Multifunctional Bacteria-Driven Microswimmers for Targeted Active

Multifunctional Bacteria-Driven Microswimmers for Targeted Active Drug Delivery Byung-Wook Park, Jiang Zhuang, Oncay Yasa, and Metin Sitti* Physical Intelligence Department, Max Planck Institute for Intelligent Systems, 70569 Stuttgart, Germany S Supporting Information *

ABSTRACT: High-performance, multifunctional bacteriadriven microswimmers are introduced using an optimized design and fabrication method for targeted drug delivery applications. These microswimmers are made of mostly single Escherichia coli bacterium attached to the surface of drug-loaded polyelectrolyte multilayer (PEM) microparticles with embedded magnetic nanoparticles. The PEM drug carriers are 1 μm in diameter and are intentionally fabricated with a more viscoelastic material than the particles previously studied in the literature. The resulting stochastic microswimmers are able to swim at mean speeds of up to 22.5 μm/s. They can be guided and targeted to specific cells, because they exhibit biased and directional motion under a chemoattractant gradient and a magnetic field, respectively. Moreover, we demonstrate the microswimmers delivering doxorubicin anticancer drug molecules, encapsulated in the polyelectrolyte multilayers, to 4T1 breast cancer cells under magnetic guidance in vitro. The results reveal the feasibility of using these active multifunctional bacteria-driven microswimmers to perform targeted drug delivery with significantly enhanced drug transfer, when compared with the passive PEM microparticles. KEYWORDS: biohybrid, bacteria, microswimmer, polyelectrolyte multilayer, targeted drug delivery osome,8,9 alginate,10 and maleimide-functionalized hyaluronic acid.11 The method of bacterial attachment to the surface of synthetic microparticles is one of the important design parameters for bacteria-driven microswimmers. The attachment has been enabled through various methods,12−15 including hydrophobicity, wettability, bioconjugations (e.g., biotin− streptavidin), and covalent binding (e.g., carbodiimide crosslinking reaction). However, with particular attachment methods, unfavorable conditions may alter the surface properties of the bacteria cell membrane, resulting in significant changes in the bacteria motility. To provide enhanced bacterial attachment for biohybrid microswimmer designs, single layers of polyelectrolyte coatings, such as positively charged chitosan10 and poly-L-lysine,16 have been applied as complementary secondary noncovalent attachment methods. A summary of characteristics of the bacteria-driven microswimmers is given in Table S1. Despite the improvements

A

ctive microswimmers have many potential applications in medicine and bioengineering, where they could enable targeted cargo (e.g., drug, gene, imaging agent, and RNA) delivery, biosensing, and cell transportation.1 Such microswimmers are a few micrometers in size, yet they swim through their environment using only local energy storage, and may additionally be steered by external inputs. Synthetic, catalytically powered microswimmers rely on the decomposition of a fuel solution (e.g., hydrogen peroxide or strong acids or bases) on a catalyst surface (e.g., platinum) to produce forward thrust. One potential downside of this type of microswimmer is that the catalytic decomposition produces toxic byproducts,2,3 and another is the limited onboard fuel source. Bacterial cells, on the other hand, have evolved to swim efficiently at the micrometer scale using food sources found in their environment. The bacteria can be integrated with synthetic substances to produce multiple functionalities through their biological actuation and sensing capabilities. Since the first work was demonstrated by Darnton et al.,4 significant progress has been made in designing bacteria-driven biohybrid microswimmers capable of different tasks by employing various materials,5 such as polystyrene,6,7 lip© 2017 American Chemical Society

Received: May 9, 2017 Accepted: September 5, 2017 Published: September 5, 2017 8910

DOI: 10.1021/acsnano.7b03207 ACS Nano 2017, 11, 8910−8923

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Figure 1. Bacteria-driven microswimmers based on PEM-MNP microparticles attached to an E. coli MG1655 bacterium. (a) Overview of the multifunctional bacteria-driven microswimmer design for targeted active drug delivery. PAH, positively charged poly(allylamine hydrochloride); PSS, negatively charged poly(sodium 4-styrenesulfonate); MNPs, magnetic nanoparticles; DOX, doxorubicin, an anticancer drug; PS, 1 μm diameter polystyrene microparticle. (b) SEM image of a single PS(MNP1PAH/PSS)4PAH-attached cell (scale bar, 1 μm). The inset in panel b is the optical image of a single bacteria-driven microswimmer (scale bar, 1 μm). (c) TEM images of thin sections of the microswimmers. The inset in panel c is the TEM image of monolayers of MNPs (scale bar, 50 nm).

ment incorporating PEM microparticles for drug delivery applications has not been previously reported in the literature. Herein, we propose a fabrication approach for multifunctional bacteria-driven microswimmers for targeted drug delivery applications. We demonstrate the incorporation of Escherichia coli (E. coli) with PEM microparticles fabricated by the LbL technique, which encapsulate the chemotherapeutic doxorubicin (DOX) and magnetite (Fe3O4) nanoparticles (MNPs). We experimentally tested these microswimmers for targeted drug delivery in a 4T1 model of breast cancer cells in vitro, and demonstrated higher swimming speeds than similar bacteriadriven microswimmers in the literature. Figure 1a illustrates the basic principles of our method. The deposition of PEM nanoshell layers on a polystyrene (PS) microparticle produces a surface with tunable stiffness that is in the range of Young’s modulus previously shown to facilitate bacterial adhesion. The technique also produces controlled surface charge and a surface chemistry, which enables complementary noncovalent interactions for bacterial attachment. To evaluate the performance of this type of bacteria-driven hybrid microswimmers, the stochastic motion of microswimmers was analyzed in free swimming conditions in 2D and 3D.23 Their steering control due to an external magnetic field13 and an environmental chemical gradient24 was demonstrated, which are crucial for guiding the drugs actively to specific target regions inside the body. Through two major control assessments of chemotaxis and magnetic guidance, the feasibility of the bacteria-driven biohybrid microswimmers was verified. Moreover, the results of our study demonstrate the feasibility of using our guidable bacteria-driven microswimmer design to perform single-celltargeted drug delivery in vitro. Our work provides a systematic design and fabrication method for multifunctional bacteriadriven microswimmers with increased swimming speed,

observed in such approaches, the better understanding of the bacterial attachment mechanism and optimization of the microswimmer designs remain an open area of research, which affect the performance of the microswimmer. It has been shown that positively charged surfaces increase bacterial attachment due to the net negative charge on most bacteria.17 In addition to the effect of the surface charge, mechanical stiffness of viscoelastic materials has recently been reported as a major factor for bacterial attachment.18 The yield of bacterial attachment on the surface of stiffer elastic materials (e.g., elastic modulus of 100 MPa) was shown to be higher than that on softer surfaces, which was considered as the most dominant bacteria-surface interaction factor among three major cues of mechanical, chemical, and topographical properties.19 Since Decher et al. first reported the layer-by-layer (LbL) self-assembly method using positively and negatively charged macromolecules to produce multilayered ultrathin films,20 the fabrication of polyelectrolyte multilayer (PEM) microstructures has emerged as a powerful technique in the fields of biomaterials, biophysics, tissue engineering, and drug delivery. The ability to modulate the physicochemical properties (i.e., chemical, physical, and mechanical cues) has enabled researchers to design customized microstructures for specific applications.19 In particular, the PEM microstructures have been extensively used for drug delivery applications due to ease of size control, wide selection of materials, precise design of microstructures, and controlled encapsulation and release of therapeutic agents such as DNA, siRNA, proteins, and enzymes.21,22 However, conventional drug delivery tactics using such inactive PEM templates rely on passive diffusion mechanisms to control the transport of drugs, which typically results in systemic toxicity, low selectivity in targeting area (e.g., cancer cells), and low yield in controlled release. Moreover, bacteria-driven microswimmers with optimized bacterial attach8911


