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Construction and evaluation of bacteria-driven liposome
Sensors and Actuators B 183 (2013) 395–400
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Construction and evaluation of bacteria-driven liposome Masaru Kojima a,∗ , Zhenhai Zhang a , Masahiro Nakajima b , Katsutoshi Ooe a , Toshio Fukuda a,b a b
Department of Micro-Nano Systems Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan Center for Micro-Nano Mechatronics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
a r t i c l e
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Article history: Received 5 July 2012 Received in revised form 21 March 2013 Accepted 27 March 2013 Available online 10 April 2013 Keywords: Liposome Bio actuator Bacteria Flagellar motor Drug delivery system
a b s t r a c t Many people proposed living things as actuators, for example microbes, cell and protein molecules. To address these problems for biomedical application, especially drug delivery system, a novel, miniature, and energy-efficient propulsion concept is proposed in this paper. Some bacteria have flagellar motor for swimming in water environment. The bacterial flagellar motor is a molecular machine that converts ionmotive force into mechanical force. Liposome is well known component for drug delivery. This vesicle can contain biologically active compounds. We proposed a new integration method of these two functions, bacteria motility and liposome carrier. Bacteria and liposome are combined through antibody binding technique and we have created a bacteria-driven liposome easily. Consequently, the effect of antibody when bacteria attached to liposome is studied experimentally. Furthermore, the stochastic nature of bacterial bio propulsion of liposome is investigated. It is shown that the mobility of liposome with bacteria was higher than that of liposome without bacteria and demonstrate potential for highly-functional drug delivery system. © 2013 Elsevier B.V. All rights reserved.
1. Introduction One of the primary goals of biomedical micro-robots is to reach currently inaccessible areas of the human body and carry out a host of complex operations. Potential targeted medical applications for these micro-robots include highly localized minimally invasive surgery, drug delivery, and screening for diseases at their early stages. Recent developments in micro/nano-scale engineering have led to realization of various miniature mobile robots [1–5]. However, two of the most difficult problems are the miniaturization of the actuators and power sources required for mobility and realization of high-mobility delivery vehicles. Bio-actuators are deemed to be one of the most promising choices for actuation [6–12]. There are many advantages over man-made actuators because they are much smaller and are capable of producing more complicated motions depend on environmental information [13–15]. More importantly, they convert chemical energy to mechanical energy very efficiently. This property realizes miniaturization of the actuators and power sources. Flagellar motor is a complex assembly of hundreds of protein molecules, spanning the inner (cytoplasmic) and outer membrane of the bacteria [16–20]. The bacterial flagellar motor is a
∗ Corresponding author. Present address: Division of Systems Science, Department of Systems Innovation, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan. Tel.: +81 6 6850 6367; fax: +81 6 789 6367. 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.03.127
molecular machine that converts ion-motive force into mechanical force. The energy source of the rotation is the electrochemical gradient of Na+ or H+ ions across the cytoplasmic membrane. The rotation is conferred to a helical protein filament, flagellum, which propels the bacterium forward. The flagellum is a propulsive organelle that includes a reversible rotary motor embedded in the cell membrane and a filament that extends into the external medium. It consists of more than 20 parts and is about 45 nm in diameter. The bacterial flagellum is formed by a helical filament, which is attached to the cell through a flexible joint known as the hook. The hook is connected to a complex structure known as the basal body that spans the inner membrane and the outer membrane. Total structure of flagellar motor is similar to stepping motor [21]. Liposome as a functional delivery vesicle can contain biologically active compounds. If required chemicals can be inject into the liposome and efficiently transported to local area in human body, there will be a potential of applying them to drug delivery system. Biological micro-robot is a good choice for transporting the liposome. To address these problems for micro-robots and drug delivery systems, we proposed new novel, miniature, and energy-efficient bio propulsion concepts and method of interfacing bacteria with liposome, with the ultimate goal of using bacteria with liposome for actuation, control, sensing, and moving toward target. However, for interfacing bacteria with liposome, complicated process was used [22]. For the easy production, we developed simple method to achieve interfacing bacteria with liposome. Fig. 1 shows a
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Fig. 1. Schematic illustration. Bacteria are attached to liposome by using antibody. (1) Making the liposome. (2) Making the liposome with the antibody. (3) Bacteria attach to liposome based on antibody binding technique.
schematic diagram illustrating bacteria attach to liposome by mean of antibody. The research work presented here intends to investigate the stochastic nature of bacterial propulsion of liposome, which is important for developing next-generation bio-hybrid swimming micro-robots finding applications in diverse fields ranging from biomedical to environmental applications. Specifically, the effect of antibody when bacteria attached to liposome is studied experimentally. We attached bacteria to the liposome’s surface for enhancing liposome mobility. It is then shown that antibody plays an important role in attaching bacteria to the liposome, and the mobility of liposome with bacteria was higher than that of liposome without bacteria. As a result of this simple method, propulsion of liposome was improved.
2. Materials and methods 2.1. Bacterial strain and culturing medium Vibrio alginolyticus mutant strain VIO5 was used for the fabrication of the bacterium-driven liposome. In wild type of V. alginolyticus, two types of flagellar systems, polar and lateral flagella are used for movement in the same cell, depending on environmental conditions [16]. Polar flagella are generated at the cell pole and their filament is sheathed with a membrane structure, which is contiguous with the outer membrane. Lateral flagella are peritrichous and are not sheathed. Lateral flagella are generated when V. alginolyticus are in a viscous environment. The energy sources of polar and lateral flagellar motors are Na+ and H+ -motive force, respectively. VIO5 used in this paper has a single Na+ -driven polar flagellum and no lateral flagellum. Cell samples were cultured in the following procedure. Firstly, cells were grown overnight at 30 ◦ C with shaking in VC medium (0.5% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.4% (w/v) K2 HPO4 , 3% (w/v) NaCl, 0.2% (w/v) glucose). The overnight culture in VC medium was inoculated into VPG medium (1% (w/v) Polypeptone, 0.4% (w/v) K2 HPO4 , 3% (w/v) NaCl, 0.5% (w/v) Glycerol) at a 100-fold dilution and grown at 30 ◦ C for 3 h. For observation of bacteria swimming, we used buffer solution (TMN 300). TMN 300 was prepared with 5 mM MgCl2 , 5 mM Glucose, 300 mM NaCl and 50 mM Tris–HCl at pH 7.5.
