Journal of Oceanology and Limnology   2021, Vol. 39 issue(6): 2107-2115     PDF       
http://dx.doi.org/10.1007/s00343-021-0420-7
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

CHEN Changyou, WANG Pingping, WU Long-Fei, SONG Tao
Biocompatibility of marine magnetotactic ovoid strain MO-1 for in vivo application
Journal of Oceanology and Limnology, 39(6): 2107-2115
http://dx.doi.org/10.1007/s00343-021-0420-7

Article History

Received Oct. 29, 2020
accepted in principle Nov. 29, 2020
accepted for publication Apr. 1, 2021
Biocompatibility of marine magnetotactic ovoid strain MO-1 for in vivo application
Changyou CHEN1,2, Pingping WANG1,2, Long-Fei WU2,3, Tao SONG1,2,4     
1 Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190, China;
2 France-China Joint Laboratory for Evolution and Development of Magnetotactic Multicellular Organisms, Chinese Academy of Sciences, Beijing 100029, China;
3 Aix Marseille University, CNRS, LCB, Marseille F-13402, France;
4 University of the Chinese Academy of Sciences, Beijing 100049, China
Abstract: Magnetotactic bacteria are capable of biosynthesizing magnetic nanoparticles, also called magnetosomes, and swimming along magnetic field lines. The abilities endow the whole cells of magnetotactic bacteria with such applications as targeted therapy and manipulation of microrobots. We have shown that the intact marine magnetotactic bacteria MO-1 kill efficiently antibiotic-resistant pathogen Staphylococcus aureus in vivo, but the biocompatibility of this marine bacterium is unknown. In this study, the strain MO-1 was chosen to analyze its biocompatibility and potential for biomedicine applications. Results showed that MO-1 cells could be guided at 37 ℃ under an external magnetic field and swim in the blood plasma and urine. They could keep active locomotivity within 40 min in the plasma and urine, although their velocity slowed down. When incubated with human cells, magnetotactic bacteria MO-1 had no obvious effects on cellular viability at low dose, while the cell toxicity increased with the augmentation of the quantity of the MO-1 cells added. In the in-vivo experiments, the median lethal dose of magnetotactic bacteria MO-1 in rats was determined to be 7.9×1010 bacteria/kg. These results provided the foundation for the biocompatibility and safety evaluations of magnetotactic bacteria MO-1 and suggested that they could be basically used in clinical targeted therapy.
Keywords: magnetotactic bacteria    motility    cell interaction    median lethal dose    
1 INTRODUCTION

Magnetotactic bacteria (MTB) are a group of phylogenetically diverse and Gram-negative microorganisms with the ability of synthesizing magnetite (Fe3O4) or greigite (Fe3S4) nanoparticles in a genetically controlled manner (Blakemore, 1982; Bazylinski and Frankel, 2004; Simmons et al., 2006; Faivre and Schüler, 2008). These nanoparticles, also called magnetosomes, are arranged in a chain-like manner, which helps magnetotactic bacteria to swim along magnetic lines (Lefèvre and Wu, 2013). Magnetotactic bacteria also have the flagella that enable them to swim autonomously and efficiently (Zhang and Wu, 2020). Based on different types of swimming behavior, magnetotactic bacteria can be divided into two main categories: one is polar strain that swims persistently in one direction along the magnetic field, and the other one is axial strain that swims in either direction along magnetic field lines (Bazylinski and Frankel, 2004). The features of magnetosomes and autonomous movements along the magnetic field endow them with rapid responsibility to external magnetic field.

As a biosynthetic magnetic nanoparticle, magnetosome is coated by a biomembrane, which could be easily decorated for in vitro uses, such as DNA extraction and immunoassays (Matsunaga et al., 2004). Magnetosomes could also be used in vivo as a contrast agent (Hu et al., 2010; Heinke et al., 2017), drug carriers (Sun et al., 2007; Alphandéry et al., 2017), and a tumor photo-/magneto-thermal transmitter (Alphandéry et al., 2011, 2012; Chen et al., 2016a). However, they are not capable of autonomous swimming and do not target the deep area in the body even when an external magnetic field is applied, thereby greatly limiting their applications.