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Figure 2. Fabrication and characterization of PEM microparticles. (a) Thickness values, estimated from QCM-D analysis, as a function of number of layers deposited from a 150 mM NaCl solution on a cationic model substrate alternatively adsorbing PAH and PSS. The inset in panel a is the fluorescence images of different number of PEM layers (PAHFITC/PSS). The scale bars are 2 μm. (b) Thickness and surface profile of an (PAH/PSS)4PAH film measured with an AFM. The inset in panel b is the AFM image of (PAH/PSS)4PAH surface in the height mode. (c) Young’s modulus distribution of (PAH/PSS)20PAH film obtained from the AFM nanoindentation tests using a Gaussian fit (n = 337).

as well as surface roughness, provides insight into the structural changes of the PEM deposition process. The close agreement between the thicknesses obtained from the AFM image and those calculated from the QCM-D data suggests that the frequency (mass) and dissipation (viscoelasticity) measurements are a reliable measure of polyelectrolyte shell thickness. The measured average thickness of (PAH/PSS)4PAH using AFM was 27.6 ± 2.6 nm. This value compares favorably with the 28.4 ± 1.4 nm estimated using the QCM-D data. The rootmean-squared (RMS) roughness computed from the AFM images was 1.2 ± 0.4 nm, which is a relatively smooth surface (Figure S1b). We applied the AFM nanoindentation technique to measure mechanical properties (e.g., the apparent Young’s modulus) of the fabricated PEM films. The indentation experiments were performed on thick films (≈100 nm) in order to reduce the influence of the relatively stiffer glass substrate. The indentation depths did not exceed 10% of the film thickness, thus restricting the fit of the Hertzian deformation mechanics to a spherical tip geometry. Figure 2c shows the distribution of Young’s modulus of the PEM surface. The apparent Young’s modulus value for the entire series of PEM multilayer films analyzed was determined to be 111.7 ± 52.2 MPa (n = 337), which is close to the elasticity of a stiff elastomer.27 The value of our result agrees with the previous studies of (PSS/PAH) films,28,29 which found Young’s modulus to be in the range of 2.8 MPa to nearly 700 MPa. PEM films have been known as tunable materials, mechanical properties of which can be controlled in several ways (e.g., ionic strength, pH, cross-linking, and addition of “stiff” or “soft” layers), thus allowing the study of bacterial attachment on the films with different stiffness.30 To enable a magnetic steering control capability for the bacteria-driven microswimmers, the synthesized MNPs were embedded inside the PEM nanoshell. Briefly, MNPs were prepared as described previously31 and were used to form a single layer of MNPs in PEM multilayers (PS(PAH/ PSS)4PAH)). By inspecting single nanoparticles with highresolution transmission electron microscopy (TEM), as shown in Figure S2a, the interplanar distance between atomic planes for single MNP was found to be approximately 1.92 Å, which confirms the crystal structure of the MNPs. We further

directional guidance, and drug delivery efficiency toward nearfuture in vivo targeted active drug delivery applications.

RESULTS AND DISCUSSION Fabrication and Characterization of Engineered PEM Microparticles and Microswimmers. PEM microparticles have been widely used as biocompatible, multifunctional composite carrier systems for local release of cargo molecules (e.g., drugs) in a passive manner. To evaluate the potential use of the PEM microparticles as a part of our biohybrid microswimmers, the fabrication and structure of the PEM microparticles were characterized systematically. PAH/PSS multilayer microparticles were fabricated by using PS microparticles as a core. Alternative deposition of the polyanions (PSS, poly(styrenesulfonic acid)) and polycations (PAH, poly(allylamine hydrochloride) or PAHFITC, poly(fluorescein isothiocyanate allylamine hydrochloride)) was performed at a concentration of 1.0 mg/mL in DI water containing 150 mM NaCl, respectively. The formation of a multilayer, nanoshell ((PAHFITC/PSS)4PAHFITC) build-up on the surface was demonstrated by a confocal laser scanning microscope (CLSM) and quartz crystal microbalance (QCM-D) for dissipation analysis, as can be seen in Figure 2a. The fluorescence intensity increased with the number of the multilayers produced. QCM-D measurements reveal that PAH/PSS multilayers are linearly growing films consisting of stacked polyelectrolyte layers, which interpenetrate each other, leading to a periodic structure in the direction perpendicular to the layers.25 It was found that the shell thickness increased linearly with the number of layers constituting the wall, with a thickness increment of 6 nm per bilayer. These observations are in agreement with previous measurements in the literature.26 Moreover, atomic force microscope (AFM) measurements were performed to provide further details about local structural information on the PEM surface and to support the interpretation of the QCM-D data, because the shell thickness estimated by QCM-D only allows a global characterization of the PEM deposition process. AFM images depicting the thickness and surface morphology of the top layer of (PAH/ PSS)4PAH can be seen in Figure 2b and Supporting Information Figure S1a,b. Visualization of the shell thickness, 8912


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Figure 3. Bacterial attachment: Physicochemical effect of the PEM surface. The frequency as a function of dissipation shifts, (Δf/ΔD), during first 10 min (a) of the experiment (initial attachment) and during the last 3 h (b) of bacterial deposition (mature surface−bacteria attachment) on the modified QCM-D quartz, as a function of the overtone number for each bacterial strain. (c) ΔD/Δf plot constructed from the 7th overtone during flow of E. coli MG1655 bacteria on the PEM (soft) and AUT-modified quartz crystal (hard) surfaces for 3 h. The reported values are the averages of replicate experiments. The fluorescence images of adhered E. coli MG1655 bacteria to the (d) soft PEM and (e) hard AUT-modified quartz surfaces after 3 h at 25 °C. The scale bars are 20 μm.

multilayers of MNPs on the first layer of the PAH is attributed to the average diameter increment, which corresponds to the measured layer thickness of 40.2 ± 9.4 nm. From the AFM and QCM-D measurement results, the thickness of PS(MNP1(PAH/PSS)4PAH) is approximately 65−75 nm. This result is in agreement with the previous reports describing MNP/PEM films built on planar substrates via the LbL strategy, where a regular film growth with the layer number was observed.34 It should be noted that the number of magnetic nanoparticles in the shell could be estimated only roughly from the TEM image analysis. The average volume (νm) for the MNPs in the PEM microparticles can be calculated by35

investigated the isothermal magnetization properties of the PEM microparticles. The PEM microparticles (PS(MNP1(PAH/PSS)4PAH)) were analyzed at a room temperature, using vibrating sample magnetometer (VSM) with fields up to 18 T. Plots of magnetization versus applied magnetic field for various samples are shown in Figure S2b. We found that these PEM microparticles (PS(MNP1(PAH/PSS)4PAH)) are superparamagnetic at room temperature, reaching a saturation magnetization value (Ms), saturation remanence magnetization (Mr), and coercivity (Hc) of 0.93 emu/g, 0.02 emu/g, and 5.5 mT, respectively. The measured Ms is much lower than the reported value of 92−100 emu/g for as-synthesized bulk magnetic nanoparticles.32 This can be explained by the fact that the nonmagnetic PS core is surrounded by the low magnetic content in the first single layer of the PEM nanoshell. We utilized scanning electron microscopy (SEM), TEM, and optical microscopy to visualize the construction of the developed bacteria-driven microswimmers. Figure 1b is a SEM image showing attachment of an E. coli bacterium to a 1 μm-diameter PEM microparticle (PS(MNP 1 (PAH/ PSS)4PAH)). The surface area of the 1 μm microparticle possibly allows 1−2 bacteria to attach per particle due to adhesive area available for bacterial attachment.33 In addition, TEM images of thin-sectioned bacteria-driven biohybrid microswimmers were obtained to further characterize the deposition of negatively charged MNPs on the first layer of positively charged PAH layer, followed by the bacteriamicroparticle interface (Figure 1c). The MNP layers are found to be equivalent to around 4−5 monolayers of MNPs with a diameter of 10.1 ± 3.2 nm per single deposition. These