Fig. 2. Schematic illustration of the formation of liposome. (A) Buffer solution (TMN 300) is added in oil with lipid, and mixed by the pipette to form micro-droplets in oil. (B) The buffer solution (TMN 300) with different antibody concentration was introduced at the bottom of 1.5 ml tube, and lipid-saturated oil is poured on top of it. A lipid monolayer forms at the oil-buffer solution interface. (C) Preparation A is then gently poured on top of preparation B. The micro-droplets are heavier than the oil that contains them and sediment into the bottom buffer solution phase. As they pass through the interface where the second layer of lipid is sitting, the bilayer is completed and the final vesicles, liposome, are formed.
2.2. Preparation of liposome A phospholipid solution was prepared (10 mM in a chloroform/methanol (2:1, v/v) mixture for 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) (Sigma)) and poured into a glass tube. The organic solvent was then evaporated under nitrogen flow and dried under vacuum to make a dry film at the bottom of the tube. Mineral oil (Nakarai) was then added to the tube prior to ultra sonication for 60 min at 50 ◦ C and vortex mixing (final lipid concentrations in oil were 0.5 mM) for 15 min. Oil with lipid was prepared finally. Buffer solution (TMN 300) with different antibody concentration (0, 1/500,000, 1/50,000, 1/5000, 1/500) was prepared. This antibody, which was made by using the purified flagella (and the sheaths) of V. alginolyticus and was obtained from rabbit as a polyclonal antibody, specifically sticks to the flagellar filaments and the outer membrane (including the sheaths). [23,24]. Five hundred microliters of buffer solution (TMN 300) with different antibody concentration was introduced at the bottom of the tube and covered with 300 L of oil with lipid. To obtain micro-droplets, we added 2.5 L of buffer solution without antibody to 100 L of the oil phase containing phospholipid and then emulsified the mixture by pipetting up and down with a micropipette. The micro-droplet solution was added to the oil with lipid within a couple of minutes. The micro-droplets in the oil phase spontaneously crossed the interface between the oil phase to the buffer solution phase according to principle of gravity, driven by the difference in molar density between buffer solution and oil with lipid. During process of transferring, the micro-droplets were converted into bilayer lipid vesicles, liposomes [25,26]. The schematic illustration of synthesis of liposome is shown in Fig. 2. 2.3. Bacteria attachment to liposome Firstly, we prepared cultivated bacteria as mentioned above. And then we prepared second culture in VPG medium. Secondly, 200 L of second culture solution was introduced 1.5 ml tube. We centrifuged cell solution at 8000 rpm for 5 min (3740, Kubota) and spin down bacterial cells. After removing the VPG medium, we
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Fig. 4. Average bacteria attachment ratio versus antibody concentration (n = 10). ND means not detected.
Fig. 3. Typical figure of bacteria attached to liposome without/with antibody.
added 200 L of TMN 300 buffer solution to the tube and suspended. This solution was used as bacteria solution. And then 5 L of liposome solution and 5 L of bacteria solution were mixed on the slide glass. Five minutes after, bacteria attached liposome was observed. 2.4. Observation of bacteria attachment to liposome The experiment of bacteria attaching to liposome was observed using phase-contrast microscopy (OLYMPUS IX71). The observed images were recorded at 30 frames/s by video recorder. Recoding image was analyzed by Image J software. 2.5. Evaluation method for liposome movements In this paper, as one of the evaluation indexes for the movements of the bacterium-driven micro objects and control samples, twodimensional mean square displacements (MSDs) were calculated by the following equation [27]: MSD(n t) =
1 N−1−n
N−1−n
[x(j t + n t) − x(j t)]
j=1
+[y(j t + n t) − y(j t)]
2
2
(1)
where t is video frame interval (∼33 ms), n and j is positive integer, N is the total number of frames in the experimental data. The center positions (x, y) of the liposome were calculated by the binarization and calculation of the centroid with image processing. 3. Results and discussion 3.1. Evaluation of bacteria attachment to liposome by antibody Typical figures of bacteria attached to liposome without/with antibody are shown in Fig. 3. The elliptical shade, which diameter was about 2–3 m, was bacteria and large sphere, which diameter was about 20 m, was liposome. From the observation data, we could confirm several bacteria attached to liposome with antibody condition. The bacteria on the liposome were attached randomly.
Patterned attachment might be better for movement. The effect of patterning of the bacteria-driven microbeads movement was demonstrated S. marcescens bacterium by Behkam et al. [10]. They showed MSD of patterned, unpatterned, and control microbeads. Patterning leads to a substantial increase in the total distance traveled. By comparison of unpatterned and control microbeads, unpatterned micro beads show large MSD. Therefore we could expect some effect by random attachment. In order to confirm the effect of antibody when bacteria attached to liposome, we counted bacterial numbers attached to liposome in each antibody concentration and measure diameter of liposome for calculation of liposome surface area (m2 ). To compare different size liposome, we use the average bacteria attachment ratio (bacteria number/area of liposome surface) as the evaluation criterion. Fig. 4 shows the plot of average bacteria attachment ratio versus antibody concentration in the experiment. This result suggest that average bacteria attachment ratio increases as antibody concentration increases. However, there is a threshold value above, which the increase rate drops. When antibody concentration became more than 1/500, we could not find any liposome with good condition. We could find only aggregations of liposome and bacteria. From these results, we confirm antibody could bind bacteria to liposome surface lipid. The reason why antibody binds to liposome, we speculate as follows. In our experiment, DOPE was used as lipid for construction of liposome. The DOPE has weak negative charge and hydrophobic property. On the other hand, tail of the antibody is relatively easy to bind to various substrates. Therefore these hydrophobic interactions and electrostatic interactions might influence binding between antibody and DOPE. Furthermore, oil water droplet method was used for production of liposome. In this case, small lipid aggregations were attached liposome surface. Recent research reported that protein insertions into lipid surface were easily happen by oil water droplet method [28]. Therefore, these properties might realize association between antibody and liposome surface. However, binding mechanism between antibody and liposome surface is still not clear, to reveal this mechanism, further examination is needed. 3.2. Observation result of the liposome movement We observed the movements of liposome driven by bacteria under optical microscopy. Sometimes the unattached bacteria can propel the liposome. However, the unattached bacteria immediately go away from liposome, Therefore, we could identify liposome motion coursed by attached bacteria from observation data and liposome movement by attached was used for analysis. Fig. 5 shows sequential image of the movement of liposome with bacteria under optical microscopy. From this result, long distance movement (not Brownian motion) of liposome was confirmed. The typical trajectory (for 10 s) of liposome without/with bacteria was shown in
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Fig. 6. Movement of liposome without/with bacteria. (A) Movement of a liposome without bacteria. (B) Liposome with bacteria.
