Determination of the heating effi ciency of magnetotactic bacteria in alternating magnetic f ield*

Tongwei ZHANG , Huangtao XU , Jia LIU , Yongxin PAN , Changqian CAO ,

1 Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Innovation Academy for Earth Science,Chinese Academy of Sciences, Beijing 100029, China

2 Innovation Academy for Earth Science, Chinese Academy of Sciences, Beijing 100029, China

3 College of Earth and Planetary Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

4 France-China Joint Laboratory for Evolution and Development of Magnetotactic Multicellular Organisms, Chinese Academy of Sciences, Beijing 100029, China

Abstract Magnetotactic bacteria (MTB) intact cells have been applied in magnetic hyperthermia therapy of tumor, showing great effi ciency in heating for tumor cell inhibition. However, the detailed magnetic hyperthermia properties and optimum heat production conditions of MTB cells are still poorly understood due to lack of standard measuring equipment. The specif ic absorption rate (SAR) of MTB cells is often measured by home-made equipment at a limited frequency and magnetic f ield amplitude. In this study,we have used a commercial standard system to implement a comprehensive study of the hyperthermic response of Magnetospirillum gryphiswaldense MSR-1 strain under 7 frequencies of 144-764 kHz, and 8 f ield amplitudes between 10 and 45 kA/m. The measurement results prove that the SAR of MTB cells increases with magnetic f ield frequency and amplitude within a certain range. In combination with the magnetic measurements, it is determined that the magnetic hyperthermia mechanism of MTB mainly follows the principle of hysteresis loss, and the heat effi ciency of MTB cells in alternating magnetic f ield are mainly aff ected by three parameters of hysteresis loop, saturation magnetisation, saturation remanent magnetisation, and coercivity. Thus when we culture MTB in LA-2 medium containing sodium nitrate as source of nitrogen, the SAR of MTB LA-2 cells with magnetosomes arranged in chains can be as high as 4 925.6 W/g (in this work, all SARs are calculated with iron mass) under 764 kHz and 30 kA/m, which is 7.5 times than current commercial magnetic particles within similar size range.

Keyword: magnetotactic bacteria (MTB); hyperthermia; rock magnetism; alternating magnetic f ield (AMF)

1 INTRODUCTION

Magnetic hyperthermia is an important treatment strategy of diseases based on raising the temperature of local-regional tissues or whole body above physiological value by converting the magnetic energy into thermal energy. It has gone into clinic testing for therapy of malignant tumors due to its intrinsic high tissue penetration and non-invasion (Falk and Issels, 2001;Rosensweig, 2002; Johannsen et al., 2007; Périgo et al.,2015; Blanco-Andujar et al., 2018). Tumor tissues are more susceptible to heat than healthy tissue. When temperatures reach the 41-46 ℃ range (therapeutic window), tumor tissue tends to suff er irreversible damage, while healthy tissue is usually unharmed(Pankhurst et al., 2003; Ortega and Pankhurst, 2013). In clinical trials, it has been proved that hyperthermia combined with conventional treatment modalities (e.g.,radiotherapy, chemotherapy) can improve response and survival outcome of tumor patients (Thiesen and Jordan, 2008; Maier-Hauff et al., 2011).

During the magnetic hyperthermia treatment process, magnetic nanoparticles (MNPs) with various sizes, ranging from superparamagnetism to magnetic single-domain (roughly 10 up to 100 nm), can be employed to complete the energy conversion through hysteresis loss or Néel/Brownian relaxation process in an alternating magnetic f ield (AMF) (Rosensweig,2002; Hergt et al., 2006; Dadfar et al., 2019; Xu and Pan, 2019). Usually, the energy conversion effi ciency is expressed by specif ic absorption rate (SAR), which is def ined as the absorbed energy per unit of nanoparticle mass (Mason et al., 2000; Ortega and Pankhurst, 2013).The SAR is directly related to the initial slope of the temperature rising curves, and calculated using the next Eq.1 (Andreu and Natividad, 2013),

wheremsis the mass of solvent,mnis the mass of MNPs,Cpis the heat capacity of solvent, and ΔT/Δtis the initial slope of the temperature rising curve: f itting the curve with Box-Lucas equation [T(t)=a(1-e-bt)] to calculate the ΔT/Δtatt=0 (Kallumadil et al., 2009).Experimental results show that single-domain particles with narrow size distribution may theoretically provide the most desired heat generation effi ciency (Hergt et al., 2008). High SAR ensures enhancement of specif ic heating power and reduction of dosage applied to the tumor.