4π vm =

3

( 2 −d a ) 3

(1)

where d is the diameter of the PEM (PS(MNP1(PAH/ PSS)4PAH)) microparticle and a is the fraction factor of the MNP wall thickness (see the Supporting Information for details). After deposition of a single layer of MNPs, the average volume of MNPs in the PEM microparticles was found to be approximately 0.13 μm3, which can be potentially tailored by the number of deposition cycles. Viscoelastic Properties of the Bacteria-Surface Interface. The LbL method incorporated with the PEM has been used for developing multifunctional drug delivery carriers, as well as functional surfaces for cell attachment. It has been reported that adjusting physicochemical properties of the PEM can influence the cell attachment and behavior.30 It would be advantageous for our bacteria-driven microswimmer design, if 8913


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Figure 4. 3D and 2D motility tests of the bacteria-driven microswimmers. (a) 3D trajectories obtained by a digital holographic microscope (DHM): Sample 3D trajectories show swimming of bacteria-driven microswimmers, tracing out helical patterns. The figure shows superimposed trajectories from different samples, and all of them were collected over 10 s DHM recordings. (b) Mean speed of bacteriadriven microswimmers over time, for which the error bars indicate the standard deviation. The experiments were performed using 10 samples from each time point. One-way ANOVA indicated statistically significant differences: (∗) p-value < 0.05; (∗∗) p-value < 0.01 compared with the mean speed at 18 h. (c) 2D mean speed distribution, trajectories, and swimming heading of free swimming bacteria-driven microswimmers with no chemoattractant gradient. The angular distribution of the swimming heading is isotropic. (d) 2D mean speed distribution, trajectories, and swimming heading of the microswimmers under chemoattractant gradient. The angular distribution of the 8914


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swimming heading is biased in the direction of higher chemical gradient, meaning that the swimming heading is anisotropic. In the polar plots (c,d), θ is defined as the projected angle in the x−y plane (the gradient is along the y-axis). (e) Mean speed distribution and trajectories of magnetically guided microswimmers in 2D. The microswimmers show directional motion parallel to the direction of the applied uniform magnetic field (26 mT).

motility and potentially overall performance of the bacteriadriven microswimmers. Figure 3c shows that the relationship (dissipation vs frequency) is nearly linear for the hard quartz surface, but for the soft PEM surface, the dissipation-frequency slope exponentially decreases in magnitude at higher frequency changes (after 30 min). Even though the soft PEM surface displays a higher dissipation at the early stage of attachment, this observation indicates that the bacteria become more stable and tightly bound to interfaces over time. There might be a more complex attachment process with the soft PEM surface because of the high water content at the PEM layer−medium interface, which makes the mechanical (viscoelastic) properties play a dominant role in bacterial attachment in addition to cell motility,38,39 particularly for E. coli possessing multiple flagella. Therefore, this observation may be explained by the fact that the stiffness of the PEM surface (111.7 MPa) provides a favorable environment for bacterial attachment, compared to the hard quartz surface (around 75 GPa). It was reported that the mechanical stiffness of the substrate, among all physicochemical properties, may play the most important role in regulating attachment of flagellated bacteria (e.g., E. coli).18,40 To provide further insight into the role of mechanical stiffness on bacterial attachment, the bacteria number on the QCM-D sensor surfaces was counted using a fluorescent microscope (Figures 3d,e). Interestingly, a significantly larger amount of E. coli was observed from the soft PEM surface, compared with the bacterial attachment on the hard quartz surface, after 3 h of bacterial attachment. Thus, we can conclude that the soft PEM surface has a more viscoelastic connection for bacterial attachment, as well as higher number of adhered bacteria. On the basis of the same positively charged amine surface chemistry of the soft PEM and hard AUT-modified quartz surfaces, it is very likely that the mechanical stiffness of the material plays a significant role in the observed increases in viscoelasticity and number of adhered bacteria. 3D Motility Analysis of Bacteria-Driven Microswimmers. A digital holographic microscope (DHM) was used to study the hydrodynamic interactions of swimming bacteria in 3D.41 The motility of the bacteria-driven microswimmers was characterized in a microfluidic chamber using a T1000 transmission DHM (Lyncee Tec, Lausanne, Switzerland) with a 40× objective lens. We analyzed the trajectories of 60 microswimmers that mostly had single E. coli attached to the PEM microparticles. Figure 4a and Supporting Information Video S1 demonstrate that most of the microswimmers exhibited oscillating, helical trajectories, which is attributed to near-constant force and torques exerted by the attached bacteria.23 Since the superparamagnetic MNPs are not homogeneously distributed inside the microparticles, there is a net magnetic moment along their easy axis when an external magnetic field is applied.13 Thus, the PEM microparticles align to the external magnetic field along their fixed easy axis, which enables their magnetic guidance. If the net propulsive force of the bacterium on the particle is exactly parallel to their magnetic moment, the PEM microparticles would have only

we could tune the bacteria−surface interface in a precise and repeatable manner to provoke proper bacteria attachment by understanding the effects of mechanical properties on bacterial attachment. To provide the details about the dependence of bacterial attachment on the mechanical stiffness of the soft PEM surface, where the development of secondary noncovalent interactions takes place,17 we monitored the attachment kinetics of bacterial cells and characterized the viscoelastic property of the bacteria-surface interface on soft PEM and hard 11-amino-1-undecanethiol (AUT)-modified quartz crystal surfaces. These substrates have the same surface chemistry and charge, consisting of positively charged amine groups. The magnitude and viscoelastic properties of an E. coli attachment were estimated as a function of changes in the resonance frequency (Δf) and energy dissipation (ΔD). In general, for rigidly attached bacteria, less change in dissipation is observed as a function of attachment, whereas, for weakly attached ones, the energy dissipation increases. By monitoring ΔD, a qualitative measure of the viscoelastic properties or relative stiffness of the bacteria−surface interface can be determined. Moreover, the magnitude of Δf/ΔD (i.e., the unit mass per dissipation) has been used as an inherent characteristics of the individual cells adhered on specific surfaces.36 A relatively high magnitude of Δf/ΔD implies a more rigid elastic connection, and a lower magnitude of Δf/ΔD stands for a soft and more viscous connection. The instantaneous values of Δf/ΔD through the overtone numbers also provide a dynamic picture of the viscoelastic changes of the bacteria-surface interface during the attachment stages.37 More viscoelastic attachment results in values of Δf/ΔD that vary in magnitude with overtone numbers. The bacterial attachment on the soft PEM and hard AUTmodified quartz surfaces was monitored after 10 min to capture the initial stage of the bacterial attachment and after 3 h to capture the mature bacteria−surface attachment. Because increasing the overtone number results in higher frequency of the sensor, comparison of the results through the applied overtone range provides a stronger evidence for the bacterial adhesion mode. The magnitudes of Δf/ΔD, as a function of the overtone number, are presented in Figure 3a,b. In the case of the PEM surface, Δf/ΔD values for bacterial adhesion remain relatively steady at low overtone numbers, but become more negative through the overtone numbers at both initial and mature attachment stages, meaning a more viscous property. This behavior may be attributed to the increasing flexibility of the bacteria−PEM interface with respect to shifting Δf/ΔD to less negative values, which possibly provides an enhanced cell motility function.38 In contrast, Δf/ΔD values for bacterial adhesion on the AUT-modified quartz surface remain relatively steady through the overtone numbers suggesting an elastic, less viscous layer. Next, in comparison of Δf/ΔD values for both surfaces after 3 h of the mature bacteria−surface attachment, Δf/ΔD values for the bacterial adhesion to the quartz surface shifted with more negative value, indicating a more rigid connection. Therefore, the mechanical stiffness of the surface may have a positive effect on the bacterial attachment and 8915


