Propulsion, deformation, and confinement response of hollow nanocellulose millimotors

Micromotor and nanomotor particles are typically made using dense solid particles that can sediment or be trapped in confined flow environments. Creation of much larger motors should be possible if a very low-density system is used with sufficient strength to carry liquid and still experience propulsive motion. Light, dense millimotors should also be able to deform more than dense solid ones in constrictions. Millimotors are created from permeable capsules of bacterial cellulose that are coated with catalase-containing metal-organic frameworks, enabling reactive propulsion in aqueous hydrogen peroxide. The motion of the motors is quantified using particle tracking and the deformation is measured using microcapillary compression and flow through confined channels. Two different propulsion mechanisms are dominant depending on the motor surface chemistry: oxygen bubbles are expelled from hydrophilic millimotors, driving motion via reaction force and buoyancy. Hydrophobic millimotors remain attached to growing bubbles and move by buoyancy alone. Despite their large size, the low-density capsules compress to pass through contractions that would impede and be blocked by solid motors. The sparse structure but relatively large size of the motors enables them to transport significant volumes of liquid using minimal solid mass as a motor support structure.


Introduction
Microorganisms can propel themselves through liquid by different swimming mechanisms, and synthetic particle motors, termed "active matter", have been created that mimic microbial motion by chemical, rather than mechanical, means [1][2][3] . Particulate nanomotors and micromotors move by converting chemical fuel from their environment into kinetic energy, and can achieve remarkable speeds relative to their body length 4,5 . Propulsion can be driven by self-generated solute gradients or electric fields 1 or by formation of gas bubbles that cause buoyancy or ejection-induced recoil effects 6 . Applications for the small motors are imagined in drug delivery 2,7-9 , environmental remediation [10][11][12][13][14] , and self-assembly 15,16 while the particles' unique motion is widely studied as well 17 . Larger, millimeter-scale motors have recently been developed from clay/DNA membranes to act as synthetic protocells that move and carry out internal biochemical reactions 18 . These larger-scale motors can broaden the possible applications of active matter, motivating us to develop a millimotor capsule that is easily functionalized but can also overcome difficulties most motors face with confined space navigation and sedimentation potential 19 .
Our approach to develop these new millimotors takes inspiration from the biological cells that active matter seeks to mimic. Cells are partially permeable to water, minimizing den-sity differences, while their softness enables them to deform and pass through narrow spaces and navigate confined environments. By contrast, most synthetic active particles are made using dense solid materials, like platinum or silica, whose large density differences with water promote sedimentation. Such particles are also too rigid to deform and escape environmental confinement 19 . A recent review 20 identified low density and robust deformability as important goals for future motor particles, and this work focuses on a new approach to meeting these challenges using unconventionally large motor capsules made from a mesh of bacterial cellulose fibers.
Hollow capsules are a promising way to minimise mass use and density issues in motor particles [21][22][23][24][25] and their low density makes them behave like much smaller particles in fluid 26 .
A recently developed bacterial cellulose capsule 27 provides a unique minimalist scaffolding for millimotor particle development, and we explore their motion and response after coating them with two different MOF nanoparticles 28 . The MOFs attach onto the capsule's cellulose nanofibers and trap catalase enzyme in their structure, enabling conversion of aqueous hydrogen peroxide fuel into oxygen bubbles to drive motion 18,29 . The MOF surface polarity determines the mode of millimotor propulsion by altering oxygen bubble affinity for the capsule surface. The velocity and motion of the driven capsules are measured by optical microscopy and shown to be quite efficient compared to solid micro-and nanomotors, despite being much larger and full of liquid. The low-density shells are efficient motor bases because of their structural integrity and minimal mass, but also provide unique benefits for bubble-driven flow. The flexible nanocellulose fiber struts allow significant deformation when passing through constrictions and the permeable capsules form a low-friction gas layer on the capsules that further enhances escape from confined spaces.

