic course of the MSD with increasing delay times, confirming that magnetic fields can induce both locomotion and directionality to the swimmers (Figure 7c). This is confirmed by the distinct (directed) trajectories (Figure 7d). Please note that the continuous lines in Figure 7d illustrate the trajectories of tracked positions of individual particles over the duration of the experiment (and not aligned chains of particles). Double-Fueled Magnetotactic Swimmer. As the final goal, magnetotactic Janus swimmers with the ability to employ two different fuels simultaneously were assembled by coimmobilizing GOx/Pt-NP and Trp on the same hemisphere (Figure 8a). The successful co-deposition of the enzymes, the Pt-NPs as well as the MF-NPs, was confirmed by CLSM and TEM, respectively (Figure 8b and Supporting Information Figure S10). In order to ensure that the enzymes remained active after the co-deposition step, the individual activity of both enzymes was tested after their immobilization. It was confirmed that Trp was not able to inactivate GOx during the immobilization process because both enzymes responded positively to their individual enzymatic activity assay (Supporting Information Figure S11). Double-Fueled Swimmers. First, the performance of the double-fueled system in the absence of a magnetic field was evaluated. The diffusion properties of these Janus swimmers were assessed using different concentrations of glucose, BARho-110, and mixtures of both fuels. The calculated Deff values 3978


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Figure 8. Double-fueled Janus swimmer with magnetotactic behavior. (a) Schematic representation of the design of the swimmers consisting of a first hemisphere with MF-NPs deposited underneath the PEG and a second one with GOx/Pt-NP and Trp. (b) Representative CLSM image (2.7 μm particles, left, middle) showing the co-deposition of fluorescently labeled GOx (green) and Trp (red) on one hemisphere. Scale bar: 10 μm. Representative TEM image (800 nm particles, right) of the swimmers indicating the coating with Pt-NP and MF-NP on opposite hemispheres. Scale bar: 100 nm.

Figure 9. Double-fueled swimmer in the absence of a magnetic field. (a) Deff as a function of the different employed fuel mixtures. (b) Representative trajectories of the double-fueled swimmers at a fuel concentration of 400 mM glucose and 100 μM BA-Rho-110. Scale bars: 10 μm. (c) MSD analysis of double-fueled swimmers at different fuel concentrations (cyan, no fuel; gray, 100 mM glucose and 50 μM BA-Rho-110; green, 400 mM glucose and 100 μM BARho-110).

a consequence of having the swimmers located in the proximity of the magnet, they undergo a translational force toward regions with higher magnetic field. The strength of this force scales linearly with the total magnetic moment of the swimmer (the sum of magnetic moments of all the immobilized MFNPs) and the field gradient. As a consequence, the swimmers underwent acceleration in the direction of the magnetic field gradient toward the magnet. As shown in Figure 7b,d, a magnetic field gradient was already able to pull Janus swimmers coated with MF-NPs. However, we have gone through an optimization process in order to select the right number of MF-NP layers, which was strong enough to orient the swimmers toward the magnet but not powerful enough to pull the assembly. One layer of MF-NP fulfilled this requirement, as confirmed by the fact that, in the absence of the fuel entities, the magnetic field did not induce a change in the mobility compared to the situation without a magnetic field (Figure 9a and Figure 10a, right gray bars). The MF-NPs were deposited on the hemisphere opposite to the other one containing both GOx/Pt-NP and Trp, in order to efficiently orient the fuel-based movement of the particle when applying the magnetic field gradient. The TEM image in Figure 8b shows swimmers with homogeneous coatings of nanoparticles, suggesting the presence of the desired assembly, that is, Pt-NPs on the hemisphere with the enzymes and MF-NPs

underneath the PEG layer on the opposite hemisphere. The velocities of these swimmers were calculated upon exposure to different concentrations of glucose, BA-Rho-110, and mixtures of both fuels with an applied magnetic field (Figure 10a and Supporting Information Table S2). The velocity increased from 0.04 μm s−1 (no-fuel) to 0.72 μm s−1 (400 mM glucose and 100 μM BA-Rho-110). In previous publications dealing with enzyme-driven swimmers, maximum velocities between 10 and 60 μm s−1 were reported.23,26 However, it is difficult to compare the performances on the basis of the maximum velocity alone as the specific design of the carrier system varied quite drastically. Herein, rather heavy 800 nm solid silica particle-based swimmers are presented, whereas others employ hollow silica capsules23 or polymersomes.26 In order to compare the performance of the different assemblies less biased by the carrier system, the enhancement between the nonfueled and fueled situation was adduced. In this case, a 10× and 4-fold enhancement for urease-driven swimmers23 and catalase-driven polymeric stomatocytes26 was found, respec3979