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Figure 5. (a) Cumulative released amounts of DOX drug molecules as a function of time from the PEM microparticles at pH 5.8 and 7.2. The inset shows the fluorescence image of the nanoshell of (PS(PAH/PSS)4PAH)), where DOX is loaded. The scale bar is 2 μm. In vitro cellular uptake of free DOX drugs, DOX-loaded passive PEM microparticles, and DOX-loaded bacteria-driven active microswimmers by 4T1 breast cancer cells. (b) CLSM images of in vitro cellular uptake of DOX drug of 4T1 cells in a microfluidic channel, which were incubated with free DOX, DOX-loaded PEM particles, and bacteria-driven microswimmers for 0.5, 1, and 6 h at the DOX concentration of 0.5 μg/mL and 37 °C. The cellular uptake of free DOX drugs, DOX-loaded PEM particles, and DOX-loaded bacteria-driven microswimmers is shown by the red colored DOX drug inside the cells. The scale bars are 10 μm. The insets show the DOX-loaded passive PEM microparticles, and DOX-loaded bacteria-driven active microswimmers. (c) In vitro cellular uptake of free DOX drugs, DOX-loaded passive PEM microparticles, and DOX8916


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loaded bacteria-driven active PEM microparticles is shown by the mean fluorescence intensity of the red-colored DOX molecules inside the cells for different time periods. (d) pH change at different initial concentrations of the bacteria-driven microswimmers. One-way ANOVA indicated statistically significant differences: (∗) p-value < 0.05; (∗∗) p-value < 0.01.

where α = v−y/v+y is the ratio of the mean speeds in the −y and +y directions; β = t−y/t+y, is the ratio of the time spent moving toward −y and +y directions; and v1̅ D is the 1D mean speed along the y-axis. The 2D trajectory of the microswimmer can be decomposed into segments persistently heading toward +y (heading up) and segments persistently heading toward −y (heading down) based on the y-component of its instantaneous heading direction. Therefore, the motion of the microswimmer along the y-axis is identical to a 1D biased random walk.24 The drift velocities in the absence and presence of the linear chemoattractant gradient were found to be 0.4 μm/s and 2.0 μm/s, respectively. The chemotactic drift velocity (2.0 μm/s) is approximately five times higher than that in the absence of any chemoattractant. This value is higher than 0.66 μm/s, obtained in the previous study.24 Note that this study was conducted with multiple-flagellated S. marcescens attached onto a larger microparticle under L-serine gradient. This result also suggests that the magnitude of the chemotactic drift velocity might be affected by experimental conditions, such as bacteria type, particle diameter, swimmer motility, and type of chemoattractant. As expected, there is negligible difference in the mean speeds of microswimmers with and without chemoattractant gradient cases (13.4 ± 2.9 μm/s in the absence and 13.3 ± 4.4 μm/s in the presence of the chemoattractant gradient).24 To evaluate the steering capability of the bacteria-driven microswimmers subjected to a uniform magnetic field, we used a permanent magnet setup to generate a high enough constant magnetic field on the order of tens of mT (inset, Figure S4a). A permanent magnet-based setup was selected instead of an electromagnetic coils-based setup, because permanent magnets do not generate heat, which is necessary to avoid a temperature-induced influence on the behavior of the biohybrid microswimmers.43 We analyzed the trajectories of the microswimmers (n = 30) that displayed a persistent motion parallel or antiparallel to the direction of the applied field, when subjected to a constant magnetic field of 26 mT. Most of the motion of the microswimmers was shown to be strongly biased along the field direction. An oscillatory motion in 2D trajectories would be indicative of a helical motion in 3D as shown in Figure 4e and Video S4. This reveals that the microswimmer directionality substantially improves with the application of a uniform magnetic field. However, it was found that the 2D mean speed was measured to be 10.4 ± 2.3 μm/s over a 30 s time interval. This mean speed reduction (about 21%) is attributed to the aggregation of the microparticles during the biohybrid microswimmer formation process, resulting from attractive dipolar interactions among the nearby PEM microparticles (PS(MNP1PAH/PSS)4PAH).32 The aggregates such as doublets and triplets were observed (data not shown). Drug Loading to the PEM Microparticles and pHResponsive Drug Release. The loading of DOX anticancer drug molecules inside the multilayered nanoshell was performed under moderate buffered conditions by the incubation of a suspension of the PEM microparticles in a DOX solution. Fluorescence images directly verify the efficient

translational motion in a straight line. In the other extreme, if the bacterium net propulsive force is perpendicular to their magnetic moment, only circular motion with no translation would occur. However, in almost all cases, the bacterium net propulsive force is in between these two extremes and thus, the PEM microparticles translate straight while rotating in a helical path. The mean speeds of the microswimmers varied from 13.3 ± 3.6 μm/s to 22.5 ± 3.3 μm/s, which corresponds to applied propulsive forces ranging from 0.13 pN to 0.21 pN approximated from the Stokes drag force, Fd = 3πμvd, where μ is the dynamic viscosity of the medium, d is the diameter of the microparticle, and v is the instantaneous translational speed of the bacteria-driven microswimmer. Regardless of the size of microswimmers, these mean speeds are compatible with the swimming speed of bare E. coli bacteria and are faster than other bacteria-driven microswimmers reported with similar dimensions,7,14,33 due to the optimized physicochemical properties of the microswimmer design and fabrication. Thus, our design approach could provide higher effective translation in one direction and higher mean speed with an effective pinjoint attachment point (i.e., bacteria can apply propulsion forces at their attachment points freely in different directions, because the bacteria−surface connection is viscous and low strength).34,36 To evaluate the durability of the microswimmers, their mean speed was measured up to 18 h. The microswimmers were shown to maintain their motility for at least 2 to 6 h. The motility was somewhat decreased after 18 h of incubation (Figure 4b). This result is compatible with the previous studies and shows that the microswimmers, fabricated through the bacterial attachment on the PEM microparticles, can possibly be used for longer duration operations.13 Guiding the Bacteria-Driven Microswimmer Motion. We evaluated the chemotactic and magnetic guiding, that is, targeting capability of the bacteria-driven microswimmers using a hydrogel three-channel microfluidic chemoattractant gradient generator25,42 and a permanent magnet-based uniform magnetic field generation setup, respectively. Both guidance methods have been evaluated in the literature and proposed for specific applications. For such quantitative evaluation, we analyzed their movement in 2D using optical microscope images. We first characterized their motility and chemotactic response to the chemoattractant α-methyl-DL-aspartate (0.1 mM). Without the linear gradient of the chemoattractant, the swimming behavior of the microswimmers (sample size, n = 84) was found to be randomly distributed in the test channel, as shown in Figure 4c and Video S2. When the linear gradient was applied, the microswimmers (n = 56) drifted to the higher concentration side of the test channel (Figure 4d and Video S3). The heading bias of the free swimming bacteria is the only factor that contributes to their chemotactic drift, while both the swimming speed bias and the heading bias contributed to the drift velocity in microswimmers.42 The drift velocity of a microswimmer conducting a 1D random walk is defined as24 Vdrift =

1 − αβ v1D 1 + αβ ̅

(2) 8917


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Figure 6. Targeted in vitro drug delivery using the drug-loaded bacteria-driven microswimmers by guiding them toward target specific cancer cells by applying remotely controlled uniform magnetic fields. (a) Free swimming bacteria-driven microswimmers with no directional guidance, that is, no applied magnetic field. (b) Directional guidance of microswimmers toward a target 4T1 breast cancer cell, when a horizontal uniform magnetic field (26 mT) is applied. The scale bars are 20 μm.