Methods and Materials
Cellulose microcapsule preparation: Cellulose microcapsules with diameters in the range of 50 − 1000 µm were synthesized by a biointerfacial polymerization process we previously developed 27 . In brief, bacterial cellulose microcapsules were grown using a waterin-oil emulsion of bacterial culture droplets as templates. The bacterial culture contains purified Acetobacter xylinum concentrated from Kombucha culture (Nourishme Organics, Australia) by gradient centrifugation, coconut water (Cocobella, Indonesia), and 10% w/v table sugar. Within 10 days, the encapsulated bacteria polymerize glucose molecules into cellulose nanofibers, with a diameter of 60 − 70 nm, that entangle to form a fiber mesh shell with a total thickness of 20 − 50 µm and a pore size of 0.5 µm 27 at the oil-water interface. Subsequently, catalase-ZIFL and catalase-ZIF90 MOF crystals were grown in situ on the nanofibers, producing hydrophobic and hydrophilic millimotors, respectively. Capsules ranged between 0.2-0.8 mm in size for both hydrophilic and hydrophobic particles. Enzyme labelling: 8.5 mg of Rhodamine B isothiocyanate (RhB, Sigma Aldrich Australia) was dissolved in 0.5 mL dimethyl sulphoxide (DMSO, Sigma Aldrich). In a glass vial, 40 mg of catalase was placed in 2 mL of sodium carbonate bicarbonate buffer (0.5 M, pH 9.5). Then, the RhB solution was added slowly into CAT solution. The CAT-RhB was then mixed for 2 h at room temperature in darkness. The unreacted enzymes were separated from the labelled enzymes in an Illustra NAP-25 column (GE Healthcare). The first band eluted with Milli-Q water, which contains labeled enzymes, was collected for sample preparation.
MOF and cellulose labelling: Congo Red and FITC were used to stain cellulose fibers and MOF crystals, respectivley to enhance microscopy visualization by addition of 34 mg of 0.5 wt% aqueous Congo Red solution to 1 mL of cellulose microcapsule dispersion.
Microscopy: Confocal and light sheet microscopy imaging experiments were carried out on a Zeiss LSM 880 with Airy scan 30 , and Zeiss Lightsheet Z.1 (Germany) microscope.
A 63x oil immersion objective, with numerical aperture NA = 1.4, 5x dry objective with NA = 0.16, and 20x water immersion objective with NA = 1 were used depending on the scale of observation desired. Low-magnification optical microscopy images were acquired via stereoscope (WILD M3C, Leica, Germany) with 6.4x objective to enable individual particle tracking in microcapsule dispersions at room temperature. ImageJ software was utilized to quantify the fluorescence intensity inside and outside of the microcapsule 31        cellulose fibers, Figure 2(c-d). The addition of the MOF particles to the capsule is expected to alter the mass and inertia of the system during subsequent propulsion studies. If we assume the MOF coating is a 99% dense single layer of 100 nm particles with a density of 2 g/cm 3 , the particles add 150 ng mass to a 0.5 mm diameter cellulose capsule, at most doubling its weight while still being dominated by the mass of the liquid cargo in its capsule.
Differences can be seen in the coating morphology at higher magnifications, Figure 2 The coating is heterogeneous due to random variations in the solution during precipitation.
More detail can be resolved at higher magnifications. Enzyme distribution is a key parameter determining millimotor motion and orientation 37 .
The distribution of enzyme on the MOF-coated capsules is demonstrated in Figures 3(j-k) using a 3D reconstruction of the millimotor capsules with a color map indicating the intensity of the Rhodamine B dye labeling the catalase. We observe a heterogeneous but thorough distribution of the enzyme on the surface of the hydrophobic motor, Figure 3 Figures 4(a-c) show capsules that have been exposed to different size FITC-dextrans and the intensity of green tracer that was able to diffuse

Millimotor capsule propulsion
Oxygen gas production and bubble formation play an essential role in propulsion of both hydrophobic and hydrophilic millimotors. When exposed to an aqueous hydrogen peroxide solution, catalase enzyme immobilized in the MOFs catalyzes hydrogen peroxide decomposition into oxygen and water molecules, with the excess oxygen gas forming bubbles and driving motion. We first assess the rate of reactive oxygen generation in a bulk solution of 1% v/v% H 2 O 2 containing hydrophobic ZIFL and hydrophilic ZIF90 motors, Figure 5(a).
