Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active

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Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active Cargo Delivery Ajay Vikram Singh, Zeinab Hosseinidoust, Byung-Wook Park, Oncay Yasa, and Metin Sitti ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b02082 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active Cargo Delivery Ajay Vikram Singh, Zeinab Hosseinidoust, Byung-Wook Park, Oncay Yasa, and Metin Sitti* Physical Intelligence Department, Max Planck Institute for Intelligent Systems, Stuttgart 70569, Germany *Corresponding author: [email protected] Abstract Biohybrid cell-driven microsystems offer unparalleled possibilities for realization of soft microrobots at the micron scale. Here, we introduce a bacteria-driven microswimmer that combines the active locomotion and sensing capabilities of bacteria with the desirable encapsulation and viscoelastic properties of a soft double-micelle microemulsion for active transport and delivery of cargo (e.g., imaging agents, genes, and drugs) to living cells. Quasimonodisperse double emulsions were synthesized with an aqueous core that encapsulated the fluorescence imaging agents, as a proof-of-concept cargo in this study, and an outer oil shell that was functionalized by streptavidin for specific and stable attachment of biotin-conjugated Escherichia coli. Motile bacteria effectively propelled the soft microswimmers across a Transwell membrane, actively delivering imaging agents (i.e., dyes) encapsulated inside of the micelles to a monolayer of cultured MCF7 breast cancer and J744.A1 macrophage cells, which enabled real-time, live-cell imaging of cell organelles, namely mitochondria, endoplasmic reticulum, and Golgi body. This in vitro model demonstrates the proof-of-concept feasibility of the proposed soft microswimmers and offers promise for potential biomedical applications in active and/or targeted transport and delivery of imaging agents, drugs, stem cells, siRNA, and therapeutic genes to live tissue in in vitro disease models (e.g., organ-on-a-chip devices) and stagnant or low flow-velocity fluidic regions of the human body. KEYWORDS: microswimmers, targeted drug delivery, double emulsions, golgi tracker, soft robots

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Miniaturized soft robots have significant potential to revolutionize biomedical technology and improve the quality of human life.1-4 Despite notable advances, the realization of micron-scale soft robots is hindered by the extreme difficulty of designing functional autonomous systems at such small scales.5-8 Biological cells, however, function autonomously and efficiently at the micro/nanoscale and thus integrating them into synthetic constructs allows for the exploitation of their complex functionalities such as active locomotion and sensing for designing multifunctional autonomous microsystems. Various biological entities such as muscle cells,9 algae,10 and bacteria11,12 have been integrated with synthetic materials to design functional biohybrid microsystems.13 Bacteria are of particular interest for the development of biohybrid microsystems, such as microswimmers,14,15 microcrawlers,14 and micropumps,16 because they can be genetically engineered relatively easier than eukaryotic cells, because of their robust propulsion and sensing capability in diverse physiological environments, and their ability to harness the biochemical energy source, i.e., ATP, inside their cell membrane to propel themselves.17 The random trajectory of bacterial swimming can be guided by environmental gradients of nutrients, oxygen, or pH; a property that has enabled tumor-targeting bacteria to accumulate in and subsequently destroy tumors by using the naturally occurring gradients inside the human body.18,19 Passing the proof-of-concept stage toward realistic biomedical applications, however, requires the design of functional synthetic materials that can work in concert with the biological cells effectively and efficiently to perform the desired functions. For applications in gene and drug delivery and biomedical imaging, microemulsions have been proven to provide many significant advantages.20,21 Their soft and viscoelastic properties allow better interaction with tissue cell membranes,22 they can encapsulate a wide range of hydrophilic and hydrophobic cargo and protect their payload against harsh environments,23 and they allow for the free diffusion of the cargo at oil-water interface into the tissue cells.24,25 Compared with nanoemulsions, microemulsions can carry 3 to 6 orders of magnitude higher amount of cargo load, and they are mechanically softer; this means they can significantly deform and squeeze through micropores, conform well to the microscale topography of the cell surface when they come in to contact, and burst after contact, which enables efficient delivery of the given cargo to the cells through diffusion. Moreover, microemulsions are neutrally buoyant, biocompatible, and biodegradable,26 which are essential properties for biomedical applications. Colloidal drug carriers based on oil-in-water (o/w), water-in-oil (w/o), and water-in-oil-in-water 2 ACS Paragon Plus Environment

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(w/o/w) double microemulsions are well established.27,28 Single-emulsion systems, such as liposome-based drug delivery vehicles, have a lower cargo capacity, lower stability, and lower monodispersity.29,30 Contrary to this, stable w/o/w double emulsions have been developed as a biologically relevant alternative to w/o single emulsion for the encapsulation of hydrophilic cargo. They can carry hydrophilic cargos in their aqueous core while their external oil shell allows for a stable dispersion in aqueous media and enables a better interaction with the tissue cell membranes.31 Additionally, they can be mass-produced in various sizes in a cost-effective manner and with a reasonably narrow size distribution.32 What makes the double microemulsions particularly useful for the developing biohybrid soft microrobots is the fact that the oil-water interface at the outer shell can be decorated with specific molecules to facilitate stable conjugation of biological cells to the emulsions.33 Despite these advantages, there is no prior report of using double emulsion systems driven by motile bacteria to deliver cargo to live cells. Microswimmers (micron-scale swimming robots) have many promising biomedical applications,5,34,35 such as active and/or targeted transport and delivery cargo, e.g., drugs,19 imaging agents, genes,36 stem cells, and RNA. We report cargo delivery by a biohybrid microswimmer composed of double emulsions driven by bacteria that are steered by an external concentration gradient towards the cells of interest. The developed soft biohybrid microswimmers are composed of w/o/w microemulsions conjugated to motile Escherichia coli (E. coli) bacterial cells. We synthesized quasi-monodisperse, biocompatible, and biodegradable double emulsions and loaded their aqueous cores with various organelle-labeling dyes, as the proof-of-concept cargo demonstration in this study. Next, we decorated their outer oil shells with streptavidin for specific and strong attachment of biotin-conjugated E. coli bacteria for efficient fluidic propulsion. The biohybrid microswimmers swam across a barrier of microporous membrane and efficiently delivered organelle-tracking dyes to the living cells, which enabled real-time live-cell imaging of internal cell organelles. In the following text, the phrase microswimmer refers to bacteria plus microemulsions. RESULTS AND DISCUSSION Synthesis of double emulsions for biohybrid microrobots In this work, we developed versatile double emulsions in terms of stability, quasi monodispersity, and surface functionalization with biologically relevant adhesive molecules to 3 ACS Paragon Plus Environment