straightforward without the rotation. Under this condition, the driving force (F) is calculated by the equations, F = v
(2)
and = 6r
Fig. 5. Sequential photographs of the movement of a liposome with bacteria. (A) Time raps image of liposome movement. (B) Merged image of (A).
Fig. 6. We noticed that the liposome with bacteria moved more widely than that without bacteria, by comparing Fig. 6(A) and (B). A liposome without bacteria moved by Brownian motion, as is shown in Fig. 6(A). Liposomes without bacteria were almost not moving, on the other hand, liposomes bound with bacteria were moving long distance. The trajectories of the center positions of the liposome for 10 s, typical one each for the bacterium-driven liposome assembled using antibody are shown in Fig. 6. And, the mean values of the moving velocities were 4.2 m/s for the bacterium-driven liposome by antibody and 0.89 m/s for the liposome without bacteria (Brownian motion). From these results, the bacterium-driven liposome showed significantly better motility than the control samples (4.7 times faster than the liposome without bacteria). Here, the driving force generated by the bacterium was estimated from the moving velocity of the bacterium-driven liposome by antibody. It is assumed that the liposome (sphere) moved
(3)
where || is the parallel drag coefficient, v is the velocity of the sphere, is the viscosity of the environmental solution, r is the radius of the sphere [29]. The driving force was calculated by Eqs. (2) and (3) where v: 4.2 m/s, : 0.001 Pa s (pure water) and r: 15 m. As a result, it is concluded that the total driving force, the sum of random direction bacteria propulsions, generated by the bacterium was about 1.2 pN. From the optical microscopy observation, 8 bacteria attached to liposome randomly. Even if all bacteria attached same site, propulsion by single bacteria was estimated as 0.15 pN. This value is consistent with the value predicted as the bacterial propulsion of single S. marcescens bacterium by Behkam et al. [11]. However, in case of another bacterium-driven liposome, v: 5.5 m/s, : 0.001 Pa s (pure water) and r: 6.5 m, driving force was about 0.67 pN. From the optical microscopy observation, 18 bacteria attached to liposome randomly. Though there was much number of the bacteria, the driving force became smaller than the 8 bacteria attached case. The driving force was offset when the number of the bacteria attaching to liposome increases. Therefore the driving force might become small. As antibody concentration increases, the number of the bacteria attached increases (Fig. 4). However, the driving force was not simply increased by offsetting phenomenon. In fact, we compared same size liposome (diameter 13 m) at each antibody concentration. As a result, 1/500,000 antibody concentration case, the liposome moved at 4.3 m/s, 1/50,000
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Fig. 7. Mean square displacement from the starting location of liposome with bacteria and antibody ((1), (2)), without bacteria and antibody ((5), (6)) and with bacteria in no antibody condition ((3), (4)).
case the liposome moved at 5.5 m/s, and 1/5000 case the liposome moved at 4.6 m/s. 3.3. Evaluation of liposome movements To evaluate liposome movement, we tried image analysis. We can get each of the position of liposome coordinates from movie data by means of software Image J. For the bacteria propelled liposome, the direction of motion is decided stochastically. Based on the trajectories, the mean square displacement (MSDs) of the bacteriadriven liposome and the control samples (the liposome without bacteria, the liposome with bacteria in no antibody) were calculated by Eq. (1) as shown in above. Fig. 7 shows the typical graph of MSDs from the starting location versus time intervals in the experiment. This result also indicates that the liposome with bacteria moved more widely than that without bacteria, by comparing Fig. 7(1), (2) and (5), (6). Furthermore, we also compared liposome movement by bacteria with antibody and without antibody. In many cases most of liposome did not move as shown Fig. 7(4). However, sometimes widely moved liposome was found as shown in Fig. 7(3). In this case, detail observation by microscopy suggested that bacteria tightly bind to liposomes. We constructed liposome
by oil water droplet method, therefore bacteria might bind to liposome due to carry-on of the oil from construction phase. There were a few bacteria attached liposome and the ratio was less than 10% as compared with antibody condition. Enhancement of movement is depends on attachment position and number of bacteria. However, in all cases, MSD of the liposome with bacteria is higher than that of the liposome without bacteria. These results clearly indicate that the bacteria attached to the liposome functioned as the main source for propulsion of liposome.