Both systems rapidly produce oxygen, but the rate of O 2 production by hydrophilic ZIF90 motors was more than six times that by hydrophobic ZIFL motors, Figure 5(a), as a result of differences in enzyme activity. The catalase enzyme has a higher affinity for a hydrophobic surface, which can cause conformation changes that denature the protein, reducing its activity 38 . Oxygen production rate is important to motor performance, as sufficiently high rates can lead to bubble ejection and propulsion by recoil forces, whereas slower growth tends to favor bubbles remaining attached to a motor so that motion is dominated by buoyancy. The mode of bubble-motor interaction, however, is also affected by the motor surface chemistry and the resultant affinity of hydrophobic bubbles for a motor.
We studied the performance of these motors by observation In all motion studies, we see a clear difference between the hydrophobic ZIFL motors, that initially move in all directions as a result of rapid bubble production and ejection, and the hydrophilic ZIF90 motors that only move vertically, Figure 5(b). For example, the hydrophobic ZIFL motor in the top row of Figure 5(b) follows a completely vertical trajectory over several seconds until it reaches the top of the water volume and drifts slightly to the side. This is consistent with extensive work on mineral flotation, where hydrophobic particles are seprated from suspensions using air bubbles that flow through the system 43,44 . The hydrophilic ZIF90 motor in the bottom row of Figure 5 ejected the motor will move in the opposite direction by recoil. If bubbles remain attached, however, the motor will move up by buoyancy and its initial orientation is less significant to the subsequent motion. We summarize the mechanisms of motion by schematic in Figure   5(d).
The cellulose microcapsules used here vary in size from 200 − 600 µm, all much larger than commonly-studied micro-and nanomotors, but consistent with recently-developed protocell millimotors 18 . We are curious about the performance of these motors versus their smaller counterparts, however, as their large size but low solid mass could provide additional benefits. As noted earlier, the capsules are low-density and permeable, with the majority of their inertia due to water that permeates the interior. A useful benchmark is the mo-

Motor deformation and confinement response
The cellulose motors have a unique porosity and response to mechanical stress because of their combined strength and flexibility 27 . As a result, we are interested in how these particles affect capsule response to extensional stress. First, we need to evaluate the extent to which the modification of the motors by the formation of MOF crystals affects their mechanical properties. Song et al. 27 found unmodified capsules deformed elastically under compression, recovering after deformations as large as 20% but remaining indented or buckled at higher strains. Adding crystalline MOF particles, however, is expected to stiffen the fibers and change the capsule response to deformation. Stress-strain curves for up to five individual millimotors were measured using an extensional flow in a microfluidic channel 27 . We found that the elastic modulus, E =100 Pa, of pristine cellulose microcapsules increased ∼ 30X after coating with hydrophilic ZIF90 MOFs and ∼ 140X after coating with hydrophobic ZIFL MOFs. The difference is likely a result of variations in coating uniformity and distribution, Along with an increased elastic modulus, the capsules preserve significant ability to recover from deformation, as we demonstrate in Figure 6. Figure 6(a) shows the microscopic response of a hydrophobic ZIFL millimotor to deformation by a blunt microcapillary end.
Both hydrophobic ZIFL and hydrophilic ZIF90 motors largely recover their original profile after being indented by external stress to a strain of ε =74% and higher, while pristine capsules do not fully recover from strains higher than ε =20% 27 . The addition of MOFs clearly increases the elasticity of the capsules while adding propulsive capability. This is consistent with above estimates of a significantly increased capsule modulus after MOF coating.  Figure 6(b) indicates that the motor structure actually flexes in response to the pressure of the bubble expansion itself. Here the bubble is likely generated inside of the capsule and then squeezes out of a pore to exert the pressure seen here. Such an effect could also occur if the bubble formed on a capsule that was confined in some way.