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realize biohybrid microrobots. Double emulsions were prepared using water and soybean oil. The preparation steps are presented in Figure 1A. Vegetable oil was chosen over the more popular silicone oil because of its biocompatibility.37,38 Most double emulsions reported in the literature have been reported based on silicone oil, therefore main design parameters such as water-to-oil ratio, agitation method and duration, surfactant combinations, and surfactant ratios had to be optimized for our new design. Based on the optimized parameters, presented in Tables S2-S5, A combination of sonication and vortex mixing was used to prepare the double microemulsions. Treatment intensity and duration were optimized for each layer, namely the internal w/o phase and the final w/o/w emulsion to maximize stability and the monodispersity of the microemulsions. We fine-tuned the treatment intensity (Table S2), duration of treatment (Table S3), and the loading efficiency of the model cargo, rhodamine B (Table S4). Sonication intensity of 25 W was chosen for the preparation of the internal core of the double microemulsions because it led to w/o emulsions that were more stable through the second round of mechanical treatment (Table S2). For the second treatment step, vortex mixing was the method of choice; the optimal duration of vortex mixing was chosen to be one-third of the initial sonication treatment to avoid the distortion of the w/o core (Table S3). This preparation method was used to prepare double emulsions loaded with rhodamine B as the cargo to deliver into the tissue cells. As demonstrated in Table S4, a treatment involving a ratio of vortexing time to sonication time equal to one-third led to a higher encapsulation efficiency of rhodamine B (25%), suggesting that using a shorter vortexing duration likely limited the disruption of the w/o core during the preparation of the double emulsion and results in better retention of the cargo. The standard curve in Figure S1 was used to calculate the encapsulation efficiency of rhodamine B uptake by double emulsions, based on the measured fluorescence intensities. The concept of hydrophilic-lipophilic-balance (HLB) was used to choose appropriate surfactants. All surfactants used in optimization experiments are listed in Table S5 along with their corresponding HLB values. A high HLB index indicates a higher solubility in water whereas a low HLB index indicates a nonpolar and/or oil soluble surfactant.39 Various surfactant combinations were investigated and a range of surfactant ratios was tested for preparation of the stable double emulsions. The combined HLB index of the microemulsions was calculated as the weight average of the HLB index of the two surfactants. The chosen surfactant pair had a combined optimum HLB index equally distanced from the HLB index of its constituents.40 This 4 ACS Paragon Plus Environment

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result is presented in Table 1 for the chosen surfactants: Cetyl trimethylammonium bromide (CTAB) and Merpol® A. The lowest possible CTAB concentration in the outer shell was implemented because of the anticipation that this surfactant might negatively affect the conformation of streptavidin integrated into the outer shell (to increase bacterial attachment) or inversely affect viability of bacteria and/or cells that come into contact with the emulsions at later stages. Finally, the optimal volumetric ratio of the outer aqueous phase to the inner w/o emulsion was found to be 4:1 (Table 1). For the w/o core, an oil-to-water ratio of 2:1 was chosen based on the recommendations in the literature.41 The chosen optimal preparation conditions led to a positive zeta potential that helped to create also a slight electrostatic attraction between the negatively charged bacterial cells and the positively charged emulsions. Further, the mutual repulsion between emulsion shells prevented coalescence and stabilized the microemulsions. To further increase the efficiency of bacterial attachment to the micelles, streptavidin was added to the outer oil shell during emulsion preparation (Figure 1B). We did not observe a significant change in zeta potential after the functionalization of the outer shell with streptavidin (+1.5 ± 0.8 versus +1.1 ± /0.1; p-value = 0.08). For all subsequent experiments, the microemulsions were prepared based on the optimum conditions identified at this stage. The microemulsions, prepared using the identified optimal conditions exhibited a mean oil shell thickness of 500 ± 31 nm determined using fluorescence microscopy images of Nile redinfused oil shells (Figure 2A). Figure 2B shows the hydrodynamic diameters of the synthesized microemulsions determined using dynamic light scattering (DLS), ranging between 3 to 10 µm with an average polydispersity index of 0.3. The diameter of the double emulsions was also quantified using ImageJ, after image segmentation (clustering) of the videos obtained from fluorescent light microscopy (Figure S2). The hydrodynamic diameter was determined indirectly based on the Brownian motion and thus a complete agreement between data presented in Figure 2B and Figure S2 is not expected. Various batches of double emulsions were prepared, encapsulating Mito-, Golgi-, and ER- tracker dyes with entrapment efficiencies of 21%, 30%, and 41%, respectively. The standard curves in Figures S3-S5 were used to calculate the encapsulation efficiency of rhodamine B uptake, based on the measured fluorescence intensities. Integration of bacterial cells with microemulsions 5 ACS Paragon Plus Environment