MTBs are a kind of aquatic microorganisms that can live in oxic-anoxic interface (OAI) environment and swim along the Earth’s magnetic f ield lines(Blakemore, 1975; Frankel et al., 1997). A unique feature of this type of bacteria is that it has nanometersized (30-120 nm) magnetosomes (Fe3O4/Fe3S4)arranged in chains, which show characteristic singledomain magnetic properties (Bazylinski and Frankel,2004; Ding et al., 2010; Zhang and Pan, 2018).Magnetosome is a kind of nanomaterial with wide application potential, which has been developed into excellent nanocarriers, such as anti-cancer drug carriers (Sun et al., 2007), transgenic carriers (Yang et al., 2016), and antibody carriers (Li et al., 2010). In addition, MTB Whole cells have been developed into nanobots for antimicrobial benef iting from the unique advantage of self-propulsion capability provided by their f lagella and the guidance capabilities ensured by their magnetosome chain (Chen et al., 2019).Magnetosome chains are usually destroyed during extraction; however, the magnetosome crystals coated by membranes still have high heat effi ciency due to well-developed crystallinity and particle uniformity(Muela et al., 2016), which are regulated strictly by a series of genes (Uebe and Schüler, 2016). Hergt et al.(2005) reported a maximum SAR value of 960 W/g (calculated with magnetosome mass) for magnetosomes with a mean diameter of 30 nm, under 10 kA/m f ield amplitude and 410 kHz frequency.Muela et al. (2016) determined a SAR of 2 394 W/g (calculated with magnetosome mass) for magnetosomes of 45 nm exposed to an AMF of 28 kA/m and a frequency of 532 kHz. In addition,each MTB intact cell with magnetosomes arranged in chains can also be used as magnetic hyperthermia agent, and the SAR of the intact cells is usually much higher than that of the isolated magnetosomes because of the low magnetostatic interactions (Fdez-Gubieda et al., 2020). Alphandéry et al. (2011) obtained the SAR value of 860 W/g (calculated with Fe mass) at 88 mT and 108 kHz for MTB intact cells usingMagnetospirillummagneticumAMB-1 strain. Gandia et al. (2019) directly proved that MTB intact cells had higher SAR than the extracted magnetosomes under the same conditions usingMagnetospirillumgryphiswaldenseMSR-1 strain, and then, the MTB intact cells signif icantly inhibited the proliferation of cancer cells in AMF. Therefore, MTB whole cells are promising agent for targeted therapy of tumors.

However, the detailed magnetic hyperthermia properties and optimum heat production conditions of MTB cells are not clear. Because the AMF conditions including magnetic f ield amplitude and frequency providing for testing magnetic hyperthermia treatment are usually limited due to the lack of commercial standard systems. In addition, the SAR value of MTB cells is often measured by home-made equipment under individual conditions. The inconsistency in accuracy and background caloric value produced from magnetic f ield coil may lead to incomparable results between diff erent laboratories. Hence,benef iting from commercial standard system D5 series (NanoScale Biomagnetics, Zaragoza, Spain)providing AMF with wide range of frequency and amplitude, we implemented a comprehensive study of the hyperthermic response ofMagnetospirillumgryphiswaldenseMSR-1 strain under diff erent magnetic conditions: frequency of 144-764 kHz, and f ield amplitude of 10-45 kA/m. The heating effi ciency was measured using calorimetric method by dual channel f iber optic sensors. We also compared the heating rates of the samples with diff erent magnetic parameters harvested from diff erent medium and commercial magnetic nanoparticles to determine the hyperthermia mechanisms of MTB cells.

2 MATERIAL AND METHOD

2.1 Preparation of sample

Three samples used in this work, including two MSR-1 samples and a kind of commercial magnetic particle (Fe3O4nanoparticles) with mean size about 30 nm bought from Sangon Biotech (Shanghai), are named MTBLA-1, MTBLA-2, and commercial magnetic particle (Scmp), respectively. MTBLA-1was cultured using medium LA-1 and method reported by Sun et al. (2008). MTBLA-2was harvested from medium LA-2 with sodium nitrate instead of ammonium chloride as sources of nitrogen referred to LA-1 and literature of Heyen and Schüler (2003).

Cell growth and magnetism were assessed by OD565(optical density at 565 nm) andCmagas described by literatures (Schüler et al., 1995). The iron concentrations of the samples were determined by the spectrophotometric ferrozine assay based on the standard curve (0, 0.5, 1, 2, 4, 6, 8, and 10 μg/mL)(Stookey, 1970; Han et al., 2020).

Fresh whole cells were collected by centrifugation at 8 000 r/min for 10 min at 4 ℃ after culturing for 24 h in medium LA-2, and then suspended in water with needed cell concentrations. Samples S1, S2, and S3were prepared from cells with concentration of OD565=8 through ultrasonic treatment for 0, 10, and 50 min in an ice-water mixture, respectively.