4. Conclusions In this paper, we reported the delivery system based on bacteriadriven liposome by simple binding method. Antibody binding technique was developed and used to attach bacteria to liposome’s surface. It was shown that bacteria and liposome were combined through antibody. Appropriate antibody concentration, 1/5000, was found according to experimental results. When antibody concentration became more than 1/500, we found unmoving aggregations of liposome and bacteria. In this case, antibody concentration was too high, both bacteria and liposome congregate together and was difficult to move. The results of MSD evaluation
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suggested that the liposome with bacteria moved broader compared to the liposome without bacteria. The mobility of liposome with bacteria was higher than that of liposome without bacteria. The bacteria attached to the liposome were the main source of propulsion. We used oil water droplet method for making liposome. This method have some advantages, especially inner contents of liposome could be change freely. Therefore we could package any good candidate for therapy or environmental control and so on. Furthermore, we also used bacteria as actuator. Some bacteria have several properties, for example taxis systems, bacteria do not move randomly but show tactic movements toward or away from environmental stimuli, such as chemical substances (chemotaxis), temperature (thermotaxis), light (phototaxis), magnetic field (magnetotaxis) and so on. Our proposed configuration realizes to add these bacterial properties to liposome delivery systems. Acknowledgements This work was partially supported by Grant-in-Aid for the Global COE Program “COE for Education and Research of Micro-Nano Mechatronics”, Grant-in-Aid for Scientific Research on Innovative Areas (23106005, 23106006, 24115507) and the Hori Foundation. We thank M. Homma and S. Kojima for providing the V. alginolyticus strains, K. Nogawa, K. Sekiyama and K. Takiguchi for useful discussions. References [1] A. Cavalcanti, B. Shirinzadeh, T. Fukuda, S. Ikeda, Nanorobot for brain aneurysm, International Journal of Robotics Research 28 (2009) 558–570. [2] S. Martel, O. Felfoul, J.-B. Mathieu, A. Chanu, S. Tamaz, M. Mohammadi, M. Mankiewicz, N. Tabatabaei, MRI-based medical nanorobotic platform for the control of magnetic nanoparticles and flagellated bacteria for target interventions in human capillaries, International Journal of Robotics Research 28 (2009) 1169–1182. [3] G.M. Patel, G.C. Patel, R.B. Patel, J.K. Patel, M. Patel, Nanorobot, A versatile tool in nanomedicine, Journal of Drug Targeting 14 (2006) 63–67. [4] R.A. Freitas Jr., The ideal gene delivery vector: chromallocytes, cell repair nanorobots for chromosome replacement therapy, Journal of Evolution and Technology 16 (2007) 1–97. [5] D.A. LaVan, T. McGuire, R. Langer, Small-scale systems for in vivo drug delivery, Nature Biotechnology 21 (2003) 1184–1191. [6] H. Hess, V. Vogel, Molecular shuttles based on motor proteins: active transport in synthetic environments, Reviews in Molecular Biotechnology 82 (2001) 67–85. [7] Y. Yoshida, R. Yoshida, R. Yokokawa, H. Suzuki, K. Atsuta, H. Fujita, S. Takeuchi, Biomolecular linear motors confined to move upon micropatterns, Journal of Micromechanics and Microengineering 16 (2006) 1550–1554. [8] N. Mori, K. Kuribayashi, S. Takeuchi, Artificial flagellates analysis of advancing motions of biflagellate micro-objects, Applied Physics Letters 96 (2010) 083701. [9] R.K. Soong, G.D. Bachand, H.P. Neves, A.G. Olkhovets, H.G. Craighead, C.D. Montemagno, Powering an inorganic nanodevice with a biomolecular motor, Science 290 (2000) 1555–1558. [10] B. Behkam, M. Sitti, Effect of quantity and configuration of attached bacteria on bacterial propulsion of microbeads, Applied Physics Letters 93 (2008) 223901–223903. [11] B. Behkam, M. Sitti, Bacteria flagella-based propulsion and on/off motion of microscale objects, Applied Physics Letters 90 (2007) 23902–23904. [12] Y. Hiratsuka, M. Miyata, T. Tada, T.Q.P. Uyeda, A microrotary motor powered by bacteria, Proceedings of the National Academy of Sciences of the United States of America 103 (2006) 13618–13623. [13] N. Darnton, L. Turner, K. Breuer, H.C. Berg, Moving fluid with bacterial carpets, Biophysical Journal 86 (2004) 1863–1870. [14] S. Martel, M. Mohammadi, O. Felfoul, Z. Lu, P. Pouponneau, Flagellated magnetotactic bacteria as controlled MRI-trackable propulsion and steering systems for medical nanorobots operating in the human microvasculature, International Journal of Robotics Research 28 (2009) 571–582. [15] K. Nogawa, M. Kojima, M. Nakajima, S. Kojima, M. Homma, T. Fukuda, Rotational speed control of Na+-driven flagellar motor by dual pipettes, Ieee Transactions on Nanobioscience 8 (2009) 341–348.
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Biographies Masaru Kojima received Doctor of Science degree from Nagoya University in 2006. He had been Research Fellow with the Department of Biology, Nagoya University in 2007, Research Fellow on Global Center Of Excellence (GCOE) with the Department of Biology, Nagoya University in 2008, Assistant Professor on GCOE with the Department of Micro-Nano Systems Engineering, Nagoya University from 2008, Assistant Professor in Department of Systems Innovation, Osaka University from 2012. His main research interest is biophysics, in particular, using the biological object and proteins as bio actuator. Zhenhai Zhang received doctor degree from Beijing Institute of Technology in 2008. He had been postdoctoral researcher in Beijing Institute of Technology from 2008, he joined Department of Micro-Nano Systems Engineering, Nagoya University as a Visiting researcher from 2010 to 2011. His research interest is MEMS/NEMS sensors and micro/nano bio robot. Masahiro Nakajima received Doctor of engineering degree from Nagoya University. He had been a Research Fellow of the Ministry of Education, Culture, Sports, Science and Technology, Nagoya University in 2006, Research Associate in Department of Micro-Nano Systems Engineering, Nagoya University from 2006, Assistant Professor in Department of Micro-Nano Systems Engineering, Nagoya University from 2007, Assistant Professor in Center For Micro-Nano Mechatonics, Nagoya University from 2009. He has interest in the research field of the applications of micro/nano manipulation, micro/nano-mechanics, nano-biology. Katsutoshi Ooe received Doctor Degree from Nagoya University in 2001. He joined Osaka Institute of Technology as a Postdoctoral researcher from 2001, Ritsumeikan University as a Associate Professor from 2004, From 2009, as a Associate Professor on GCOE with the Department of Micro-Nano Systems Engineering, Nagoya University, Associate Professor in Department of machine system engineering, Daiichi Institute of Technology from 2012. He is mainly engaging in the research field of biomedical engineering. Toshio Fukuda received the Doctor of Engineering from the University of Tokyo in 1977. He joined in the National Mechanical Engineering Laboratory, in Japan, from 1977. He joined the Science University of Tokyo in Japan from 1982. He joined Department of Mechanical Engineering, Nagoya University in Japan as a professor from 1989. He is mainly involved in the research field of intelligent robotic and mechatronic system, celler robotic system, micro and nano robotic system.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Construction and evaluation of bacteria-driven liposome Masaru Kojima a,∗ , Zhenhai Zhang a , Masahiro Nakajima b , Katsutoshi Ooe a , Toshio Fukuda a,b a b
Department of Micro-Nano Systems Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan Center for Micro-Nano Mechatronics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
a r t i c l e
i n f o
Article history: Received 5 July 2012 Received in revised form 21 March 2013 Accepted 27 March 2013 Available online 10 April 2013 Keywords: Liposome Bio actuator Bacteria Flagellar motor Drug delivery system
a b s t r a c t Many people proposed living things as actuators, for example microbes, cell and protein molecules. To address these problems for biomedical application, especially drug delivery system, a novel, miniature, and energy-efficient propulsion concept is proposed in this paper. Some bacteria have flagellar motor for swimming in water environment. The bacterial flagellar motor is a molecular machine that converts ionmotive force into mechanical force. Liposome is well known component for drug delivery. This vesicle can contain biologically active compounds. We proposed a new integration method of these two functions, bacteria motility and liposome carrier. Bacteria and liposome are combined through antibody binding technique and we have created a bacteria-driven liposome easily. Consequently, the effect of antibody when bacteria attached to liposome is studied experimentally. Furthermore, the stochastic nature of bacterial bio propulsion of liposome is investigated. It is shown that the mobility of liposome with bacteria was higher than that of liposome without bacteria and demonstrate potential for highly-functional drug delivery system. © 2013 Elsevier B.V. All rights reserved.