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Figure 10. Double-fueled swimmer in the presence of a magnetic field. (a) Velocity of doubled-fueled swimmers as a function of the different employed fuel mixtures. (b) Representative trajectories of the double-fueled swimmers at a fuel concentration of 400 mM glucose and 100 μM BA-Rho-110. The schematic shows the position of the magnet in order to appreciate the gradient of the magnetic field which causes the magnetic force acting on the swimmers. Scale bars: 10 μm. (c) MSD analysis of double-fueled swimmers at different fuel concentrations (cyan, no fuel; gray, 100 mM glucose and 50 μM BA-Rho-110; green, 400 mM glucose and 100 μM BA-Rho-110).

The position of the magnet and the gradient of the magnetic field, which induced the magnetic force acting on the swimmers, were schematically added to the trajectory plot. The swimmers closer to the magnet were located in positions with larger gradient of the magnetic field. As a consequence, swimmers with the same total magnetic moment experience a larger magnetic force, which increased the closer the swimmers were located to the magnet. The trajectories of the swimmers labeled with (I) and (II) appear very similar, despite the fact that one swimmer was closer to the magnet than the other one. On the other hand, although swimmers (II) and (III) were located at approximately the same distance to the magnet, swimmer (II) was much more mobile than swimmer (III). Both observations might be explained by the difference in their total magnetic moment due to variability in the deposited amount of MF-NPs per silica particles, as a consequence of the employed coating procedures. Further, the directionality of the swimmers’ mobility was reflected by the transition from a linear to a ballistic course of the MSD curve when the magnetic field was applied at the same fuel condition (compare Figure 9c and Figure 10c). The enhancement of the diffusion properties of the Janus swimmers is explained by the asymmetric generation of a concentration gradient. Admittedly, symmetric systems or swimmers, which were homogeneously equipped with enzymes, were also able to establish a concentration gradient. However, this concentration gradient was homogeneously distributed around the swimmer, and an osmotic flow profile evolved homogeneously pointing toward the swimmer. As a result of the environmental flow, symmetric swimmers are expected to exhibit an enhancement of their diffusion properties, as well. In an effort to compare the enhancement of both systems individually, magnetically guided symmetric and Janus swimmers were assembled and their diffusion properties were assessed. In this context, the term symmetric refers to the deposited enzymes only. The MF-NPs remained arranged in a Janus fashion. Supporting Information Figure S14 summarizes the Deff of symmetric and Janus swimmers in the absence and presence of an externally applied magnetic field gradient when exposed to 400 mM glucose and 100 μM BA-Rho-110. In comparison to the symmetric swimmers, the Deff values were ∼3 times (absence of a magnetic field) and ∼5.5 times higher (presence of a magnetic field) when the enzymes were immobilized on just one hemisphere. The results underlined undoubtedly the benefit of the Janus architecture for the efficiency of the self-propelled mobility.

tively. In comparison, the enhancement of the double-fueled swimmers described herein increased by 18 times. The evaluated diffusion properties in the presence of (single or both) fuels were considerably higher in the presence of the magnetic field gradient compared to their counterparts in the absence of the external force. The beneficial effect of this external stimulus on the diffusion properties of the swimmers yielded up to 2 times higher velocities in contrast to the situation where no magnetic field was applied. As a consequence of the magnetic field, the rotation of the particles was likely limited, and thus, changes of the direction of movement caused by the particle’s rotation was minimized. Hence, the Janus swimmers exhibited directional mobility when a magnetic field gradient was applied (Movie S3 and Supporting Information Figure S13). This aspect was supported by the extracted traces of the swimmers (Figure 10b), which featured extended trajectories and a clear direction.