Applications of the whole cell of magnetotactic bacteria have recently attracted considerable attention. The whole magnetotactic bacteria could not only be guided to target the deep area but also perform as energy converters, which are conducive to microrobot construction and targeted therapy (Fdez-Gubieda et al., 2020). Magnetotactic bacteria are suitable actuators of bacterial microrobot due to their autonomous swimming ability. Magnetosopirrillum gryphiswalense (MSR-1), an axial strain, was constructed as a guidable delivery vehicle for cargo transport and release (Alsaiari et al., 2016). It was also integrated with ciprofloxacin-loaded microtubes to build controllable microrobots, and the robots could target an infectious biofilm under the guidance of an external magnetic field and then release the antibiotics (Stanton et al., 2017). A polar magnetotactic bacterium may be more suitable as an actuator than the above axial strain because it swims persistently in one direction along the magnetic field. A polar marine magnetotactic coccus, designated strain MC-1 is one of the best actuators for building microrobots. MC-1 bacteria pushed a microsphere and swam along the magnetic field, thus showing a great potential in manipulated movement under large weight loading (Martel et al., 2006). Further, MC-1 cells were used to transport drug-loaded nanoliposomes to target the tumor in the blood under the control of magnetic field (Taherkhani et al., 2014; Felfoul et al., 2016). Another polar magnetotactic bacterium, magnetotactic ovoid strain MO-1, is also a highly suitable candidate for constructing microrobots. Magnetotactic ovoid strain MO-1 was isolated from the Mediterranean Sea, and it is a kind of chemoautotrophic bacterium (Lefèvre et al., 2009). MO-1-derived microrobots were constructed by attaching microbeads to the cell surface, and the microrobots could swim in the microfluidic channel under the control of magnetic field (Ma et al., 2012). MO-1 cells have also been fabricated as a separating microrobot to target separate pathogens and then kill them using different external magnetic fields (Chen et al., 2014, 2017b). By using magnetosomes in the cell body, intact magnetotactic bacteria could produce positive magnetic resonance imaging contrast for visualizing mouse tumor xenografts, thereby providing a potential tool for improved cancer imaging visualization in preclinical and translational studies (Benoit et al., 2009). The whole cell of magnetotactic bacteria could also transmit the magnetic field energy into the thermal energy through the intracellular nanoparticles. When applied in the alternating magnetic field, they produced heat and then strongly affected cancer cell proliferation on clinical conditions (Gandia et al., 2019). Magnetotactic bacterium-based magnetic hyperthermia was also used to target and kill pathogenic cells, thus could potentially treat infectious diseases (Chen et al., 2016b). Aside from thermal energy, magnetotactic bacteria could transmit the magnetic field energy to the mechanical force, which could inhibit the viability of pathogen (Chen et al., 2017a).

Although the whole magnetotactic bacteria show many potentials in the construction of microrobots and targeted therapy, their biocompatibility and biosecurity should be considered seriously. On the one hand, when magnetotactic bacteria enter into the body fluid, such as blood and urine, for drug delivery and targeted treatment, their status and swimming ability require sufficient evaluation. When incubated in the blood, the terminal velocity of Magnetococcus marinus strain MC-1 decayed for a duration of 40 min (Martel et al., 2009). On the other hand, magnetotactic bacteria, as foreign substances, are certainly attacked by the immune system in the human body, and the toxicity should be assessed. As for magneto-ovoid strain MO-1, which has been used for the construction of bacterial microrobots and targeted therapy, the biocompatible studies in the blood and urine are still unknown until now, and they need to be carried out.

In the present study, magneto-ovoid strain MO-1 was used to investigate its biocompatibility for in vivo applications. The swimming ability of MO-1 bacteria in the blood and urine was first assessed. The interaction of bacteria and human cells was examined to show the biocompatibility and the cellular safe dose. Finally, the median lethal dose (LD50) of magnetotactic bacteria in rats was determined for in vivo biomedical utilization.

2 MATERIAL AND METHOD 2.1 Material

Chemical regents containing NaCl, MgCl2, Na2SO4, CaCl2, KCl, NH4Cl, NaHCO3, K2HPO4, and NaNO3 were purchased from BBI Co., Ltd. Succinic acid, L-cysteine, tryntone, and yeast extract were bought from AMRESCO Commercial Finance, LLC. MTT was purchased from Sigma-Aldrich. The mineral solution and vitamin solution for magnetotactic ovoid strain MO-1 were prepared in accordance with previous reports (Frankel et al., 1997). Ferric quinate was also prepared. Artificial urine was purchased from Qingdao Jisskang Biotechnology Co., Ltd. (China), and its pH was adjusted to 7.5 by using NaOH solution.