loading of fluorescent DOX molecules in the interior of the nanoshell (PS(PAH/PSS)4PAH) with its complete structure intact (the inset image in Figure 5a). The mechanism of DOX loading inside the nanoshell is probably due to the strong adsorption of the negatively charged PSS layers, which interacts with the positively charged DOX molecules, hence leading to their efficient loading. The encapsulation efficiency is 69% and the loading capacity is 2.4%, which is comparable to the previous reports.44 Moreover, the release mechanism for the loaded DOX in the PEM nanoshell is crucial for drug delivery applications. The PEM microparticles were exposed to two different pH media to assess the pH responsiveness. Figure 5a

presents the release profile of DOX molecules from the PEM microparticles at pH 5.8 and 7.2 over a period of 120 h. The data indicate that the released DOX molecules at pH 5.8 (65.3%) are more than that at pH 7.2 (54.9%) after 48 h of incubation. Moreover, a considerable burst release within the first 2 h was found to be 30.9% at pH 5.8 and 20.2% at pH 7.2, respectively. This demonstrates that the release rate is pH dependent and increases with the decrease of pH, which leads to a swelling of PEM nanoshell and thus increases its permeability and the diffusion of DOX molecules. The pHsensitive release of PAH/PSS films is mostly related to the variation in charge density of polyelectrolyte molecules in 8918


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ACS Nano response to the solution pH.45 Amino groups of PAH become charged when the pH decreases, causing an increase in the osmotic pressure. Consequently, the PEM nanoshell swells since water molecules diffuse into it. This phenomenon, together with the electrostatic interaction between DOX molecules and the PAH/PSS multilayers, facilitates the pHsensitive DOX release, which can be significantly enhanced by acidic conditions in the presence of cancer cells or bacteria. Intracellular Release and Cellular Uptake of Drug Molecules. The mechanism of DOX internalization and the intracellular distribution of our bacteria-driven DOX-loaded PEM particle-based microswimmers in 4T1 cancer cells was investigated by a CLSM at different incubation times for 0.5, 1, and 6 h (Figure 5b). The cellular uptake of DOX in 4T1 cancer cells was measured quantitatively by the fluorescence mean intensity, after cells were incubated with free DOX, DOXloaded passive PEM particles, and DOX-loaded active microswimmers at DOX concentration of 0.5 mg/mL at 37 °C (Figure 5c). As expected, the cellular uptake of free DOX, DOX-loaded PEM particles, and DOX-loaded microswimmers increased over time. Interestingly, free DOX rapidly entered into 4T1 cells and accumulated in the nuclei after 0.5 h incubation, whereas DOX-loaded PEM particles and microswimmers exhibited a slower and prolonged DOX release in the nuclei and cytoplasm after 0.5 h incubation, indicating slower cellular uptake. The long-term accumulation of DOX in the cell nuclei may enhance the anticancer efficiency with spatiotemporal-controlled delivery.46 However, the amount of the cellular uptake of DOX with the microswimmers was found to be remarkably larger than that of the passive PEM microparticles. In the case of the microswimmers, the pH drop caused by the presence of bacteria in the microenvironment may explain the enhanced DOX release, compared with the inactive PEM microparticles. To prove our hypothesis, we examined the effect of the initial concentration of the microswimmers on pH change (Figure 5d). It is clearly shown that pH decreases with increasing microswimmer concentration over time. This effect has been observed in a previous study, where the presence of bacteria in a medium causes a rapid decrease in pH.47 It was also reported that the magnitude of the rapid pH drop strongly depends on the concentration of the carbon source (i.e., 5 mM glucose as mean normal blood glucose level), followed by the glucose metabolism of the bacteria generating citric, pyruvic, lactic, and acetic acids.48,49 Another possible explanation for the enhanced DOX release is the active convection of the active PEM particles caused by the bacteria propulsion, compared with the passive PEM particles. In Vitro Targeted Drug Delivery Using the Proposed Biohybrid Microswimmers. The in vitro delivery of DOX drug molecules to 4T1 cancer cells with the bacteria-driven microswimmers is shown in Figure 6. Although our results clearly show both chemotactic and magnetic guidance capabilities of the proposed microswimmers, the magnetic guidance was selected for in vitro targeted drug delivery demonstration because the microswimmers can be delivered to the targeted area much faster from a much remote location with magnetic guidance with compared to the chemotactic one. Two experiments were conducted: a control delivery experiment with no exerted magnetic field and a delivery experiment with a uniform magnetic field strength of 26 mT. The 2D trajectories of the microswimmers display a persistent motion parallel to the direction of the magnetic field. The motion of the microswimmers is strongly biased according to the field

direction. Therefore, the result shows the feasibility of using the developed microswimmers that are guidable with an external magnetic field to perform cancer-cell-targeted drug delivery in vitro. Previous reports have demonstrated microswimmers using a commercial 6-μm diameter superparamagnetic microparticles guided by an electromagnetic coil setup,13 but our proposed customized and controlled magnetic material encapsulation design inside the PEM shells provides magnetic guiding capability in addition to chemotactic guidance for the microswimmers.

CONCLUSIONS In summary, we have developed a systematic design and fabrication methodology for creating PEM microparticlesbased, anticancer drug-loaded, and bacteria-driven microswimmers for targeted drug delivery purposes. This methodology involves the fabrication of PEM drug carriers and the optimization of their physicochemical properties for a favorable bacteria−surface attachment interface. We found that tuning the viscoelastic properties of the bacterial−surface interaction may be the most critical factor for bacterial attachment and the motility of microswimmers. We also showed that the biohybrid microswimmers exhibit biased and directional motion under chemoattractant gradients and magnetic guidance, respectively, to demonstrate the guiding capability of such microswimmers. Moreover, our microswimmer design was able to deliver the cancer drug molecules, encapsulated inside PEM carriers, with high efficiency to target breast cancer cells in vitro. Such in vitro demonstration shows the potential targeted drug delivery capability of the proposed microswimmers toward future in vivo applications. As a future work, we will encapsulate other biomolecules, such as siRNA and enzymes, potentially combined with biosensing platforms,50 inside our microswimmers and examine the long-term cell viability through some analytical methods, such as Live/Dead cell assay, LDH cytotoxicity assay, and a real-time polymerase chain reaction for gene expression markers. Also, the reported promising results demonstrate the potential use of such microswimmers as in vivo next-generation targeted active drug delivery systems in the near future.5 METHODS Preparation of Magnetite (Fe3O4) Nanoparticles. Aqueous dispersions of magnetic nanoparticles (MNPs) were prepared, based on the coprecipitation of ferrous and ferric ion solutions (1:2 molar ratio). Briefly, 20 mL of aqueous 1 M FeCl3 and 5 mL of 2 M FeSO4· 7H2O in 2 M HCl were added to 250 mL of 0.7 M NH4OH (28−30%, Sigma-Aldrich) under rapid mechanical stirring. Stirring was allowed to continue for 1 h, and the dark-brown colloidal particles were allowed to precipitate. The sediment was redispersed in 50 mL of distilled water, and subsequently three aliquots of 10 mL of tetramethylammonium hydroxide solution (10% in water, Sigma-Aldrich) (1 M) were added, again with rapid stirring. Preparation of Polyelectrolyte Multilayers (PEMs) on Microparticles and Surfaces. The polyelectrolyte (PAH/PSS)nPAH on the PS microparticles and the surfaces, where n represents the number of bilayers, were prepared by the layer-by-layer (LbL) technique. Poly(allylamine hydrochloride) (PAH, Mw = 58 000), poly(fluorescein isothiocyanate allylamine hydrochloride) (PAHFITC), and poly(sodium 4-styrenesulfonate) (PSS, Mw = 70 000) were purchased from SigmaAldrich. The polyelectrolyte solutions were prepared at a concentration of 1.0 mg/mL in 150 mM NaCl solution. Before use, polyelectrolyte solutions were filtered through a 0.22 μm filter. First, PEM microparticles were fabricated by alternative deposition of PAH and PSS on anionic 1 μm PS (Sigma-Aldrich) or 1 μm fluorescent PS 8919