CONCLUSION In summary, we present GOx/Pt-NP and Trp as two nanomotors capable of inducing particle locomotion. The former nanomotor relied on the interplay between the enzyme GOx and the high catalytic activity of inorganic Pt-NPs without requiring H2O2 as a fuel. Moreover, Trp was utilized as enzymatic engine, demonstrating that bioavailable proteins/ peptides could effectively be employed as fuel to propel submicrometer-sized particles. Both systems enhanced the diffusion properties by a factor of 8 and 16, respectively. In the next step, we combined both engines on the same carrier and showed Janus swimmers exhibiting self-induced locomotion by exploiting simultaneously two different nanoengines, which employed two different biocompatible fuel species. When MF-NPs were incorporated into the assemblies, the swimmers featured a magnetotactic behavior. While an applied magnetic field gradient did not induce motion in the swimmers 3980


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mL−1 of DyLight 633 maleimide solution in DMSO was added. The solution was shaken for 6 h. The labeled enzyme was purified by dialysis for 2 days against ultrapure water and lyophilized. Manganese Ferrite Nanoparticles. MF-NPs were synthesized by chemical thermodecomposition of the manganese and iron metal precursors, following a procedure published elsewhere.58 Briefly, Fe(acac)3 (1.33 mmol), Mn(acac)2 (0.67 mmol), 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol), and benzyl ether (20 mL) were mixed and magnetically stirred under a flow of nitrogen. The mixture was heated to reflux up to 200 °C for 2 h, followed by stirring at 300 °C for 1 h. The black mixture was allowed to cool to room temperature. The colloids were washed three times in ethanol by centrifugation and dried at 60 °C. For particles with a diameter of 10 nm, the procedure was repeated by adding 84 mg of the presynthesized magnetic nuclei. The mixture was initially heated to reflux at 200 °C for 1 h and then stirred at 300 °C for 30 min. Afterward, the colloids were washed in ethanol by centrifugation and dried at 60 °C. Trypsin Swimmer. The fabrication of the trypsin swimmer followed a procedure similar to that for the GOx/Pt-NP swimmer. However, after the first PLL layer the Janus particles were released from the wax droplets and were dispersed in 200 μL PEG solution (0.5 mg mL−1 in buffer, 15 min) and then washed 3× in buffer. Next, the particles were suspended in 200 μL of trypsin solution (1 mg mL−1 in buffer, 40 min) and washed 3× in buffer. Double-Fueled Janus Particles with Magnetic Control. Twenty milligrams of wax droplets obtained from the Pickering emulsion of 800 nm silica particles was dispersed in 200 μL of MnFe2O4 (MQ water, 1 mg mL−1, 40 min at 8 °C) followed by filtering and washing in buffer. When the wax droplets were dry, one layer of PMA (buffer, 4 mg mL−1, 20 min at 8 °C) and PEG (buffer, 0.5 mg mL−1, 20 min at 8 °C) was deposited, followed by filtering, washing, and drying. Next, the particles were released from the wax as described above. The released Janus particles were equipped with trypsin and GOx/Pt-NP (1 mg mL−1 in buffer, 1:1 mol ratio, 40 min) as described above. Motion Experiments. The 800 nm sized swimmers were dispersed in 100 μL of buffer or buffer containing varying concentrations of glucose (50 to 400 mM), BA-Rho-110 (5 to 100 μM), or different mixtures of both substrates and 20 wt % glycerol. Glycerol increased the viscosity of the fluid and diminished diffusion caused by Brownian motion (D ∼ 1/η, with η being the viscosity).18,25,61 The dispersions were transferred into PLL-coated microfluidic slides and mounted on a microscope. In contrast to the bare polystyrene slides, their PLL modification resembled a biorelevant environment. The samples were allowed to stabilize for 15 min, during which the light of the microscope was switched off. The movies were recorded with 16.67 fps. For motion experiments in which a magnetic field was applied, a cubic NdFeB magnet was placed at the side of the ibidi slide. The trajectories of each individual particle were tracked by using the Fiji plugin “TrackMate”.62 The trajectories were extracted and imported to Matlab in which the mean squared displacement analysis was performed, using a protocol published elsewhere.63 The effective diffusion coefficients were extracted with Deff = MSD(2iΔt)−1 (i = 2, for two-dimensional analysis), following a procedure published earlier.61 The linear fit was exercised for 1/10th of the duration of the experiment. In the case where the ballistic thrust of the propelled Janus particles was not randomized by the rapid reorientation of the swimmer (when a static magnetic field was applied), a nonlinear fit was applied at short delay times according to the equation MSD = 4DΔt + v2Δt2. The instantaneous velocity was estimated from the length of a given trajectory of a swimmer to a known time point (>20 s). For each experimental condition, between 20 and 150 particles were analyzed and two independent repeats were conducted. The error bar given in the plots is the standard deviation of the mean. Instrumentation. Transmission electron microscopy images were taken with the Tecnai TEM from FEI. Bright-field microscopy was conducted with an inverted Olympus IX81 microscope using a 60× immersion objective. Confocal laser scanning fluorescence microscopy was conducted using a Zeiss Axiovert microscope coupled to an LSM