1. Introduction One of the primary goals of biomedical micro-robots is to reach currently inaccessible areas of the human body and carry out a host of complex operations. Potential targeted medical applications for these micro-robots include highly localized minimally invasive surgery, drug delivery, and screening for diseases at their early stages. Recent developments in micro/nano-scale engineering have led to realization of various miniature mobile robots [1–5]. However, two of the most difficult problems are the miniaturization of the actuators and power sources required for mobility and realization of high-mobility delivery vehicles. Bio-actuators are deemed to be one of the most promising choices for actuation [6–12]. There are many advantages over man-made actuators because they are much smaller and are capable of producing more complicated motions depend on environmental information [13–15]. More importantly, they convert chemical energy to mechanical energy very efficiently. This property realizes miniaturization of the actuators and power sources. Flagellar motor is a complex assembly of hundreds of protein molecules, spanning the inner (cytoplasmic) and outer membrane of the bacteria [16–20]. The bacterial flagellar motor is a
∗ Corresponding author. Present address: Division of Systems Science, Department of Systems Innovation, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan. Tel.: +81 6 6850 6367; fax: +81 6 789 6367. 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.03.127
molecular machine that converts ion-motive force into mechanical force. The energy source of the rotation is the electrochemical gradient of Na+ or H+ ions across the cytoplasmic membrane. The rotation is conferred to a helical protein filament, flagellum, which propels the bacterium forward. The flagellum is a propulsive organelle that includes a reversible rotary motor embedded in the cell membrane and a filament that extends into the external medium. It consists of more than 20 parts and is about 45 nm in diameter. The bacterial flagellum is formed by a helical filament, which is attached to the cell through a flexible joint known as the hook. The hook is connected to a complex structure known as the basal body that spans the inner membrane and the outer membrane. Total structure of flagellar motor is similar to stepping motor [21]. Liposome as a functional delivery vesicle can contain biologically active compounds. If required chemicals can be inject into the liposome and efficiently transported to local area in human body, there will be a potential of applying them to drug delivery system. Biological micro-robot is a good choice for transporting the liposome. To address these problems for micro-robots and drug delivery systems, we proposed new novel, miniature, and energy-efficient bio propulsion concepts and method of interfacing bacteria with liposome, with the ultimate goal of using bacteria with liposome for actuation, control, sensing, and moving toward target. However, for interfacing bacteria with liposome, complicated process was used [22]. For the easy production, we developed simple method to achieve interfacing bacteria with liposome. Fig. 1 shows a
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Fig. 1. Schematic illustration. Bacteria are attached to liposome by using antibody. (1) Making the liposome. (2) Making the liposome with the antibody. (3) Bacteria attach to liposome based on antibody binding technique.
schematic diagram illustrating bacteria attach to liposome by mean of antibody. The research work presented here intends to investigate the stochastic nature of bacterial propulsion of liposome, which is important for developing next-generation bio-hybrid swimming micro-robots finding applications in diverse fields ranging from biomedical to environmental applications. Specifically, the effect of antibody when bacteria attached to liposome is studied experimentally. We attached bacteria to the liposome’s surface for enhancing liposome mobility. It is then shown that antibody plays an important role in attaching bacteria to the liposome, and the mobility of liposome with bacteria was higher than that of liposome without bacteria. As a result of this simple method, propulsion of liposome was improved.
2. Materials and methods 2.1. Bacterial strain and culturing medium Vibrio alginolyticus mutant strain VIO5 was used for the fabrication of the bacterium-driven liposome. In wild type of V. alginolyticus, two types of flagellar systems, polar and lateral flagella are used for movement in the same cell, depending on environmental conditions [16]. Polar flagella are generated at the cell pole and their filament is sheathed with a membrane structure, which is contiguous with the outer membrane. Lateral flagella are peritrichous and are not sheathed. Lateral flagella are generated when V. alginolyticus are in a viscous environment. The energy sources of polar and lateral flagellar motors are Na+ and H+ -motive force, respectively. VIO5 used in this paper has a single Na+ -driven polar flagellum and no lateral flagellum. Cell samples were cultured in the following procedure. Firstly, cells were grown overnight at 30 ◦ C with shaking in VC medium (0.5% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.4% (w/v) K2 HPO4 , 3% (w/v) NaCl, 0.2% (w/v) glucose). The overnight culture in VC medium was inoculated into VPG medium (1% (w/v) Polypeptone, 0.4% (w/v) K2 HPO4 , 3% (w/v) NaCl, 0.5% (w/v) Glycerol) at a 100-fold dilution and grown at 30 ◦ C for 3 h. For observation of bacteria swimming, we used buffer solution (TMN 300). TMN 300 was prepared with 5 mM MgCl2 , 5 mM Glucose, 300 mM NaCl and 50 mM Tris–HCl at pH 7.5.