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ACS Nano (Thermo Fisher) microparticles at 10 min of incubation time for each PEM layer. The dispersions (0.1 wt %) were then centrifuged, the supernatant was replaced by 150 mM NaCl solution, and the particles were redispersed. This washing procedure was repeated three times before the next PEM layer deposition. For the MNPs deposition, the prepared MNPs were adsorbed from a 0.5 mg/mL suspension, replacing the first PSS layers. For the surface samples, the films were deposited on microscopy cover glasses (14 mm in diameter, Marienfeld GmbH, Germany). Before deposition, the glass slides were cleaned by consecutive incubation in hot solutions (60 °C) of 2% (w/v) Hellmanex III (Hellma GmbH, Germany), 0.01 M sodium dodecyl sulfate, and 0.1 M HCl during 15 min for each solution followed by multiple rinsing with pure water. The film build-up was performed at 25 °C by alternating dipping of the glass slides into PAH and PSS solutions over 10 min with an intermediate washing step with the buffer for 10 min. The PEM samples were stored in 150 mM NaCl at 4 °C and never allowed to dry during the measurements. Bacteria Culturing. Escherichia coli, strain MG1655, cultured on Luria broth (LB) agar plates (Sigma-Aldrich) were transferred to 5 mL of LB broth (Sigma-Aldrich) and allowed to divide overnight at 37 °C and 150 rpm. Five microliters of concentrated MG1655 solution was diluted in 5 mL of fresh LB broth and allowed to culture another 4 h until the measured optical density at 600 nm (OD600) using a BioTek Gen5 Synergy 2 plate reader (Bad Friedrichshall, Germany) was 0.5. An aliquot of 2.0 μL of the liquid culture was transferred to an agar swarm plate (0.5% Eiken Agar, 2.0% LB broth, 0.5% D-(+)-glucose), followed by an incubation of the agar plate at 30 °C for 15 h. After the culturing period, bacteria on the leading edge of the colony was either suspended in motility buffer (0.01 M KH2PO4, 0.067 M NaCl, 10−4 M EDTA, pH = 7.2) or suspended in a PEM solution for the fabrication of the biohybrid microswimmers. Biohybrid Microswimmer Formation. There are two methods used for biohybrid microswimmer formation. First, the bacteria were harvested by centrifugation (1500g for 5 min) and washed three times with 1× PBS (phosphate buffered saline, pH = 7.2). A 10 μL bacterial solution was mixed with 10 μL of PEM microparticle solution (0.1 wt %, 1 mL) and incubated for 10 min at room temperature, allowing for initial attachment of the bacteria to the microparticles. The second approach is performed in an agar swarm plate. The microswimmers were assembled by placing an aliquot of 5 μL PEM microparticle solution onto the leading edge of the swarm plate and gently pipetting 3−5 times to incubate the bacteria with the PEM microparticles sufficiently. The solution was collected back immediately and incubated at room temperature for 10 min. Then, the solution was diluted by adding 190 μL of motility buffer to the solution. The final solution was further diluted to achieve an appropriate concentration for vision tracking of the microswimmers. Atomic Force Microscope Characterization. A biological AFM system (NanoWizard 4, JPK Instruments, Berlin, Germany) was used to characterize the PEM surface roughness, thickness, and Young’s modulus. The AFM head was mounted on a Zeiss Axio Observer A1 inverted biological optical microscope. All experiments were performed in a liquid environment of 150 mM NaCl aqueous solution. At the end of the PEM build-up experiment, the crystal containing the PEM surface was carefully collected and placed in a wet chamber for AFM measurements. Roughness and thickness images were collected in intermittent (tapping) mode with HQ:NSC18/CRAuBS cantilevers (Mikromash, Estonia) with a resonance frequency of 33 kHz. The root-mean-square roughness (Rq) was determined from 1.5 μm × 1.5 μm squares using JPK image processing software. The PEM surface was indented with a razor blade and topographic images were recorded. The thickness of the film was taken as the difference between the height of the PEM surface and the bottom surface of the crystal. The mechanical properties of the (PAH/PSS)20PAH surface were characterized by the nanoindentation method. A spherical colloidal tip was prepared by gluing a 6-μm diameter SiO 2 microparticle (Microparticles GmbH, Germany) to the end of a tipless rectangular silicon nitride AFM cantilever (HQ:CSC38) using epoxy. The effective spring constant of each cantilever was calibrated using the thermal noise fluctuations method. Calibrated spring

constants for the colloidal tip cantilever used in experiments was 0.079 to 0.083 N/m. The recorded force−distance curves were analyzed using a custom MATLAB procedure, and the Hertz model was used to calculate the viscoelastic properties of the PEM surfaces. Young’s modulus was then calculated from51 F(δ) =

4 R E δ 3/2 3 1 − ν2

(3)

where F is the measured force, E is the local Young’s modulus, R is the cantilever’s tip radius (for a spherical tip), ν is the Poisson’s ratio of the sample (assumed as 0.5 for an incompressible material), and δ is the sample indentation. The statistical analysis was done using the software OriginPro 2016 (OriginLab, Northampton, USA). Scanning Electron Microscope Imaging. Bacteria-driven microswimmers were imaged with a Zeiss Ultra 55 Gemini scanning electron microscope (Carl Zeiss Inc., Oberkochen, Germany) using an accelerating voltage of 5 keV and an in-lens detector. To prepare samples for SEM, briefly, biohybrid microswimmers suspended in PBS were placed on membrane filters (Sartorius, Germany) for 5 min. Filters were incubated in 2.5% glutaraldehyde in 0.2 M Cacodylate buffer (pH 7.2) overnight at 4 °C, and then rinsed with 0.2 M Cacodylate buffer. Postfix was conducted with cells in 1% osmium tetroxide in 0.2 M Cacodylate buffer for 30 min. Biohybrid samples were dehydrated in a series of increasing aqueous ethanol concentrations for 10 min in each solution. The samples were further dehydrated and preserved using a series of increasing hexamethyldisilazane concentrations in ethanol.52 The samples were coated with 10 nm of gold using a Leica EM ACE600 sputer coater (Leica Microsystems, Wetzlar, Germany). Transmission Electron Microscope Imaging. An ARM200F microscope (JEOL, Japan) operating at 80 kV was used to obtain TEM images of thin-sectioned slices (100 nm) of biohybrid microswimmer samples. After a dehydration process, the samples were embedded in Epofix (Struers GmbH, Germany), then the thin sections were cut with a diamond knife 35° using a Leica EM UC6/ FC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and mounted on a 200-mesh Lacey-Carbon grid (Plano GmbH, Germany). Quartz Crystal Microbalance for Dissipation Characterization. The PEM build-up, structure, and properties were characterized by QCM-D. The QCM-D measurements were performed with AT-cut quartz crystals mounted in an E4 system (Q-sense AB, Gothenburg, Sweden). All QCM-D experiments were performed under flow-through conditions at 100 μL/min using a digital peristaltic pump (IsmaTec Peristaltic Pump, IDEX). All quartz crystals (QSX-303, Q-sense AB, Sweden) were subsequently cleaned by ultrasonication in acetone, ethanol, and DI water, followed by immersion in a 5:1:1 H2O/NH3/H2O2 solution at 75 °C for 10 min. For polyelectrolyte multilayer formation, the crystals were modified with mercaptopropionic acid (MPA, Sigma-Aldrich) self-assembled monolayer, introducing negatively charged carboxyl groups, which are able to interact with the first PAH layer. For a positively charge surface, the quartz crystals were modified with 11-amino-1undecanethiol (AUT, Sigma-Aldrich). The quartz crystals were incubated in an absolute ethanol solution of 1 mM MPA and AUT for 12 h to obtain a surface modified with −COOH and −NH2 of MPA and AUT, respectively, followed by rinsing with ethanol and DI water and drying with nitrogen.53 To investigate the bacteria−surface interactions with the soft PEM ((PAH/PSS)4PAH) and hard AUT-modified quartz surfaces with respect to mechanical stiffness, bacterial cultures were grown at 150 rpm and 30 °C until the measured optical density at 600 nm was about 0.3. The bacteria were harvested by centrifugation (1500g for 5 min) and washed three times with PBS. The bacterial suspension (2 × 107 cells/mL) in PBS was injected into the sensor chambers at 25 °C with a flow rate of 100 μL/min until the resonant frequencies of the sensors became stable. At the end of the experiment, the bacteria adhered on crystals were stained by SYTO 9 and propidium iodide and bacterial numbers were counted with a Zeiss fluorescent microscope. All 8920