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The outer oil shell of the double emulsions was functionalized with streptavidin to allow for specific attachment of chemically biotinylated E. coli MG1655 with their cell membrane, not with their flagella, the appendage used for bacterial swimming function. For this purpose, the cargo-loaded w/o emulsions were dispersed into aqueous medium containing different concentrations of streptavidin and 0.1% CTAB. Figure 2C shows the total streptavidin adsorbed to the oil shell as a function of the concentration of streptavidin in the medium. Nearly half of the protein was adsorbed to the microemulsion surface at 0.5 weight percent (wt%) streptavidin concentration. Presence of the high HLB surfactant CTAB is expected to be one of the limiting factors affecting protein adsorption by creating spatial hindrance in the oil phase.42 Protein adsorption at oil/water interface is a complex process and therefore it is difficult to identify which characteristics associated with streptavidin are important for the interfacial adsorption at oil shell. The triglyceride polarity and high viscosity of oil phase could additionally relate the inverse adsorption of protein to the protein concentration.27 The w/o/w double emulsions were subsequently washed and diluted into motility media containing biotinylated E. coli MG1655 (OD600 = 0.5) (Figure 1B). The bacteria were labeled with SYTO9 for facile imaging and tracking. Once attached, the bacteria-emulsion construct was imaged using fluorescence microscopy to confirm the attachment of bacteria to the microemulsions (Figures 2D and 2E). At this stage, the microemulsions were separated into fractions using membranes with various pore sizes and only the fraction with a size range of 5 to 7 µm were used for active cargo delivery experiments (Figure 1C). The microemulsions in this size range bound to 1-2 bacterial cells, as shown in Figures 2D and 2E. The size fractionation allowed for more uniform swimming patterns and thus a more controlled investigation in the developmental stage of the system. Propulsion performance of bacteria-driven emulsion-based microswimmers The swimming trajectories of bacteria-propelled microemulsions (microswimmers) are shown in Figures 2F and 2G. Microemulsions without bacteria exhibited an average speed of 0.5 ± 0.2 µm/s, which can be attributed to the Brownian diffusion.43 Microemulsions attached to a single bacterium swam with an average speed of 6.5 ± 0.8 µm/s in the motility media (Figure 2F and Supplementary Movie S1), whereas microemulsions attached to two bacteria swam at 4.5 ± 0.2 µm/s (Figure 2G and Supplementary Movie S1). The reduction of average speed could be attributed to the cancelling of propulsion force vectors by multiple bacteria attaching randomly 6 ACS Paragon Plus Environment

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around the micelle. In case of one bacterium attached to the emulsion surface, the microswimmer exhibits a random zigzag movement due to the rotation of the particle and the stochastic “run” and “tumble” swimming mode of the attached E. coli, as observed in our previous studies.44,45 Microswimmers with two attached bacteria showed a smoother swimming trajectory. Further details of the mean speed and effective diffusion of the microswimmers and their associated parameters are given in Table 2. The effective diffusion constant of bacteria-driven and passive microemulsions in Table 2 is one of the essential parameters that determine their cargo delivery efficiency. If the carriers diffuse fast, then they could reach to and contact tissue cells in their vicinity faster in high numbers and deliver the cargo in high amounts after the contact. Thus, because the effective diffusion constant of bacteria-driven emulsions is 3 to 4 orders of magnitude higher than the passive emulsions, their cargo delivery efficiency would be significantly higher than the passive emulsions, widely used in the literature for cargo delivery. Therefore, the effective diffusion constant of the microswimmers should be maximized as much as possible for the optimal cargo delivery by selecting the type of bacteria with high mobility and optimizing the emulsion size and the number and attachment locations of multiple bacteria.45 To assess the stability of bacteria attachment on the microemulsions under the prevailing physicochemical conditions, a fraction of double emulsions with a larger hydrodynamic diameter of 10 µm and 4- to 5-attached bacterial cells per microemulsion were monitored for 4 hours. The results for microemulsions with or without streptavidin indicate that streptavidin increased the stability of bacterial attachment to the microemulsions but no significant decrease in detachment was observed in either case (Table S6). However, when we compared the average number of E. coli attached at each time points in absence and presence of streptavidin with increasing time from 0 to 4 hours using student t-test, we see a significant difference (t-test = 0.03; p-value ≤ 0.05). This indicates stable biohybrid bonding over time. In vitro delivery of imaging contrast agents to cells by the soft microswimmers We created a concentration gradient of glucose across a transwell membrane, where the bacteriadriven microswimmers then swam up the glucose concentration gradient toward the cancer cells. The control, in this case, is microswimmers randomly swimming in the absence of the 7 ACS Paragon Plus Environment

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concentration gradient. Such gradients are naturally present in the body; for example, tumor environments possess a higher local glucose and a lower oxygen concentration. To investigate the performance of the developed microswimmers for in vitro delivery of imaging agents to a monolayer of cultured MCF7 breast cancer and J744.A1 macrophage cells, the soft microswimmers, composed of E. coli MG1655 bacterial cells and double microemulsions loaded with dyes (e.g., Mito-, ER-, and Golgi-trackers), were introduced into a Transwell (Figure 3A). Here, a Transwell membrane is used for creating quasi-monodisperse emulsion swimmers, as a size filter. The microswimmers actively swam through the Transwell’s bottom membrane and reached the cells at the bottom plate guided and steered by an external glucose gradient. The pore size of the Transwell membrane was selected to be 8 µm so that microswimmers with diameters of 5 to 7 µm could pass through the membranes with high efficiency (Figure S2). The microemulsions had a lower density than the cell culture media, hence, in the absence of bacterial propulsion, the buoyancy was dominant and a very small number of passive microemulsions could pass through the membrane and come into contact with the cells at the bottom. Figures 3B and 3C show bacterial cells clustered around the pores of the membrane, where the microemulsions could not be visualized using a scanning electron microscope (SEM). As shown in supplementary Movie S2, the same E. coli clusters were evident in fluorescence time-lapse videos. This movie was recorded from bottom plane of the culture dish and showed an increase in florescence intensity over time, indicating the clustering of microswimmers (containing SYTO9-labelled bacteria) around the pores of the membrane. We verified that the congregation of bacteria around pores is not passive drying effect during SEM sample preparation by running the same experiment in the absence of a glucose concentration gradient. We observed random attachment of E. coli to the membrane (Figures S6A,B), which indicates that bacterial congregation around the pores in Figure 3 was most likely driven by the bacteria actively swimming up the glucose gradient. The biohybrid soft microswimmers and their individual components were assessed for potential adverse bio-toxicity effects on the viability of the cultured MCF7 cells with an MTT assay, which quantified the mitochondrial activity of the cells and thus indirectly measured their potential toxicity towards the cell line. The results in Figure 3D indicate that the microswimmers had a lower adverse effect on the mitochondrial activity of the cultured cell lines than the free 8 ACS Paragon Plus Environment