Fig. 2. Schematic illustration of the formation of liposome. (A) Buffer solution (TMN 300) is added in oil with lipid, and mixed by the pipette to form micro-droplets in oil. (B) The buffer solution (TMN 300) with different antibody concentration was introduced at the bottom of 1.5 ml tube, and lipid-saturated oil is poured on top of it. A lipid monolayer forms at the oil-buffer solution interface. (C) Preparation A is then gently poured on top of preparation B. The micro-droplets are heavier than the oil that contains them and sediment into the bottom buffer solution phase. As they pass through the interface where the second layer of lipid is sitting, the bilayer is completed and the final vesicles, liposome, are formed.
2.2. Preparation of liposome A phospholipid solution was prepared (10 mM in a chloroform/methanol (2:1, v/v) mixture for 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) (Sigma)) and poured into a glass tube. The organic solvent was then evaporated under nitrogen flow and dried under vacuum to make a dry film at the bottom of the tube. Mineral oil (Nakarai) was then added to the tube prior to ultra sonication for 60 min at 50 ◦ C and vortex mixing (final lipid concentrations in oil were 0.5 mM) for 15 min. Oil with lipid was prepared finally. Buffer solution (TMN 300) with different antibody concentration (0, 1/500,000, 1/50,000, 1/5000, 1/500) was prepared. This antibody, which was made by using the purified flagella (and the sheaths) of V. alginolyticus and was obtained from rabbit as a polyclonal antibody, specifically sticks to the flagellar filaments and the outer membrane (including the sheaths). [23,24]. Five hundred microliters of buffer solution (TMN 300) with different antibody concentration was introduced at the bottom of the tube and covered with 300 L of oil with lipid. To obtain micro-droplets, we added 2.5 L of buffer solution without antibody to 100 L of the oil phase containing phospholipid and then emulsified the mixture by pipetting up and down with a micropipette. The micro-droplet solution was added to the oil with lipid within a couple of minutes. The micro-droplets in the oil phase spontaneously crossed the interface between the oil phase to the buffer solution phase according to principle of gravity, driven by the difference in molar density between buffer solution and oil with lipid. During process of transferring, the micro-droplets were converted into bilayer lipid vesicles, liposomes [25,26]. The schematic illustration of synthesis of liposome is shown in Fig. 2. 2.3. Bacteria attachment to liposome Firstly, we prepared cultivated bacteria as mentioned above. And then we prepared second culture in VPG medium. Secondly, 200 L of second culture solution was introduced 1.5 ml tube. We centrifuged cell solution at 8000 rpm for 5 min (3740, Kubota) and spin down bacterial cells. After removing the VPG medium, we
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Fig. 4. Average bacteria attachment ratio versus antibody concentration (n = 10). ND means not detected.
Fig. 3. Typical figure of bacteria attached to liposome without/with antibody.
added 200 L of TMN 300 buffer solution to the tube and suspended. This solution was used as bacteria solution. And then 5 L of liposome solution and 5 L of bacteria solution were mixed on the slide glass. Five minutes after, bacteria attached liposome was observed. 2.4. Observation of bacteria attachment to liposome The experiment of bacteria attaching to liposome was observed using phase-contrast microscopy (OLYMPUS IX71). The observed images were recorded at 30 frames/s by video recorder. Recoding image was analyzed by Image J software. 2.5. Evaluation method for liposome movements In this paper, as one of the evaluation indexes for the movements of the bacterium-driven micro objects and control samples, twodimensional mean square displacements (MSDs) were calculated by the following equation [27]: MSD(n t) =
1 N−1−n
N−1−n
[x(j t + n t) − x(j t)]
j=1
+[y(j t + n t) − y(j t)]
2
2
(1)
where t is video frame interval (∼33 ms), n and j is positive integer, N is the total number of frames in the experimental data. The center positions (x, y) of the liposome were calculated by the binarization and calculation of the centroid with image processing. 3. Results and discussion 3.1. Evaluation of bacteria attachment to liposome by antibody Typical figures of bacteria attached to liposome without/with antibody are shown in Fig. 3. The elliptical shade, which diameter was about 2–3 m, was bacteria and large sphere, which diameter was about 20 m, was liposome. From the observation data, we could confirm several bacteria attached to liposome with antibody condition. The bacteria on the liposome were attached randomly.
Patterned attachment might be better for movement. The effect of patterning of the bacteria-driven microbeads movement was demonstrated S. marcescens bacterium by Behkam et al. [10]. They showed MSD of patterned, unpatterned, and control microbeads. Patterning leads to a substantial increase in the total distance traveled. By comparison of unpatterned and control microbeads, unpatterned micro beads show large MSD. Therefore we could expect some effect by random attachment. In order to confirm the effect of antibody when bacteria attached to liposome, we counted bacterial numbers attached to liposome in each antibody concentration and measure diameter of liposome for calculation of liposome surface area (m2 ). To compare different size liposome, we use the average bacteria attachment ratio (bacteria number/area of liposome surface) as the evaluation criterion. Fig. 4 shows the plot of average bacteria attachment ratio versus antibody concentration in the experiment. This result suggest that average bacteria attachment ratio increases as antibody concentration increases. However, there is a threshold value above, which the increase rate drops. When antibody concentration became more than 1/500, we could not find any liposome with good condition. We could find only aggregations of liposome and bacteria. From these results, we confirm antibody could bind bacteria to liposome surface lipid. The reason why antibody binds to liposome, we speculate as follows. In our experiment, DOPE was used as lipid for construction of liposome. The DOPE has weak negative charge and hydrophobic property. On the other hand, tail of the antibody is relatively easy to bind to various substrates. Therefore these hydrophobic interactions and electrostatic interactions might influence binding between antibody and DOPE. Furthermore, oil water droplet method was used for production of liposome. In this case, small lipid aggregations were attached liposome surface. Recent research reported that protein insertions into lipid surface were easily happen by oil water droplet method [28]. Therefore, these properties might realize association between antibody and liposome surface. However, binding mechanism between antibody and liposome surface is still not clear, to reveal this mechanism, further examination is needed. 3.2. Observation result of the liposome movement We observed the movements of liposome driven by bacteria under optical microscopy. Sometimes the unattached bacteria can propel the liposome. However, the unattached bacteria immediately go away from liposome, Therefore, we could identify liposome motion coursed by attached bacteria from observation data and liposome movement by attached was used for analysis. Fig. 5 shows sequential image of the movement of liposome with bacteria under optical microscopy. From this result, long distance movement (not Brownian motion) of liposome was confirmed. The typical trajectory (for 10 s) of liposome without/with bacteria was shown in
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Fig. 6. Movement of liposome without/with bacteria. (A) Movement of a liposome without bacteria. (B) Liposome with bacteria.