2.2 Animal

Adult male Sprague-Dawley (SD) rats weighing 180–200 g were used in the LD50 experiments and purchased from Beijing HFK Bio-Technology Co., Ltd. (China). Blood was acquired after the rats were anesthetized, and then the plasma was obtained by centrifuging the rat blood.

2.3 Bacterial culture and transmission electron microscopy (TEM)

The magnetotactic ovoid strain MO-1 was grown at room temperature in EMS2 medium as previously reported (Lefèvre et al., 2009). The EMS2 medium was first added with (per liter of deionized water; pH 6.8) 19.45-g NaCl, 5.9-g MgCl2, 3.24-g Na2SO4, 1.8-g CaCl2, 0.55-g KCl, 0.2-g NH 4Cl, 1.26-g NaHCO3, 0.6-g L-cysteine, 3-g HEPES, and 0.35-g agarose. The medium was then autoclaved at 120 ℃ for 20 min. After the medium was cooled to approximately 45 ℃, the following solutions were added (per liter) to previously sterile-filtered stock solutions in the following order: 0.5-mL vitamin solution, 0.5-mL ferric quinate (0.4 mmol/L), 3-mL sodium thiosulfate (40% w/v), 4-mL dipotassium hydrogen phosphate (1.86% w/v), and 5-mL modified Wolfe's mineral solution. Ultrathin copper grids were immersed in the bacterial suspensions to observe the MO-1 cell morphology, and then the grids were dried in a clean bench after retrieval for observation using a transmission electron microscope (Hitachi H-7650B; Hitachi, Japan).

2.4 Speed analysis of magnetotactic bacteria in plasma and urine

Speed analysis of magnetotactic bacteria MO-1 in the plasma and urine was carried out in a microfluidic chip. The microfluidic channel contains four corners, and its plane geometry was shown in the previous report (Ma et al., 2012). The width and depth of the channel were 200 μm and 20 μm, respectively. The rat plasma or artificial urine was pushed into a microfluidic channel. After one of the micropools in the chip was blocked, the MO-1 solution was injected into the other micropool. A magnetic field was applied to guide the MO-1 cells in the microfluidic channel. The movement of MO-1 swarm in the plasma or artificial urine was observed under 40×magnification in the dark-field mode. The swimming behavior was recorded using a Canon 500D camera that recorded 1 920×1 080 pixel images at 20 frames per second. The experiments were conducted at 37 ℃ to mimic the in vivo biomedical applications of human physiological temperature and repeated thrice. The temperature controlling instrument of Live Cell Imaging System was used to keep the microfluidic chip with MO-1 cell samples at 37 ℃. All the blood and urine solutions were at human physiological pH. The average swimming velocities were calculated using the MTrackJ plugin for ImageJ (Murat et al., 2015; Zhang et al., 2017).

2.5 Cell culture and MTT assay

Human immortalized epidermal cell HaCaT (No. 3111C0001CCC000373) and human cervical carcinoma cell Hela (No. 3111C0001CCC000011; China Infrastructure of Cell Line Resources, Beijing, China) were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Australian origin, Hyclone Laboratories, Logan, UT, USA), 100-IU/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 100-μg/ mL streptomycin (Sigma-Aldrich) at 37 ℃ in 5% CO2. The HaCaT and Hela cells were seeded at 10 000 cells/well in 100-μL DMEM in a 96-well plate and incubated for 24 h. The medium was then replaced with the magnetotactic bacteria MO-1 working solution for the succeeding experiments. The magnetotactic bacteria MO-1 working solution (per well in 100-μL DMEM) contains 0 (control sample), 2×105, 4×105, 8×105, 1.6×106, 3.3×106, 6.5×106, 1.3×107, 2.5×107, and 5×107 bacteria.

After incubation was performed for 24 h, the viability of the HaCaT and Hela cells was analyzed by MTT method. In brief, the cell medium was replaced with 100-μL DMEM without phenol red (Gibco) containing MTT agent and incubated at 37 ℃ in the dark for 4 h. The medium was discarded, and 100 μL of dimethyl sulfoxide (Sigma-Aldrich) was added to the well. After shaking for 15 min, the cell plates were centrifuged at 3 000 r/min for 5 min, and the supernatant was transferred to a new 96-well plate. The absorbance was measured at 570 nm with a reference at 630 nm by using a microplate absorbance reader (168-1130 model, Bio-Rad Laboratories, Hercules, CA, USA). The relative cell viability (%) relative to control was calculated using the following equation: (Asample/Acontrol)×100% (where A is the absorbance). The experiments were repeated thrice.