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bacteria, possibly because of the decrease in the rate of uptake by the cells compared to free bacteria. Once they crossed the membrane, the microswimmers were further able to swim and deliver the organelle tracker dyes to a monolayer of cultured MCF7 breast cancer and J744.A1 macrophage cell lines. First, the microswimmers carrying organelle tracker dyes (Mito-trackers) were incubated with the MCF7 cell line overnight to investigate their performance for mitochondria-labeling dye delivery, as shown in Figures 4A,B. Next, the same experiment was repeated with the microswimmers carrying and delivering the Mito-, ER-, and Golgi-trackers to the monolayer of J744.A1 macrophage cells to label mitochondria, endoplasmic reticulum, and Golgi body, respectively (Figure S7). For positive control, we treated both cell cultures with given tracker dyes as per the manufacturer’s routine direct labeling protocols, which relied on delivering a large amount of dyes to the cells via passive diffusion (Figures 4A-C and Figure S7). We also incubated cells with bare microemulsions (without bacterial attachment) as negative control and we found fluorescence intensity was significantly less compared with microswimmers (t < 0.03, p ≤ 0.05) and positive control (t = 0.01, p ≤ 0.05). Representative fluorescence images for macrophages are shown in Figure S7 and S8 for each dye and the results for mean fluorescence intensities, based on 20 frames from each sample are tabulated in Table 3. Here, as indicated by comparable mean fluorescence intensities calculated from histograms, microswimmers effectively labeled the internal organelles of the live cells, comparable with standard direct staining methods. On the other hand, active dye-loaded microswimmers labeled the cells with much higher efficiency with compared to the passive dye-loaded microemulsions without E. coli attachment (see Figures S7 and S8 and Table 3). We did not demonstrate selectivity in the presence of other cells. Such selectivity can be achieved by using genetically engineered bacterial cells that display surface proteins/peptides targeting biomarkers of interest expressed over tumor cells. To understand the mechanism of the dye/cargo delivery to the cells by the soft microswimmers,

interactions,

i.e.,

internalization,

of

the

rhodamine

B-encapsulated

microswimmers with macrophages were recorded as a time-lapse series of fluorescence images for 30 min after the addition of the microswimmers onto the cell monolayer. Supplementary Movies S3 shows the internalization of the microswimmers by macrophages. The movie 9 ACS Paragon Plus Environment

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demonstrates that, after the physical contact with the cell membrane, soft microswimmers conform to the cell surface and collapse/burst, due to the osmotic pressure from extracellular to intracellular regions.46 The released dye/cargo molecules then diffuse inside the cells efficiently. In the case of macrophages, the bacteria that were attached to the swimmers are also internalized by macrophages. Furthermore, Figures S9 and S10 show time-lapse fluorescence microscope images of the microswimmers and passive microemulsions, respectively, uptaken by macrophage cells, as indicated by the fluorescence intensity detected inside the cells. Internalization of the microswimmers could be triggered by sensing of bacteria or their secreted product at the surface of macrophage cells.47,48 Supplementary Movie S4 shows the 3D confocal microscope reconstruction of the macrophages subjected to the rhodamine B-encapsulated microswimmers after 2 hours and overnight incubation, respectively, confirming the internalization of the delivered cargo. Our team is currently investigating the pathway that could be leading the cascade for the uptake. Rhodamine B-labelled microemulsions incubated with macrophages show uptake and phagocytosis, which might be triggered by sensing of bacteria or their secreted product at the surface of macrophage cells.47,48 Once internalized, the in vitro double-emulsion systems, shown here, exhibited successful mitochondria, endoplasmic reticulum, and Golgi body staining with the delivered fluorescent dyes. The delivery of cell tracers might occur on account of bursting of internalized emulsion drops due to difference in osmolarity inside drops versus intracellular environment, which facilitates the intracellular free diffusions of the dyes towards organelles dispersed into cytoplasm.49,50 As shown in supplementary Movie S3, microemulsion drops attached over cell surface may also undergo membrane rupture due to emulsion shell instability arising from surrounding hyper osmolar cell culture media, and thus releasing organelle tracer dye on cell surface freely diffusing through the J744.A1 membrane and leading to staining specific organelle. Further improvement of the system could be achieved by functionalizing emulsion shells with organelle specific antibodies and cell recognition sites to facilitate the intracellular trafficking and delivery. To show that bacteria-driven cargos could be delivered across future in vivo barriers, we assessed the shape deformation and squeezing capability of our soft microswimmers using Transwells with membrane pore diameters of 3, 5, and 8 µm, where 3- and 5-µm pores were smaller than the diameter of the microemulsions (around 5-7 µm). We quantified the number of fluorescently stained microswimmer emulsions passing through the membrane pores by 10 ACS Paragon Plus Environment