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to 86 microswimmers with the errors representing as the standard deviation. The MSD was calculated from

measurements were performed in duplicate. The obtained data are evaluated by using the Voight model and the Sauerbrey relation54 Δm = − CQCM

Δfn n

MSD(t ) = ⟨r 2(t )⟩ = ⟨∥( r (⃗ t0 + t ) − r (⃗ t0))∥2 ⟩t0

(4)

(5)

where r ⃗ is a two-dimensional position vector and MSD(t) is averaged by running t0 from 0 to tend − t. Drug Loading and pH-Responsive Drug Delivery. To assess the potential of the fabricated PS(PAH/PSS)4PAH microparticles as drug delivery carriers, their drug-loading capacity was investigated using an anticancer drug, Doxorubicin (DOX, Sigma-Aldrich). The loading of DOX inside the PEM nanoshell layers was performed under ambient conditions in closed containers by the mixture of PS(PAH/ PSS)4PAH microparticles and a solution of DOX. A 0.1 g of PS(PAH/ PSS)4PAH microparticles was added to 1 mL of DOX solution (0.1 mg/mL) at 25 °C and then the solution was stirred for 12 h under dark conditions. The DOX-loaded microparticles were centrifuged at 10 000g for 5 min to remove unloaded DOX. The supernatant and the washing solutions were collected together to determine the DOX loading using a BioTek Gen5 Synergy 2 plate reader (Bad Friedrichshall, Germany) at a wavelength of 490 nm. For the in vitro drug release test, the DOX-loaded microparticles (5 mg) were suspended in 1 mL of release medium at different pH values. The pH of all conditions was determined using a pH meter (inoLab pH 7110, WTW, Germany). Samples with a volume of 0.5 mL were periodically withdrawn for analysis and replaced by the equal volume of fresh medium. The stirring rate and temperature of release medium were adjusted to 300 rpm at 37 °C. The loading and release of DOX in the shells was quantified by calculating the difference between the loaded amount and the released amount from the supernatant liquids, combined with a standard curve of DOX solutions ranging in concentrations from 10 to 160 μg/mL. For comparison, the loading content (LC) and encapsulation efficiency (EE) of DOX in the PEM microparticles were calculated by44

where Δm is the adsorbed mass on the surface, CQCM is the mass sensitivity constant (17.7 ng cm−2 Hz−1 at f = 5 MHz), and Δf n is the change in the resonance frequency at the nth harmonic. Since the Sauerbrey relationship is only valid for acoustically rigid films with low dissipation, it is important to use the Voight model as a viscoelastic model55 to quantify the changes in mass and effective viscoelastic properties of the PEM surface. Vibrating Sample Magnetometer (VSM) Characterization. The magnetic properties of the PEM-MNP microparticles were characterized by a model 10 Vibrating Sample Magnetometer (MicroSense, USA). Field-dependence hysteresis loops of PEMMNP particles were obtained. Fabrication of Microfluidic Chemoattractant Gradient Generator. An agarose hydrogel three-channel microfluidic chemoattractant gradient generator was applied to characterize the chemotaxis behavior of E. coli MG1655 over a quasi-steady-state chemical gradients.56 The microfluidic gradient generator was prepared by assembling a molded agarose hydrogel containing the three channels, which were typically 100 μm tall and 500 μm wide, as shown in Figure S3a. The channel hydrogel chips were fabricated using standard techniques of soft lithography. The chips were molded by placing 4% agar solution (VWR Chemicals, USA) onto the silicon master mold, where the channel patterns were enclosed by a polydimethylsiloxane (PDMS) spacer. To complete the assembly of the gradient generator, the agarose hydrogel was sandwiched between two acrylic panels. The gradient profile in the middle channel was probed using a 0.1 mM fluorescein (Sigma-Aldrich), and a linear gradient in the device was calibrated. The fluorescein solution was applied to the source channel, and the fluorescence intensity profiles of the middle channel were monitored using a Zeiss Axio Observer A1 inverted microscope with a N-Achroplan 20× objective lens and Axiocam 503 CCD camera every 10 s. The images were analyzed using Zeiss ZEN software. The quasi-steady concentration gradient was achieved in 20 min with a continuous flow of the fluorescein (diffusion coefficient, D = 4.25 × 10−6 cm2/s, at 25 °C) and the PBS in the source/sink channels sustained by a Legato 210 syringe pump (KD Scientific, USA) at 5 μL/min. The established linear chemical concentration gradient in the center channel over the width of the test channel is demonstrated in Figure S3b. Magnetic Guidance Setup. We designed a one-directional magnetic guidance setup consisting of two disc-shaped permanent magnets separated by a distance of 10 cm, axially magnetized, and 60 mm in diameter (NdFeB, N42, Webcraft GmbH, Germany), built onto a custom-made microscope stage surrounding a microfluidic chamber, which was placed onto a Zeiss Axio Observer A1 inverted microscope. This setup is suitable for generating a uniform field in the region of interest (ROI), so that the microswimmers can be oriented along one direction. The magnetic field surrounding two permanent magnets at the distance of 10 cm in the x-axis were modeled using COMSOL Multiphysics 5.2a (COMSOL, Sweden). The simulated magnetic field at the center of the ROI and the field intensity are displayed in Figure S4. Magnetically guided microswimmers were exposed to a uniform magnetic field at 26 mT. The magnetic field in the ROI was measured with a Lake Shore Cryotronics (Darmstadt, Germany) model 460 three-channel Gaussmeter. Microswimmer Tracking and Motility Analysis. Tracking of bacteria-driven microswimmers was performed by both a Zeiss Axio A1 inverted microscope and a T1000 transmission digital holography microscope (DHM, Lyncee Tec, Lausanne, Switzerland) with a 40× objective lens. All motility characterization parameters including mean speed, velocity, and mean square displacement (MSD) were obtained by computational analysis of recorded video using an in-house tracking program developed in MATLAB R2015a (Mathworks, Natick, USA). The details of this algorithm are described in our previous publications.42,57−60 The mean speed calculated is an average of 30

loading content (%) mass of DOX in PEM microparticles = × 100 mass of DOX‐loaded PEM microparticles (6)

encapsulation efficiency (%) =

mass of loaded DOX × 100 initial mass of DOX (7)

In vitro Intracellular Uptake of Free DOX, DOX-Loaded Passive PEM Microparticles, and DOX-Loaded Active Microswimmers. A mouse mammary carcinoma cell line 4T1 (CRL-2539) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). This tumor cell line is a model for stage IV human breast cancer in growth patterns. 4T1 cells were cultured in RPMI-1640 (Gibco, Life technologies, USA) medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. All cultures were incubated at 37 °C at 100% humidity and 5% CO2. The DOX concentration in 4T1 cells was examined by detecting the fluorescence of the intercellular DOX. 4T1 cells were treated with free DOX, DOX-loaded PEM, and biohybrid microswimmers at DOX concentration of 0.5 μg/mL. To examine the intercellular uptake of DOX, 4T1 cells were seeded in collagen-coated 8-well imaging chambers (zell-kontakt, Nörten-Hardenberg, Germany) at a density of 1 × 104 cells/well and incubated for 24 h at 37 °C. After that, the culture medium was removed and the cells were incubated with free DOX, DOX-loaded PEM, and biohybrid microswimmers at DOX concentration of 0.5 μg/mL at 37 °C for specific time points. After washing twice in PBS, cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature and observed by confocal laser scanning microscope (CLSM, Nikon Eclipse Ti confocal microscope with Yokogawa CSU-W1 spinning disk) using a Plan Fluor 40× objective lens. The images were analyzed using ImageJ to calculate the fluorescence intensities. 8921


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ACS Nano Statistical Analysis. The reported values obtained from the AFM image, QCM-D, and 2D motility analyses were the averages of replicate experiments. Young’s modulus of (PAH/PSS)20PAH film was the average value obtained from the AFM nanoindentation tests using a Gaussian fit (n = 337). The 3D motility tests over time (n = 10), DOX cumulative release (n = 3), in vitro cellular uptake (n = 5), and pH change at initial concentration (n = 3) data were compared using one-way ANOVA followed by post hoc Tukey’s multiple comparison test. The statistical analysis was performed using the software OriginPro 2016 (OriginLab, Northampton, USA). The differences were considered significant for p value < 0.05, and very significant for p value < 0.01.