2.6 LD50 of magnetotactic bacteria in rats

The magnetotactic ovoid strain MO-1 in exponential growth was collected using a magnetic block and suspended in phosphate buffer solution (PBS, pH7.0). Then, 16 SD rats were randomly assigned to four groups (n=4), and 5×108, 5×109, 5×1010, and 5×1011 MO-1 cells in 3-mL PBS were injected into four groups of rats intravenously. The animals in each group were observed for 1 week. Afterwards, Behrens-Kärber method was used to calculate the LD50 and 95% confidence interval (CI) (Zhang et al., 2015):

where Xm is the logarithm of the maximum dose; i is the difference in the logarithm of adjacent concentrations; p and q are the lethal success and failure rates in each group, respectively; and n is the number of rats in each group.

3 RESULT AND DISCUSSION 3.1 Characterization of magnetotactic bacteria MO-1

Magneto-ovoid strain MO-1 is a marine species affiliated with zeta proteobacterial strains, and it grew at aerobic–anaerobic interfaces in the culture flasks. The TEM results showed that magnetotactic bacteria MO-1 owned two flagellar bundles on the long hemisphere of the cell (Fig. 1a, black arrows). They have an autonomous swimming ability toward North Pole in a magnetic field. TEM images showed that they also synthesized bio-membrane-coated magnetosomes with an average number of 11.1±2.94 (Fig. 1b), which assisted them to feel and move along the magnetic fields. The morphologies of most MO-1 magnetosome particles have an elongated cubo-octahedral shape, with the length of 77.06±11.1 nm, width of 71.54±10.59 nm respectively (n=149). The features of fast autonomous movement and magnetotaxis may lead them to reach the deep site inside the organism body for drug delivery and targeted therapy.

Fig.1 Magnetotactic ovoid strain MO-1 a. magnetotactic ovoid strain MO-1. Black arrows indicated the two flagellar bundles and white arrow indicated the magnetosome chain; b. number of magnetosome (1 432) in 129 MO-1 cells.
3.2 Speed of magnetotactic bacteria MO-1 in rat plasma and artificial urine

Movement ability is an important characteristic for MO-1 cells to perform a task. Generally, in vivo application scenarios in biomedical areas are always accompanied with the body liquid environment, such as blood and urine. Here, the magnetotactic velocity of magnetotactic bacteria MO-1 in the blood plasma and artificial urine was assessed at the human physiological temperature of 37 ℃ to mimic the in vivo biological application scenarios. The rat plasma was acquired, and the MO-1 cells and the plasma were injected into the microfluidic chip. At 37 ℃, when a magnetic field with a strength of 2.2 mT was applied, MO-1 cells could swim along the magnetic field in the blood plasma liquid, the fresh MO-1 cells swam at the beginning speed of about 300 μm/s, while the swimming speed decreased as the time pasted (Fig. 2a & b). In the first 4 min, the MO-1 cells swam at the velocity of above 200 μm/s and then travelled at more than 100 μm/s in the following 20 min. In the next time, the MO-1 cells continued to swim at a lower speed below 100 μm/s. During the entire process, the MO-1 cells were capable of responding to the external magnetic field guidance at 37 ℃ (Fig. 2b). The experimental results showed that magnetotactic bacteria MO-1 could survive and keep their swimming motility in the plasma at the human physiological temperature for at least 45 min.

Fig.2 Speed of magnetotactic bacteria MO-1 in rat plasma under external magnetic field a. the swimming speed of magnetotactic bacteria MO-1 in the plasma at 37 ℃ based on 450 bacterial cells; b. the movement trajectories of MO-1 cells along magnetic field in plasma at 37 ℃.

When artificial urine was used as another model of body fluid, the state of magnetotactic bacteria MO-1 at the human physiological temperature (37 ℃) was similar to that in the blood plasma (Fig. 3a). The MO-1 cells could be navigated by an external magnetic field and swim in the urine at a speed of above 200 μm/s during the first 6 min. The bacteria over time continued swimming at the speed of more than 130 μm/s for 20 min. Finally, their speed further decreased in the next time but still moved in the urine at a lower speed below 100 μm/s (Fig. 3a & b). The swimming experimental results confirmed that the MO-1 cells could survive and swim for at least 40 min in the urine at physiological temperature.