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measuring the mean fluorescent intensity of swimmers under the membrane region over 30 minutes. As shown in Figure 5A, only around 18% less bacteria-driven microemulsions could squeeze and pass through the 5-µm pores, compared with the case of 8-µm pores. However, we saw much less microswimmers passing through 3-µm pores (around 65% less than the case of 8µm pores), indicating that reducing the pore diameter by around 50% of the size of the microswimmers significantly reduced their barrier passing capability. Therefore, to be able to pass through significantly (50% or more) smaller microbarriers, relative to the microswimmer diameter, the synthesized emulsion size needs to be reduced accordingly. To further verify the shape deformation and squeezing capability of the microemulsions, we passed the larger microemulsions through microfluidic channels that were smaller than the emulsion diameter. We observed the shape deformation of the microemulsions (Figure 5B). The developed soft microswimmers thus offer the efficient transfer of the cargo to the cell membrane, enabled by the soft double emulsions to achieve an easily detectable level of organelle labeling. Use of these microswimmers for delivery of therapeutic and imaging agents can decrease the administered dosage, and therefore reduce the potential adverse effects of the cargo on the living tissues. Further investigations are needed for to gain a better understanding of the interactions of bacterial cells with the fluid mosaic structures and to assess the stability and functionality of the developed system under varying environmental conditions, relevant to biomedical applications. Conclusions The proposed bacteria-driven emulsions-based microswimmers demonstrated the feasibility and promise of soft, biocompatible, and active carriers to deliver cargo with high efficiency to tissue cells. Due to their active propulsion, they had three to four orders of magnitude larger effective diffusion constant, which means they could reach and contact the cells in their vicinity significantly faster and more efficiently than the emulsions diffusing passively with no propulsion. Moreover, they had highly efficient cargo delivery mechanism, close to routine direct staining methods, due to their soft emulsion bodies, where the mechanically soft swimmers are believed to conform to the tissue cell surfaces well when they contacted them and collapse to deliver the cargo inside their cores to the cells in high amounts through diffusion. Potential future in vivo active or targeted cargo transport and delivery applications of these soft, biocompatible, biodegradable, and neutrally buoyant microswimmers can be implemented in 11 ACS Paragon Plus Environment

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organ-on-a-chip devices51 and stagnant or low flow-velocity fluidic regions of the human body, such as inside the cerebrospinal fluid, inside the eye, in the urinary tract, in the bile duct, and in capillaries of the vascular system. As demonstrated in the porous membrane experiments, soft emulsion-based microswimmers can squeeze and pass through hollow microtubular structures, such as small capillaries or bile duct branches, and microporous, heterogeneous regions inside complex biological fluids, robustly with no mechanical damage (as long as the diameter of the pores/tubes is not smaller than around 50% of the emulsion diameter). Similarly, in our vascular system, soft 6-8 µm diameter red blood cells can pass through capillaries by deformation.52 Depending on the microporosity of these environments, the diameter of our quasi-monodisperse microswimmers can be tuned to be from hundreds of nanometers up to tens of microns. Swimming next to the tissue cell regions, the cargo would be delivered by mechanical contact and collapsing of the emulsions at the cell surface (Supplementary Movies S3) so that the cargo molecules can diffuse inside the tissue cells. Such in vivo demonstrations would require medical imaging and localization of microswimmers and their physiological environment and optimization of the microswimmers for their motility, immunoresponse, biodegradability, size, cargo amount and type, and (magnetic and taxis-based) steering capability, in their given physiological operation environment and desired medical cargo delivery objective. As potential specific medical applications, such microrobotic active or targeted cargo delivery systems can be used for applications such as active or targeted cancer chemotherapy, gene therapy, highresolution medical imaging, in situ tissue scaffolding and growth.53 Microswimmer optimization, medical imaging, and localization issues for such specific medical uses would be investigated in small animal models in the future. There is certainly a safety risk associated with using microorganisms as therapeutics. The risk, however, is minimized if the microorganism is used in body niches that are not sterile. Oral delivery appears to be the safest route, as demonstrated by the success of probiotics on the market. We have outlined the associated risks in our recent review.17 Materials and Methods Preparation and characterization of w/o/w double emulsions 12 ACS Paragon Plus Environment

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The double emulsions were prepared using a modified version of a previously reported method.54 Briefly, Millipore water and soybean oil (VWR) were added to a vial with water-to-oil ratio of 1:2 along with the chosen anionic surfactant and mixed by sonication (intensity and duration were optimized). The mixture was then supplemented with water and cationic surfactant of choice (ratios optimized) and mixed by sonication or vortexing. Size distribution and stability of microemulsions were optimized by tweaking water-to-w/o ratio, surfactant pairs, sonication intensity and duration, and the duration of vortexing. The appropriate surfactant pair was chosen based on the calculated composite HLB index as the weight average values of the respective HLB of each component. The hydrodynamic diameter of the microemulsions was determined using DLS (Mastersizer analyzer, Malvern) and the zeta potential was calculated from measurements of electrophoretic mobility performed by the same equipment. Nikon Eclipse Ti Confocal Microscope with Yokogawa CSU-W1 spinning disk was used for imaging double emulsions loaded with rhodamine B (Ex/Em 553/627). The images were analyzed using ImageJ to calculate the thickness of the outer shell (indicated by fluorescent Nile red dye). Assembly of double emulsions and bacterial cells Escherichia coli (E. coli) MG1655 (ATCC700926) was inoculated from glycerol stock onto 0.5% LB-agar plate and after overnight incubation transferred to motility medium (0.01 M potassium phosphate, 0.067 M sodium chloride, 10-4 M EDTA, 0.01 M glucose, and 0.002% Tween-20, pH 7.0) containing 3 mg of dissolved biotin (Sulfo-NHS-LC-Biotin, Life technologies) and grown overnight at 37°C in a shaker incubator. The bacteria were subsequently stained with SYTO9 (Ex/Em 485/ 498) by mixing the dye (prepared as per instructions from the vendor) and the bacterial suspension 1:1, incubating for 15 minutes and gently washing the cells three times with PBS to remove excess SYTO9. The streptavidin-coated double emulsion was added to 1 mL of the overnight bacterial suspension (108 cfu/mL) and incubated at room temperature for 45 minutes with gentle shaking to allow for biotin–streptavidin binding. Loading and external functionalization of the double emulsions To load the microemulsions, the fluorescence dye of choice, namely Mito-tracker™ Red FM (Invitrogen, Ex/Em: 581/ 644), ER-tracker™ (Invitrogen, Ex/Em: 587/ 615), Cell light GolgiGFP (Invitrogen, Ex/Em: 488/ 510), rhodamine B (Sigma, Ex/Em: 553/627) or Nile-red (Sigma, 13 ACS Paragon Plus Environment