The result indicates the motors could actually be changing shape during motion, increasing their flexibility and responsiveness, and offering a mechanism for more complex movements in future work. As pointed out earlier, there is a need for motors to navigate confined environments while adapting and recovering to the different conditions they encounter. We now evaluate whether such deformation and response can occur when the millimotors encounter confinement during their propelled movement. We do this by studying the performance of the motors during propulsion in a constricted channel and note whether the combination of propulsion and flexibility enhance their mobility. Figure 7(a) shows a series of images of a hydrophilic ZIF90 motor as it moves vertically through a gradually constricting capillary. The spherical millimotor initially has a diameter of 2.9 mm so can enter the capillary but must compress in order to exit, as the capillary internal diameter reduces from 3.5 mm, to 2.2 mm at its outlet. The motor has been artificially colored red in the image and the surrounding bubbles have been colored blue to enhance visibility. The motor is initially driven by buoyancy but slows in the second frame in response to the narrowed passage. The narrowing of the channel causes compression of the motor, increasing its strain to more than 25%, Figure 7(c). However, the motor continues to produce bubbles behind itself that grow as the reaction proceeds. A solid particle at this point would be unable to proceed and simply block the channel, but the flexible motor is able to deform much more in response to stress, Figure 6(a), and continues to move, albeit in a more cyclical way as seen in the plot of vertical position in Figure 7(b). Despite the small variations in velocity, the overall progress of the motor is relatively constant as a result of its flexibility and reactive propulsion. The growth of surface bubbles likely aids movement by reducing drag on the capsule. Figure 7(a) also shows the formation of a large bubble behind the motor that aids in pushing it through the constriction. Interestingly, the bubble driving the capsules through the capillary is pinned to the capillary wall, allowing it to push against the capsule. Such behavior is beneficial for this additional propulsion mechanism to act, as a non-wetting capillary surface would reduce the ability of the bubble to push the motor. The results in Figure 7 show that the production of deformable responsive motors with the low-density cellulose capsules developed here enable use in confined environments that solid particle counterparts can not handle. A high level of deformation might be expected to cause erosion of the MOFs from the capsules surface; however, no detectable fragments were observed following the deformation experiments in Figure 6(a). In addition, the results in Figure 7 indicate propulsion is maintained even during quite robust deformation in confinement, further confirming the motors largely remain attached.

Conclusions
In this study, enzyme-powered millimotors have been made by crystallization of MOF particles encapsulating catalase enzymes onto soft cellulose microcapsules. The large size, but low mass of the motors makes them a unique addition to the range of existing particle motors as they are able to carry significant volumes of liquid cargo because of their capsule permeability. The large capsules are able to move using chemical fuel thanks to the numerous nanomotors attached to their surfaces, analogous to a large tanker ship driven by multiple tugboats.
Two mechanisms of propulsion result from enzyme-mediated production of oxygen gas bubbles in the presence of hydrogen peroxide. Bubbles grow on and remain attached to hydrophobic motors, driving vertical motion as buoyancy rapidly grows. For hydrophilic motors, bubbles are initially expelled from the structures, driving randomly-directed motion by recoil force, but as more bubbles grow motion becomes dominated by buoyancy and the motors rise similarly to the hydrophobic motors. Despite their large size, the millimotors move with surprising speeds, reaching levels of 80 motor diameters per second for the hydrophilic motors, when recoil force dominates movement, and 8 motor diameters per second when buoyancy dominates. The low density of the millimotors makes them highly efficient at movement, exhibiting similar or faster relative velocities than much smaller solid particle motors. The flexible response to mechanical stress of the millimotors enables them to squeeze through significant constrictions, making their mobility viable even in confined spaces and environments where solid motor particles would clog and jam. The millimotors can compress their diameter by as much as 30% in a constriction and their continuous generation of gas bubbles creates a low-slip surface layer and a back-pressure that enhances movement through tight spots. Such flexibility also offers a possible way to rapidly release the liquid cargo of the capsules and we plan to study this in future work.