Fig.3 Speed of magnetotactic bacteria MO-1 in artificial urine under external magnetic field a. the swimming speed of magnetotactic bacteria MO-1 in the urine at 37 ℃ based on 296 bacterial cells; b. the movement trajectories of MO-1 cells along magnetic fields in the urine at 37 ℃.

Targeted therapy or drug delivery in the human body mediated by magnetotactic bacteria probably need the ability of these bacteria to swim autonomously in the body fluid. The results of this study demonstrated that magnetotactic bacteria MO-1 are capable of surviving and swimming in the blood and urine. According to the average speed and surviving time, the distance that magnetotactic bacteria MO-1 swam in the blood for 45 min or in the urine for 40 min at physiological temperature could even reach up to approximately 40 cm. Considering the size of human organ and the principle of proximal intervention with external carriers, the magnetotactic bacteria MO-1 swimming distance is enough to satisfy the requirements of drug delivery and targeted therapy. The blood results are also similar to those when other magnetotactic bacteria of MC-1 were used (Martel et al., 2009). Other factors, such as blood flow velocity and vascular branch, were not considered in the present study. However, it was reported that magnetotaxis enabled MTB to overcome counter-directional flow and migrate effectively through porous micromodels (Rismani Yazdi et al., 2018a, b). As to the scene that the velocity of MO-1 cells actually decreased as the incubation time increased, the main reason is possibly the osmotic pressure induced by different salt concentrations. For example, the NaCl concentrations in the blood and urine are 8.5 and 4.2 g/L respectively, much lower than those (19.45 g/L) needed by MO-1 to grow. Consistently, it has also been implied that the velocity of the bacteria could also be controlled by varying the concentration of Na+ in the medium (Lefèvre et al., 2009). Besides, a magnetic field with a higher strength could slow down the speed of magnetotactic bacteria (Yang et al., 2012). It is also suggested from the survival and movement abilities that magnetotactic bacteria do not proliferate spontaneously inside an animal nor cause severe infection and pathogenicity. Thus, their safety for the in vivo application is much better than that of other pathogenic bacteria.

3.3 Interaction of magnetotactic bacteria MO-1 with human HaCaT and Hela cells

Although the infection and pathogenicity of magnetotactic bacteria as a therapeutic tool may be negligible according to the analysis, the dose effect at the cellular level and the in vivo toxicity of magnetotactic bacteria MO-1 remained to be studied. In the present study, the biocompatibility and biological safety dose of magnetotactic bacteria MO-1 at the cellular level were examined via MTT assay. HaCaT and Hela cells (1×104 cells) were treated with magnetotactic bacteria MO-1 at different bacterial cell concentrations ranging from 0 to 5×107 bacteria/well in 100-μL DMEM in a 96-well plate for 24 h. As shown in Fig. 4a, the incubation of MO-1 cells at the concentration of less than 3.3×106 bacteria/well had no effect on the viability of the HaCaT cells. The cellular viability decreased by approximately 40% at the concentration of 6.5×106 bacteria/well and continued to decrease as the number of MO-1 cells was increased to 5×107 bacteria/well (Fig. 4a). As for Hela cells, co-culture of MO-1 cells did not induce cell toxicity at the bacterial concentration of less than 1.6×106 bacteria/well (Fig. 4b). However, when the MO-1 concentration was at 3.3×106 bacteria/well, the viability of Hela cells decreased to about 80% and continued to decrease with the increased concentration of magnetotactic bacteria MO-1. A potential reason for the increased cytotoxicity may be that increased magnetotactic bacteria touched with cells and then were phagocytosed, leading to increases in oxidative stress and reactive oxygen species inside the cell (Wang et al., 2014, 2017).

Fig.4 The cell viability of HaCaT (a) and Hela (b) cells after interaction with magnetotactic bacteria MO-1

Previous reports showed that 6.5×106 bacteria of Magnetococcus marinus strain MC-1 incubated with 1×105 murine J774A.1 and NIH/3T3 cells in a 96-well plate induced a huge decrease in cellular viability to approximately 30% (Taherkhani et al., 2014). The decrease in cellular viability caused by magnetotactic bacteria MO-1 in the present study was much smaller than the results of strain MC-1. The difference may be attributed to the different cell lines and added bacterial species. The method of coating nanoliposome on the surface of MC-1 bacteria was proposed to reduce the toxicity of magnetotactic bacteria (Taherkhani et al., 2014).