in the absence of the fuel entities, the addition of fuel molecules led to directed double-powered swimmers with the highest obtained velocity of ∼0.72 μm s−1. These double-fueled enzyme-based motors with enhancement of the swimmer’s directional locomotion will considerably contribute to the application of biocompatible nanobots in many different perspectives in a biomedical context. Future developments will aim at overcoming current limitations of the swimmers including the need for high glucose, weight of the carrier, stability and long-term performance of the motors, among others by, for instance, considering alternative (artificial) enzymes or polymeric carriers.

MATERIALS AND METHODS Materials. Paraffin wax (melting point 56−58 °C) was commercially available from Merck KGaA (Germany). Aqueous dispersions of 50 mg mL−1 silica particles (800 nm or 2.7 μm in size) were purchased from Microparticles GmbH (Germany). 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), poly(Llysine) (PLL, MW 30000−70000 g mol−1), trypsin from bovine pancreas (10000 BAEE U mg−1), hexadecyltrimethylammonium bromide (CTAB), fluorescein 5(6)-isothiocyanate (FITC), manganese(II) acetylacetonate (Mn(acac)2), iron(III) acetylacetonate (Fe(acac) 3 ), 1,2-hexadecanediol, oleic acid (OA), tris(hydroxymethyl)aminomethane (TRIS), oleylamine, benzyl ether (98%), hydrogen hexachloroplatinate(IV), trisodium citrate, and sodium borohydride were purchased from Sigma-Aldrich. αMethoxy-ω-carboxylic acid succinimidyl ester poly(ethylene glycol) (PEG-MW 2000 Da) was available from Iris Biotech GmbH (Germany). BCA reagent A (protein assay), Amplex Red, DyLight 633 maleimide, and (benzyloxycarbonyl-Ile-Pro-Arg)2-R110 (BA-Rho110) were obtained from Thermo Fisher Scientific. Copper(II) sulfate was purchased from Alfa Aesar. Poly(methacrylic acid) (PMA) was acquired from PolySciences. Chloroform and ethanol were obtained from VWR. Ultrapure water (18.2 MΩ cm−1 resistance) was provided by an ELGA Purelab Ultra system (ELGA waterlabs). μ-Slides VI 0.4 coated with PLL were purchased from ibidi GmbH (Germany). FITClabeled PLL,46 poly(L-lysine)-graf t-poly(ethylene glycol) (PEG; grafting ratio g = 3.5),25 and citrate-capped Pt-NPs60 were synthesized according to the procedure published previously. Buffer Solutions. HEPES buffer (10 mM) was adjusted to pH 7.4 using ultrapure water. Pickering Emulsion for 2.7 and 0.8 μm Particles. Two initial bilayers of (PLL/PMA)2 were deposited on 200 μL (250 μL) of a 50 mg mL−1 aqueous solution of 2.7 μm (0.8 μm) silica particles by incubating the particles in the polymer solutions (1 mg mL−1 in HEPES buffer, 10 min), followed by three washing steps with HEPES buffer. Next, 200 μL of these modified particles was dispersed in 1.1 mL of an aqueous CTAB (0.1 cmc) solution and stirred at 250 rpm in a preheated water bath at 75 °C. Then, 80 and 160 mg for 2.7 and 0.8 μm particles, respectively, of paraffin wax was added. When the wax was completely melted, the mixture was stirred for 10 min at 1600 rpm. The emulsion was allowed to cool to room temperature. The solid wax droplets were filtered, washed thoroughly with Milli-Q water, and dried. GOx/Pt-NP Swimmer. 20 mg of wax droplets obtained from the Pickering emulsion of 800 nm silica particles were dispersed in 200 μL aqueous Pt-NP (40 min at 8 °C). The droplets were filtered, washed and dried. The wax droplets were dispersed in 200 μL of a solution containing PLL solution (1 mg mL−1 in buffer, 20 min at 8 °C) and filtered, washed and dried. The wax was removed using chloroform. The particles were separated by centrifugation and washed with ethanol and buffer. The released Janus particles were dispersed in 200 μL PEG (0.5 mg mL−1 in buffer, 20 min) and then washed 3× in buffer. GOx (1 mg mL−1 in buffer, 40 min) were immobilized on the particles followed by 3 washing cycles in buffer. DyLight 633 Maleimide-Labeled Trypsin. Trypsin (5 mg, 0.21 μmol) was dissolved in 190 μL HEPES buffer, and 10 μL of a 10 mg 3981