straightforward without the rotation. Under this condition, the driving force (F) is calculated by the equations, F = v
(2)
and = 6r
Fig. 5. Sequential photographs of the movement of a liposome with bacteria. (A) Time raps image of liposome movement. (B) Merged image of (A).
Fig. 6. We noticed that the liposome with bacteria moved more widely than that without bacteria, by comparing Fig. 6(A) and (B). A liposome without bacteria moved by Brownian motion, as is shown in Fig. 6(A). Liposomes without bacteria were almost not moving, on the other hand, liposomes bound with bacteria were moving long distance. The trajectories of the center positions of the liposome for 10 s, typical one each for the bacterium-driven liposome assembled using antibody are shown in Fig. 6. And, the mean values of the moving velocities were 4.2 m/s for the bacterium-driven liposome by antibody and 0.89 m/s for the liposome without bacteria (Brownian motion). From these results, the bacterium-driven liposome showed significantly better motility than the control samples (4.7 times faster than the liposome without bacteria). Here, the driving force generated by the bacterium was estimated from the moving velocity of the bacterium-driven liposome by antibody. It is assumed that the liposome (sphere) moved
(3)
where || is the parallel drag coefficient, v is the velocity of the sphere, is the viscosity of the environmental solution, r is the radius of the sphere [29]. The driving force was calculated by Eqs. (2) and (3) where v: 4.2 m/s, : 0.001 Pa s (pure water) and r: 15 m. As a result, it is concluded that the total driving force, the sum of random direction bacteria propulsions, generated by the bacterium was about 1.2 pN. From the optical microscopy observation, 8 bacteria attached to liposome randomly. Even if all bacteria attached same site, propulsion by single bacteria was estimated as 0.15 pN. This value is consistent with the value predicted as the bacterial propulsion of single S. marcescens bacterium by Behkam et al. [11]. However, in case of another bacterium-driven liposome, v: 5.5 m/s, : 0.001 Pa s (pure water) and r: 6.5 m, driving force was about 0.67 pN. From the optical microscopy observation, 18 bacteria attached to liposome randomly. Though there was much number of the bacteria, the driving force became smaller than the 8 bacteria attached case. The driving force was offset when the number of the bacteria attaching to liposome increases. Therefore the driving force might become small. As antibody concentration increases, the number of the bacteria attached increases (Fig. 4). However, the driving force was not simply increased by offsetting phenomenon. In fact, we compared same size liposome (diameter 13 m) at each antibody concentration. As a result, 1/500,000 antibody concentration case, the liposome moved at 4.3 m/s, 1/50,000
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Fig. 7. Mean square displacement from the starting location of liposome with bacteria and antibody ((1), (2)), without bacteria and antibody ((5), (6)) and with bacteria in no antibody condition ((3), (4)).
case the liposome moved at 5.5 m/s, and 1/5000 case the liposome moved at 4.6 m/s. 3.3. Evaluation of liposome movements To evaluate liposome movement, we tried image analysis. We can get each of the position of liposome coordinates from movie data by means of software Image J. For the bacteria propelled liposome, the direction of motion is decided stochastically. Based on the trajectories, the mean square displacement (MSDs) of the bacteriadriven liposome and the control samples (the liposome without bacteria, the liposome with bacteria in no antibody) were calculated by Eq. (1) as shown in above. Fig. 7 shows the typical graph of MSDs from the starting location versus time intervals in the experiment. This result also indicates that the liposome with bacteria moved more widely than that without bacteria, by comparing Fig. 7(1), (2) and (5), (6). Furthermore, we also compared liposome movement by bacteria with antibody and without antibody. In many cases most of liposome did not move as shown Fig. 7(4). However, sometimes widely moved liposome was found as shown in Fig. 7(3). In this case, detail observation by microscopy suggested that bacteria tightly bind to liposomes. We constructed liposome
by oil water droplet method, therefore bacteria might bind to liposome due to carry-on of the oil from construction phase. There were a few bacteria attached liposome and the ratio was less than 10% as compared with antibody condition. Enhancement of movement is depends on attachment position and number of bacteria. However, in all cases, MSD of the liposome with bacteria is higher than that of the liposome without bacteria. These results clearly indicate that the bacteria attached to the liposome functioned as the main source for propulsion of liposome.