3.4 Determination of LD50 of magnetotactic bacteria in rats

From an application perspective, the effect of magnetotactic bacteria on the whole human body must be considered. Different doses of magnetotactic bacteria MO-1 were injected into rats in each test group to acquire the LD50, and the rats were observed persistently for 1 week. As shown in Fig. 5, the survival rate decreased as the doses of magnetotactic bacteria MO-1 were increased. The experimental animals did not die in the groups of 5×108 bacteria/ (3 mL), one died in the 5×109 bacteria/(3 mL) group, three died in the 5×1010 bacteria/(3 mL) group, and all animals died in the 5×1011 bacteria/(3 mL) group. Behrens-Kärber method was used to calculate the LD50 of the rats (0.2 kg) at 7.9×1010 bacteria/kg, with a 95% CI of 1.99×1010–3.155×1011 bacteria/kg. According to a guidance (U.S. Food and Drug Administration, 2005), the dose per unit body weight of an adult (60 kg) is equivalent to 0.16 of that of rats (0.2 kg). Therefore, the LD50 of an adult human (60 kg) was calculated to be 1.264×1010 MO-1 bacteria/kg.

Fig.5 Determination of LD50 of magnetotactic bacteria in rats

With the abilities of magnetic nanoparticle synthesis and autonomous swimming, magnetotactic bacteria have been used as contrast agents and in vivo drug delivery. As a contrast agent, Magnetospirillum magneticum AMB-1 was intravenously injected into mice with 1×109 cells (Benoit et al., 2009). The number of magnetotactic bacteria MC-1 intravenously injected into rats for drug delivery was reported to be 1×108 cells (Felfoul et al., 2016). Thus, the actual quantity of magnetotactic bacteria for in vivo uses was much less than its LD50 (7.9×1010 MO-1 bacteria/ kg in rats) obtained in the present study. Of course, different species of magnetotactic bacteria may have different LD50 values, thereby needing further investigations. Regardless of the species of magnetotactic bacteria, after entering the body from the blood vessels or urethra, these bacteria may interact with the initially encountered organ or tissue and then interact with different kinds of cells, especially immune cells. Whether magnetotactic bacteria induced an intense immune response or not depends mainly on the immunogenicity and quantity of the bacteria. In any case, as a kind of Gram-negative bacteria, the endotoxins of magnetotactic bacteria MO-1 should be paid with sufficient attention when used in biomedical applications (Alphandéry, 2020). On one hand, agents such as poly-L-lysine could be modified to reduce or eradicate the endotoxins of magnetotactic bacteria (Hamdous et al., 2017; Mandawala et al., 2017). On the other hand, rats could actually overcome some endotoxins of MO-1 cells according to the study. Nevertheless, the endotoxins of magnetotactic bacteria / magnetosomes released from bacterial cells into the animal body should be considered seriously. Taken together, based on the LD50 obtained in the present study, the dose of MO-1 cells may be safe and it could satisfy the requirement of clinical tumor therapy.

4 CONCLUSION

The biocompatibility of magnetotactic bacteria was determined. The MO-1 cells were able to swim along magnetic field lines in blood and urine and survived 37 ℃ for approximately 40 min. When HaCaT and Hela cells were incubated with magnetotactic bacteria MO-1, their cellular effect was dependent on the dose below 3.3×107 bacteria/mL, which was well tolerated by human cells. In the animal experiments, an LD50 of 7.9×1010 bacteria/kg in rats was determined. These data provided the key to understand the biocompatibility and safety of magnetotactic bacteria to a certain extent and suggested that magnetotactic ovoid strain MO-1 could be used in targeted therapy. However, the distribution of whole bacteria in the body should be further studied.

5 DATA AVAILABILITY STATEMENT

The data supporting the findings of this study are shown in this article. Otherwise, please contact the corresponding author on reasonable request.

6 ACKNOWLEDGMENT

We thank Dr. Haitao CHEN for the assistance in analyzing the swimming speed of magnetotactic bacteria MO-1 in plasma and urine by using the MTrackJ plugin for ImageJ.

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