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Ex/Em: 485/525) for measuring oil shell thickness was dissolved in Milli-Q water or vegetable oil at a concentration of 10 µg/mL, each. Merpol® A was added to this solution at a final concentration of 0.5% (v/v). The dye solution was subsequently added to soybean oil and mixed with probe ultrasonication (Branson Sonifier 250) for 7.5 minutes. This w/o emulsion was washed to remove excess dye and added to 2.5 volumes of phosphate buffered saline (PBS, pH 7.2) containing 0.1% (v/v) of CTAB and 0.5 mg/mL streptavidin. The mixture was mixed using a vortex for 2.5 minutes. The durations and concentrations were determined through a rigorous optimization process. Loading efficiency of each dye was determined by breaking up the micelles with addition of 50% (v/v) ethanol and measuring the fluorescence intensity using a Synergy 2 plate reader (Biotek). Loading efficiency was then calculated as the weight percentage of the dye inside the microemulsions to the total dye added to the initial aqueous phase. The measured intensity was converted into concentration by using a standard curve. The standard curve was compiled for each dye by measuring the fluorescence intensity of serial dilutions of each dye (Figures S3-S5). Quantification of Mito-tracker release rate, streptavidin loading density and dye uptake by cells Water-in-oil emulsions loaded with Mito-tracker were filtered with Transwell membranes (0.4 µm pore size) to remove non-emulsified water and oil phase. The emulsified phase trapped in the membrane was then diluted into complete low glucose Dulbecco's modified Eagle's medium cell culture media and release of the dye was monitored with time using a Synergy 2 plate reader in fluorescence mode. To quantify the amount of streptavidin loaded onto the outer shell of the double emulsion, the w/o/w microemulsion was filtered through Transwell membrane (0.4 µm pore size) and re-suspended in PBS. Streptavidin release was monitored with time and quantified using the Lowry colorimetric method (Micro BCA Protein Assay Reagent Kit, Pierce). Dye uptake in control cells stained with conventional methods were quantified measuring cell fluorescence using ImageJ and compared with the dye delivery by microswimmers to MCF7 cells with the following formula: Corrected total cell fluorescence (CTCF) = Integrated Density – ((Area of selected cell) x (Mean fluorescence of background readings)) 14 ACS Paragon Plus Environment

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In brief, the cell of interest was selected using freeform drawing/selection tools to outline stained cell periphery in ImageJ. From the analyze menu, we selected the “set measurements” and made sure to check area, integrated density and mean gray value. “Measure” plugin was selected from the analyze menu which prompts up a popup box with a stack of values for that first cell. Now a region next to the cell of interests was selected that has no fluorescence, and this will be the background, to be statistically accurate, more than three selections from around the cell are made. This step is repeated for the other cells in the field of view that show fluorescence and dye uptake. All the data in results window were selected and saved into a new excel worksheet and CTFT calculated as mentioned above. Cell culture and toxicity assessment MCF7 breast cancer cell line (ATCC HTB-22) and J774.A1 (ATCC TIB67) were seeded in ibidi® 8 chambered microwells with glass bottom at a seeding density of 5 × 104 cells/mL in complete low glucose DMEM (Life) and grown for 3 days (60% confluency, ~106 cells per well) at 37°C in a humidified incubator containing 5% CO2. MTT assay was used for cytotoxicity assessment as described in the literature.54 Briefly, bacteria-driven soft microswimmers, double microemulsions, and E. coli MG1655 were incubated with the cell monolayer. The concentration of double emulsions and free bacterial cells was equivalent to the estimated concentration of these components in the suspension of the soft microswimmers. After 8 hours of incubation, the mitochondrial activity was quantified by measuring the absorbance of colored formazan product at 570 nm. Percent cell viability was determined by normalization of the absorbance of test samples by control. Experimental setup for delivery of dyes to cell monolayer using the soft microswimmers Prior to addition of the microswimmers, cell culture media was removed, and the cells were washed twice with PBS and covered with high glucose DMEM with 0.1% (v/v) penicillinstreptomycin without phenol red. Freshly prepared soft microswimmers (1 mL) containing different dyes were added into the Transwell (8-µm pore size) placed inside each well. The microwell plate was incubated for 1h in a humidified incubator at 37oC and 5% CO2. Subsequently the Transwell was removed and cell culture plate was incubated for another 1 hour 15 ACS Paragon Plus Environment

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after which the plate was transferred to a confocal microscope (Nikon Ti) with a humidified chamber and the cells were imaged using the appropriate filters. Positive control samples were prepared by staining the cells as per manufacturer’s instructions. Microscopy and image analysis The trajectory of the soft microswimmers were monitored far from the chamber walls at 20 frames per second (fps) using a Zeiss Axio Observer microscope with EC Plan Neofluar 63x/1.25 Ph3 oil immersion objective and a CCD camera (Andor Scientific, Belfast, UK). The images were post-processed using an in-house MATLAB (Mathworks) script to obtain the 2D microswimmer trajectories. The trajectories were adjusted for any background fluid flow by subtracting the displacements caused by fluid drift from the microemulsions displacements. The fluid drift velocity was computed from the average velocity of all non-motile bacteria, typically around 6–10 per frame. The instantaneous 2D speeds were measured by first smoothing each trajectory using a five-frame moving average and then computing the speeds from adjacent frames of the smoothed trajectory. Mean square displacement (MSD) of the swimming trajectories was calculated by MSD(Δt) = ⟨[xi(t + Δt) – xi(t)]2⟩