4. Conclusions In this paper, we reported the delivery system based on bacteriadriven liposome by simple binding method. Antibody binding technique was developed and used to attach bacteria to liposome’s surface. It was shown that bacteria and liposome were combined through antibody. Appropriate antibody concentration, 1/5000, was found according to experimental results. When antibody concentration became more than 1/500, we found unmoving aggregations of liposome and bacteria. In this case, antibody concentration was too high, both bacteria and liposome congregate together and was difficult to move. The results of MSD evaluation
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suggested that the liposome with bacteria moved broader compared to the liposome without bacteria. The mobility of liposome with bacteria was higher than that of liposome without bacteria. The bacteria attached to the liposome were the main source of propulsion. We used oil water droplet method for making liposome. This method have some advantages, especially inner contents of liposome could be change freely. Therefore we could package any good candidate for therapy or environmental control and so on. Furthermore, we also used bacteria as actuator. Some bacteria have several properties, for example taxis systems, bacteria do not move randomly but show tactic movements toward or away from environmental stimuli, such as chemical substances (chemotaxis), temperature (thermotaxis), light (phototaxis), magnetic field (magnetotaxis) and so on. Our proposed configuration realizes to add these bacterial properties to liposome delivery systems. Acknowledgements This work was partially supported by Grant-in-Aid for the Global COE Program “COE for Education and Research of Micro-Nano Mechatronics”, Grant-in-Aid for Scientific Research on Innovative Areas (23106005, 23106006, 24115507) and the Hori Foundation. We thank M. Homma and S. Kojima for providing the V. alginolyticus strains, K. Nogawa, K. Sekiyama and K. Takiguchi for useful discussions. References [1] A. Cavalcanti, B. Shirinzadeh, T. Fukuda, S. Ikeda, Nanorobot for brain aneurysm, International Journal of Robotics Research 28 (2009) 558–570. [2] S. Martel, O. Felfoul, J.-B. Mathieu, A. Chanu, S. Tamaz, M. Mohammadi, M. Mankiewicz, N. Tabatabaei, MRI-based medical nanorobotic platform for the control of magnetic nanoparticles and flagellated bacteria for target interventions in human capillaries, International Journal of Robotics Research 28 (2009) 1169–1182. [3] G.M. Patel, G.C. Patel, R.B. Patel, J.K. Patel, M. Patel, Nanorobot, A versatile tool in nanomedicine, Journal of Drug Targeting 14 (2006) 63–67. [4] R.A. Freitas Jr., The ideal gene delivery vector: chromallocytes, cell repair nanorobots for chromosome replacement therapy, Journal of Evolution and Technology 16 (2007) 1–97. [5] D.A. LaVan, T. McGuire, R. Langer, Small-scale systems for in vivo drug delivery, Nature Biotechnology 21 (2003) 1184–1191. [6] H. Hess, V. Vogel, Molecular shuttles based on motor proteins: active transport in synthetic environments, Reviews in Molecular Biotechnology 82 (2001) 67–85. [7] Y. Yoshida, R. Yoshida, R. Yokokawa, H. Suzuki, K. Atsuta, H. Fujita, S. Takeuchi, Biomolecular linear motors confined to move upon micropatterns, Journal of Micromechanics and Microengineering 16 (2006) 1550–1554. [8] N. Mori, K. Kuribayashi, S. Takeuchi, Artificial flagellates analysis of advancing motions of biflagellate micro-objects, Applied Physics Letters 96 (2010) 083701. [9] R.K. Soong, G.D. Bachand, H.P. Neves, A.G. Olkhovets, H.G. Craighead, C.D. Montemagno, Powering an inorganic nanodevice with a biomolecular motor, Science 290 (2000) 1555–1558. [10] B. Behkam, M. Sitti, Effect of quantity and configuration of attached bacteria on bacterial propulsion of microbeads, Applied Physics Letters 93 (2008) 223901–223903. [11] B. Behkam, M. Sitti, Bacteria flagella-based propulsion and on/off motion of microscale objects, Applied Physics Letters 90 (2007) 23902–23904. [12] Y. Hiratsuka, M. Miyata, T. Tada, T.Q.P. Uyeda, A microrotary motor powered by bacteria, Proceedings of the National Academy of Sciences of the United States of America 103 (2006) 13618–13623. [13] N. Darnton, L. Turner, K. Breuer, H.C. Berg, Moving fluid with bacterial carpets, Biophysical Journal 86 (2004) 1863–1870. [14] S. Martel, M. Mohammadi, O. Felfoul, Z. Lu, P. Pouponneau, Flagellated magnetotactic bacteria as controlled MRI-trackable propulsion and steering systems for medical nanorobots operating in the human microvasculature, International Journal of Robotics Research 28 (2009) 571–582. [15] K. Nogawa, M. Kojima, M. Nakajima, S. Kojima, M. Homma, T. Fukuda, Rotational speed control of Na+-driven flagellar motor by dual pipettes, Ieee Transactions on Nanobioscience 8 (2009) 341–348.
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Biographies Masaru Kojima received Doctor of Science degree from Nagoya University in 2006. He had been Research Fellow with the Department of Biology, Nagoya University in 2007, Research Fellow on Global Center Of Excellence (GCOE) with the Department of Biology, Nagoya University in 2008, Assistant Professor on GCOE with the Department of Micro-Nano Systems Engineering, Nagoya University from 2008, Assistant Professor in Department of Systems Innovation, Osaka University from 2012. His main research interest is biophysics, in particular, using the biological object and proteins as bio actuator. Zhenhai Zhang received doctor degree from Beijing Institute of Technology in 2008. He had been postdoctoral researcher in Beijing Institute of Technology from 2008, he joined Department of Micro-Nano Systems Engineering, Nagoya University as a Visiting researcher from 2010 to 2011. His research interest is MEMS/NEMS sensors and micro/nano bio robot. Masahiro Nakajima received Doctor of engineering degree from Nagoya University. He had been a Research Fellow of the Ministry of Education, Culture, Sports, Science and Technology, Nagoya University in 2006, Research Associate in Department of Micro-Nano Systems Engineering, Nagoya University from 2006, Assistant Professor in Department of Micro-Nano Systems Engineering, Nagoya University from 2007, Assistant Professor in Center For Micro-Nano Mechatonics, Nagoya University from 2009. He has interest in the research field of the applications of micro/nano manipulation, micro/nano-mechanics, nano-biology. Katsutoshi Ooe received Doctor Degree from Nagoya University in 2001. He joined Osaka Institute of Technology as a Postdoctoral researcher from 2001, Ritsumeikan University as a Associate Professor from 2004, From 2009, as a Associate Professor on GCOE with the Department of Micro-Nano Systems Engineering, Nagoya University, Associate Professor in Department of machine system engineering, Daiichi Institute of Technology from 2012. He is mainly engaging in the research field of biomedical engineering. Toshio Fukuda received the Doctor of Engineering from the University of Tokyo in 1977. He joined in the National Mechanical Engineering Laboratory, in Japan, from 1977. He joined the Science University of Tokyo in Japan from 1982. He joined Department of Mechanical Engineering, Nagoya University in Japan as a professor from 1989. He is mainly involved in the research field of intelligent robotic and mechatronic system, celler robotic system, micro and nano robotic system.