REFERENCES (1) Carlsen, R. W.; Sitti, M. Bio-Hybrid Cell-Based Actuators for Microsystems. Small 2014, 10, 3831−3851. (2) Elgeti, J.; Winkler, R. G.; Gompper, G. Physics of MicroswimmersSingle Particle Motion and Collective Behavior: A Review. Rep. Prog. Phys. 2015, 78, 056601. (3) Sánchez, S.; Soler, L.; Katuri, J. Chemically Powered Micro- and Nanomotors. Angew. Chem., Int. Ed. 2015, 54, 1414−1444. (4) Darnton, N.; Turner, L.; Breuer, K.; Berg, H. C. Moving Fluid with Bacterial Carpets. Biophys. J. 2004, 86, 1863−1870. (5) Hosseinidoust, Z.; Mostaghaci, B.; Yasa, O.; Park, B.-W.; Singh, A. V.; Sitti, M. Bioengineered and Biohybrid Bacteria-Based Systems for Drug Delivery. Adv. Drug Delivery Rev. 2016, 106, 27−44. (6) Akin, D.; Sturgis, J.; Ragheb, K.; Sherman, D.; Burkholder, K.; Robinson, J. P.; Bhunia, A. K.; Mohammed, S.; Bashir, R. BacteriaMediated Delivery of Nanoparticles and Cargo into Cells. Nat. Nanotechnol. 2007, 2, 441−449. (7) Behkam, B.; Sitti, M. Bacterial Flagella-Based Propulsion and On/Off Motion Control of Microscale Objects. Appl. Phys. Lett. 2007, 90, 23902. (8) Nguyen, V. D.; Han, J.-W.; Choi, Y. J.; Cho, S.; Zheng, S.; Ko, S. Y.; Park, J.-O.; Park, S. Active Tumor-Therapeutic Liposomal Bacteriobot Combining a Drug (Paclitaxel)-Encapsulated Liposome with Targeting Bacteria (Salmonella Typhimurium). Sens. Actuators, B 2016, 224, 217−224. (9) Felfoul, O.; Mohammadi, M.; Taherkhani, S.; de Lanauze, D.; Zhong Xu, Y.; Loghin, D.; Essa, S.; Jancik, S.; Houle, D.; Lafleur, M.; Gaboury, L.; Tabrizian, M.; Kaou, N.; Atkin, M.; Vuong, T.; Batist, G.; Beauchemin, N.; Radzioch, D.; Martel, S. Magneto-Aerotactic Bacteria Deliver Drug-Containing Nanoliposomes to Tumour Hypoxic Regions. Nat. Nanotechnol. 2016, 11, 941−947. (10) Li, D.; Choi, H.; Cho, S.; Jeong, S.; Jin, Z.; Lee, C.; Ko, S. Y.; Park, J. O.; Park, S. A Hybrid Actuated Microrobot using an Electromagnetic Field and Flagellated Bacteria for Tumor-Targeting Therapy. Biotechnol. Bioeng. 2015, 112, 1623−1631. (11) Uthaman, S.; Zheng, S.; Han, J.; Choi, Y. J.; Cho, S.; Nguyen, V. D.; Park, J. O.; Park, S. H.; Min, J. J.; Park, S.; Park, I. K. Preparation of Engineered Salmonella Typhimurium-Driven Hyaluronic-Acid-Based Microbeads with Both Chemotactic and Biological Targeting Towards Breast Cancer Cells for Enhanced Anticancer Therapy. Adv. Healthcare Mater. 2016, 5, 288−295. (12) Fernandes, R.; Zuniga, M.; Sassine, F. R.; Karakoy, M.; Gracias, D. H. Enabling Cargo-Carrying Bacteria via Surface Attachment and Triggered Release. Small 2011, 7, 588−592. (13) Carlsen, R. W.; Edwards, M. R.; Zhuang, J.; Pacoret, C.; Sitti, M. Magnetic Steering Control of Multi-Cellular Bio-Hybrid Microswimmers. Lab Chip 2014, 14, 3850−3859. (14) Park, S. J.; Park, S.-H.; Cho, S.; Kim, D.-M.; Lee, Y.; Ko, S. Y.; Hong, Y.; Choy, H. E.; Min, J.-J.; Park, J.-O.; Park, S. New Paradigm for Tumor Theranostic Methodology using Bacteria-Based Microrobot. Sci. Rep. 2013, 3, 3394. (15) Zhuang, J.; Park, B.-W.; Sitti, M. Propulsion and Chemotaxis in Bacteria-Driven Microswimmers. Adv. Sci. 2017, 1700109. (16) Sahari, A.; Headen, D.; Behkam, B. Effect of Body Shape on the Motile Behavior of Bacteria-Powered Swimming Microrobots (BacteriaBots). Biomed. Microdevices 2012, 14, 999−1007. (17) Bos, R.; van der Mei, H. C.; Busscher, H. J. Physico-Chemistry of Initial Microbial Adhesive Interactions-its Mechanisms and Methods for Study. FEMS Microbiol. Rev. 1999, 23, 179−230. (18) Lichter, J. A.; Thompson, M. T.; Delgadillo, M.; Nishikawa, T.; Rubner, M. F.; Van Vliet, K. J. Substrata Mechanical Stiffness Can Regulate Adhesion of Viable Bacteria. Biomacromolecules 2008, 9, 1571−1578. (19) Saha, N.; Monge, C.; Dulong, V.; Picart, C.; Glinel, K. Influence of Polyelectrolyte Film Stiffness on Bacterial Growth. Biomacromolecules 2013, 14, 520−528. (20) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277, 1232−1237.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03207. Figures S1−S4 and Table S1; section on calculation of the average volume of the MNP1 layer (PDF) Movie S1: 3D motility analysis of the bacteria-driven microswimmers using a digital holographic microscope (40× magnification) (AVI) Movie S2: 2D motility analysis: swimming bacteriadriven microswimmers with no chemoattractant gradient (20× magnification) (AVI) Movie S3: 2D motility analysis: swimming bacteriadriven microswimmers under a linear chemoattractant gradient (20× magnification) (AVI) Movie S4: 2D motility analysis: swimming bacteriadriven microswimmers under a uniform (26 mT) magnetic field (20× magnification) (AVI) Movie S5: In vitro targeted active drug delivery demonstration of the bacteria-driven microswimmers on cultured breast cancer cells (40× magnification) (AVI) Movie S6: In vitro targeted active drug delivery demonstration of the bacteria-driven microswimmers on cultured breast cancer cells under a uniform magnetic field (40× magnification) (AVI)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Oncay Yasa: 0000-0002-0282-6127 Metin Sitti: 0000-0001-8249-3854 Author Contributions

B.P. and M.S. conceived the ideas and designed the study. B.P., J.Z., and O.Y. performed the fabrication, experiments, and analyzed the data. All authors wrote the manuscript together. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors thank Prof. Peter van Aken and Prof. Joachim Spatz for their instrument support, Dr. Hunter Gilbert and Dr. Lindsey Hines for their thorough review of the manuscript, and Joshua Giltinan and Donghoon Son for their help in magnetization measurements and calculations. This work is funded by the Max Planck Society. 8922


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ACS Nano

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