[1]

where Δt is the displacement, xi is the ith position, and t is the time. In addition, for each channel, images of 10-20 random microscope fields (0.1 mm x 0.1 mm) were captured and used to analyze and quantify the intensity of stained cells with ImageJ. For each single and double bacteria-attached double emulsions, at least 30 videos were captured and analyzed for measuring their mean speeds and trajectories. Statistical analysis All experiments were performed in triplicates. Quantitative data were expressed as a mean with ± standard deviation. Statistically significant difference between samples and control was evaluated using one-way analysis of variance (ANOVA) followed by post hoc tukey’s test for pairwise mean comparison in GraphPad Prism 6 and p-value < 0.05 was chosen as the threshold for statistical significance. 16 ACS Paragon Plus Environment

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Acknowledgements The authors thank T. Endlein for development of the 2D particle tracking and image analysis algorithms. This study is funded by the Max Planck Society.

Supplementary Information Electronic supporting information is available online and covers Figures S1 to S8, Tables S1 to S5, and Movies S1 to S4. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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33. Bourouina, N.; Husson, J.; Hivroz, C.; Henry, N., Biomimetic Droplets for Artificial Engagement of Living Cell Surface Receptors: The Specific Case of the T-cell. Langmuir 2012, 28, 6106-6113. 34. Khare, M.; Singh, A.; Zamboni, P., Prospect of Brain-machine Interface in Motor Disabilities: The Future Support for Multiple Sclerosis Patient to Improve Quality of Life. Annals of Medical and Health Sciences Research 2014, 4, 305-312. 35. Nelson, B. J.; Kaliakatsos, I. K.; Abbott, J. J., Microrobots for Minimally Invasive Medicine. Annu. Rev. Biomed. Eng. 2010, 12, 55-85. 36. Qiu, F.; Fujita, S.; Mhanna, R.; Zhang, L.; Simona, B. R.; Nelson, B. J., Magnetic Helical Microswimmers Functionalized with Lipoplexes for Targeted Gene Delivery. Adv. Funct. Mater. 2015, 25, 1666-1671. 37. Fattaccioli, J.; Baudry, J.; Henry, N.; Brochard-Wyart, F.; Bibette, J., Specific Wetting Probed with Biomimetic Emulsion Droplets. Soft Matter 2008, 4, 2434-2440. 38. Bottier, C.; Fattaccioli, J.; Tarhan, M. C.; Yokokawa, R.; Morin, F. O.; Kim, B.; Collard, D.; Fujita, H., Active Transport of Oil Droplets along Oriented Microtubules by Kinesin Molecular Motors. Lab Chip 2009, 9, 1694-1700. 39. Kanouni, M.; Rosano, H.; Naouli, N., Preparation of a Stable Double Emulsion (W1/O/W2): Role of the Interfacial Films on the Stability of the System. Adv. Colloid Interface Sci. 2002, 99, 229-254. 40. Leal-Calderon, F.; Schmitt, V.; Bibette, J., Emulsion Science: Basic Principles. Springer Science & Business Media: 2007. 41. Matsaridou, I.; Barmpalexis, P.; Salis, A.; Nikolakakis, I., The Influence of Surfactant HLB and Oil/Surfactant Ratio on the Formation and Properties of Self-emulsifying Pellets and Microemulsion Reconstitution. AAPS PharmSciTech 2012, 13, 1319-1330. 42. Courthaudon, J. L.; Dickinson, E.; Christie, W. W., Competitive Adsorption of Lecithin and Beta-casein in Oil in Water Emulsions. J. Agric. Food Chem. 1991, 39, 1365-1368. 43. Zöttl, A.; Stark, H., Emergent Behavior in Active Colloids. J. Phys.: Condens. Matter 2016, 28, 253001. 44. Patteson, A.; Gopinath, A.; Goulian, M.; Arratia, P., Running and Tumbling with E. coli in Polymeric Solutions. Sci. Rep. 2015, 5, 15761. 45. Arabagi, V.; Behkam, B.; Cheung, E.; Sitti, M., Modeling of Stochastic Motion of Bacteria Propelled Spherical Microbeads. J. Appl. Phys. 2011, 109, 114702. 46. M'Barek, K. B.; Molino, D.; Quignard, S.; Plamont, M.-A.; Chen, Y.; Chavrier, P.; Fattaccioli, J., Phagocytosis of Immunoglobulin-coated Emulsion Droplets. Biomaterials 2015, 51, 270-277. 47. Möller, J.; Luehmann, T.; Hall, H.; Vogel, V., The Race to the Pole: How High-aspect Ratio Shape and Heterogeneous Environments Limit Phagocytosis of Filamentous Escherichia coli Bacteria by Macrophages. Nano Lett. 2012, 12, 2901-2905. 48. Liu, P.; Wu, X.; Liao, C.; Liu, X.; Du, J.; Shi, H.; Wang, X.; Bai, X.; Peng, P.; Yu, L.; Wang, F.; Zhao, Y.; Liu, M., Escherichia coli and Candida albicans Induced Macrophage Extracellular Trap-like Structures with Limited Microbicidal Activity. PloS One 2014, 9, e90042. 49. Omotosho, J. A.; Whateley, T. L.; Law, T. K.; Florence, A. T., The Nature of the Oil Phase and the Release of Solutes from Multiple (W/O/W) Emulsions. J. Pharm. Pharmacol. 1986, 38, 865-870. 19 ACS Paragon Plus Environment

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Figures

Figure 1. Schematic representation of the development process for double emulsion-based soft bacteria-driven microswimmers: (A) Preparation of w/o/w double microemulsions loaded with cargo inside their water cores and decorated with streptavidin on their outer oil shells. (B) Attachment of motile bacteria to the double microemulsion outer oil shells through the specific biotin-streptavidin interaction. (C) Collection of quasi-monodisperse bacteria-driven double microemulsions using Transwell membranes with given pore sizes.