Article

ACS Nano 700 confocal scanning module (Carl Zeiss, Germany) and a 100× oilimmersion objective. CLSM measurements were done with 2.7 μm sized assemblies. We would like to emphasize that the orientation of the measured Janus particles toward the optical path of the microscope is important because in CLSM (focal) planes are scanned; that is, the Janus balance between individual particles may appear uneven. This observation was thoroughly discussed by our group previously.46 Quartz crystal microbalance with dissipation monitoring (QCM-D): QCM-D was performed using a Q-Sense E4 (Sweden). Silica crystals (QSX300, Q-Sense) were cleaned by immersion into a 2 wt % SDS solution, rinsed in ultrapure water, and dried. The crystals were exposed to UV/ozone for 20 min and mounted onto the liquid exchange chamber of the device. The frequency and dissipation measurements were monitored at 22 ± 0.02 °C. When a stable baseline in HEPES buffer solution was achieved, the aqueous polymer solution (PLL or PMA, 1 mg mL−1 in HEPES buffer) was introduced into the chamber and allowed to adsorb onto the crystal. After the surface was saturated, the chamber was rinsed with buffer solution in order to remove excess polymer. After two bilayers of PLL/PMA, the polymer-coated surface was exposed to a solution of MF-NP (1 mg mL−1 in ultrapure water) and left to absorb. After the surface was saturated, excess MF-NPs were removed by rinsing with buffer. This procedure was repeated until the desired number of (polymer/MFNP) layers were deposited.

authors thank M. Bañobre-López (from INL, Braga, Portugal) for the magnetic hysteresis loops.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00441. Experimental details, motility distribution of Trp swimmers, characterization of platinum nanoparticles and manganese ferrite nanoparticles, characterization of the activity of immobilized trypsin, MSD vs delay plots for bio-inorganic and trypsin swimmer including the linear fit, quartz microbalance characterization of the layer-by-layer deposition of MF-NP multilayer films, characterization of the co-deposition of GOx and Trp and their activity, effective diffusion coefficients and velocities of dual-fueled swimmers without and with magnetic field, characterization of the diffusion properties of symmetric and Janus swimmer, time-lapse images of magnetotactic swimmers (PDF) Movie S1: Glucose-driven GOx/Pt-NP swimmer (MPG) Movie S2: Peptide-driven trypsin swimmer (MPG) Movie S3: Dual-fueled swimmer with magnetotactic behavior with and without magnetic field and with and without fuel (MPG)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Verónica Salgueiriño: 0000-0002-9396-468X Brigitte Städler: 0000-0002-7335-3945 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by a research grant from the Danish Council of Independent Research, Technology and Production Sciences, Denmark (B.S., P.S.S.), the Xunta de Galicia (Regional Government, Spain) under project EM2014/035 and the Spanish Ministerio de Economiá y Competitividad under project CTM2014-58481-R (M.A.R.-D., V.S.). The 3982


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