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Figure 2. Size distribution and protein adsorption for the synthesized double microemulsions: (A) Fluorescence confocal micrograph overlay with differential interference contrast micrograph, showing thin-shelled w/o/w double microemulsions; inset: a higher magnification image of single microemulsion, showing the shell thickness. Scale bar represents 10 µm. (B) Particle size distribution of w/o/w double microemulsions, as determined by DLS. Data compiled with a bin width of 1. (C) Percent fraction of streptavidin adsorbed at the oil droplet shell as a function of the weight percent (concentration) of the protein in the final aqueous phase. Error bars represent standard deviation from the mean for 3 independent measurements. Fluorescent microscope images of quasi-monodisperse microemulsions attached to (D) one and (E) two bacteria, stained with SYTO9 dye. 2D swimming trajectories (red line) of the microswimmers propelled by (F) one and (G) two bacteria are shown on phase-contract optical microscope images. Scale bar represents 5 µm. 22 ACS Paragon Plus Environment

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Figure 3 (A) Schematic diagram of the in vitro test setup used to investigate the active cargo transport and delivery of the microswimmers. (B) Scanning electron micrograph from top surface of the Transwell membrane, showing bacteria accumulation around the pores of the membrane. Scale bars represent 20 µm. (C) A single cluster of bacteria around the membrane pores, imaged from bottom. Scale bars represents 20 µm. (D) Results of the MTT assay showing decrease in viability of MCF7 cells as a result of incubation with the microswimmers and their components. Error bars represent standard deviation from the mean for 3 independent measurements.


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Figure 4. (A) Control fluorescent images represent cell cultures treated with Mito-tracker dyes as per the manufacturer’s routine protocols. In the fluorescent images, stained nuclei are shown with blue color (DAPI); mitochondria are shown with red color (Mito-tracker); and overlay images show combined color for the nuclei and mitochondria. (B) Microswimmers, loaded with Mito-tracker imaging dyes, delivered the dyes to the breast cancer cell line MCF7 after overnight incubation with cells. Scale bar represents 40 µm.


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Figure 5. Demonstration of Squeezing and deformation capability of rhodamine B-labelled bacteria-driven microemulsions through Transwell membrane pores with 3, 5, and 8 µm diameters. (A) Rhodamine intensity measured by incubating cells with bacteria-driven double emulsions (White columns) and passive (no bacteria-attached) double emulsions (grey columns), as control. (B) Optical microscope image of a soft double emulsion squeezing and passing through a microfluidic channel smaller than its diameter via shape deformation, when high enough flow or pressure is applied. No bacteria were attached to the emulsion surface in this demonstration. Scale bar represents 5 µm.


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Tables

Table 1. Physicochemical properties of synthesized double microemulsions (PI: polydispersity index) Volumetric ratio of

%

%

HLB of

Emulsion

Zeta

Hydrophilic Lipophilic

surfactant

surfactant

surfactant

mixture

(high HLB)

(low HLB) ratio

4:1

1.0

0.1

10.6

7.0 ± 1.0

0.4

+4.3 ± 0.9

4:1

0.5

0.25

6.25

6.4 ± 0.5

0.3

+1.7 ± 0.3

4:1

0.25

0.50

5.5

6.1 ± 0.9

0.4

+0.7 ± 0.1

4:1

0.1

1.0

6.1

5.7 ± 0.7

0.5

+1.2 ± 0.6

5:1

1.0

1.0

16

4.9 ± 0.9

0.6

+2.9 ±/1.1

5:1

0.5

0.5

8

5.2 ± 0.1

0.4

+1.5 ±/0.8

*5:1

0.5

0.5

8

5.5 ± 0.3

0.3

+1.1 ±/0.1

aqueous phase to w/o emulsions

†

diameter

PI

(µm)†

Data = Mean ± SD (n = 5)

* with Streptavidin


potential (mV) †

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Table 2. Comparison of effective diffusion constants (Deff) and mean speeds (Vmean) for microemulsions driven by no, one, and two bacteria

Double emulsion diameter (µm)

Deff

Deff

Deff

Vmean

Vmean

Vmean

for double

for double

for double

for double

for double

for double

emulsions

emulsions

emulsions

emulsions

emulsions

emulsions

with no

with single

with two E.

with single

with two E.

with no

attached

E. coli

coli (m 2/s)

E. coli

coli (µm/s)

attached

bacteria

(m2/s)

(µm/s)

bacteria

2

(m /s)

(µm/s)

5.0 ± 0.1

2.5 x 10-14

8.3 x 10-10

7.5 x 10-10

7.7 ± 1.0

7.1 ± 0.9

0.53 ± 0.2

7.0 ± 0.3

2.1 x 10-13

1.6 x 10-10

7.7 x 10-10

6.5 ± 0.8

4.5 ± 0.2

0.50 ± 0.2


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Table 3. Comparing the cell-organelle staining using active dye-loaded microswimmers-based dye delivery versus conventional direct staining as positive control and passive dye-loaded double emulsions (without attached bacteria) as negative control

Mean intensity

Working dye concentration for

Mean

microswimmers, double

intensity for

emulsions without

positive

bacteria and direct

control

addition to culture

for samples labeled by dyeloaded emulsions without

Mean intensity for samples labeled by microswimmers

attached bacteria

Mito-tracker

500 nM

7.0 ± 1.3

2.3 ± 0.9

4.2 ± 1.1

ER-tracker

1 µM

4.1 ± 0.3

1.6 ± 0.3

3.4 ± 0.8

2.8 ± 0.3

1.2 ± 0.7

2.2 ± 0.1

Golgi-tracker

50 particles per cell (PPC)


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Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active

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