2.2 Transmission electronic microscopy analysis

Morphological characteristics of samples involved in this work were examined by transmission electron microscopy (TEM, JEOL JEM-2100, Tokyo)operating at 200 kV at the Institute of Geology and Geophysics, Chinese Academy of Sciences (Beijing).The sizes of magnetosome were analyzed using standard analytical software. The major and minor axes of magnetosomes were used as the length (L)and width (W) of the crystal, respectively, and the grain size was def ined as (L+W)/2.

2.3 Rock magnetic measurements

The measurement of room-temperature hysteresis loops, f irstorder reversal curves (FORCs) and saturation isothermal remanent magnetization (SIRM)were performed on a VSM3900 magnetometer(Princeton Measurements Corporation, USA,sensitivity 5.0×10-10Am2). Hysteresis loops were measured with f ield increments of 8 mT, ranging between maximum f ields of ±1.00 T with an average time of 500 ms. SIRM acquisition curves were obtained with logarithmic increments to a maximum f ield of 1.00 T. A total of 180 curves for each sample were measured in FORCs using an increasing f ield step of 1.2 mT with an average time of 400 ms in the range from -50 to 50 mT forBuand from 0 to 100 mT forBc.BuandBcare two parameters in the FORCs test programs, representing magnetostatic interactions and coercivity of samples. The FORC diagrams were processed using FORCinel version 3.06 software with a smooth factor of 3.

2.4 Magnetic hyperthermia analysis

Magnetic hyperthermia measurements of all samples had been performed using a commercial system D5 series (NanoScale Biomagnetics, Zaragoza,Spain) at the Institute of Geology and Geophysics,Chinese Academy of Sciences (Beijing, China).Samples suspended in water (1.0 mL) in a 2-mL glass chromatography vial were set in the middle of the coil.The temperatures during measurement were recorded by dual channel optic f iber temperature probes with response temperature of 0.1 ℃. One probe was placed in the center of samples, and the other probe was in the gap between glass and coil. Every magnetic f ield condition was f irst tested by 1.0-mL water, and the two probes would stabilize to the same temperature, which was the initial temperature of the corresponding testing condition.

The eff ect of AMF parameters on the heat production effi ciency of samples were investigated.Sample MTBLA-2with iron concentration of 48.4 μg/mL was tested at 339 kHz with varying AMF amplitudes (10, 15, 20, 25, 30, 35, 40, and 45 kA/m),and at 30 kA/m with diff erent frequencies (144, 298,339, 377, 485, 621, and 764 kHz), respectively. All the other tests were performed under the magnetic f ield condition of 764 kHz and 30 k/Am.The SAR values were calculated using the Eq.1 as mentioned above, where the solvent was 1-mLwater with the heat capacity of 4.185 J/(g·K).

3 RESULT

3.1 Structural characterizations of MTB cells and magnetosomes

3.1.1 Culture results of MSR-1

After culturing for 24 h, the cells of MTBLA-1and MTBLA-2reached the states of OD565=0.9±0.1,Cmag=0.6±0.1, and OD565=0.9±0.1,Cmag=1.8±0.1,respectively. The high OD values imply that both LA-1 with ammonium chloride and LA-2 with sodium nitrate are suitable for the growth of MSR-1 under the culture conditions. Usually, theCmagis well correlated with the average number of magnetosomes per cell and can be used for semi-quantitative assessment of magnetosome formation (Schüler et al., 1995). Thus,the largerCmagvalue of MTBLA-2indicates that it may contain more magnetosomes, and the medium LA-2 is more suitable for magnetosomes synthesis.

3.1.2 TEM characterization of MTB cells,magnetosomes, and commercial magnetic beads

Figure 1 shows the images of MTBLA-1, MTBLA-2and commercial magnetic particle Scmp. It is obvious that the magnetosomes in MTBLA-1and MTBLA-2are arranged in chains, whereas the Scmpnanoparticles are clustered together (Fig.1a-f). Size analysis reveals that one MTBLA-1cell contains an average of 7.8±3.9 magnetosomes with the average particle size of(27.9±7.9) nm (Fig.1a-b), and the cell of MTBLA-2possess an average of 17.7±5.6 magnetosomes with the average particle size of (35.7±7.7) nm (Fig.1c-d).Compared with MTBLA-2, the MTBLA-1have fewer magnetosomes per cell and bimodal size distributions with a main peak at 32 nm and a secondary peak at 16 nm on the particle size distribution histogram,indicating more immature magnetosomes (Fig.1b),while the MTBLA-2cells have more magnetosomes with larger average particle size, consistent with the results ofCmagdetermination. The true average size of Scmpnanoparticles is (21.2±6.3) nm, which is smaller than the size described in the product specif ication,possibly due to the statistical errors caused by sample aggregation (Fig.1e-f).

As displayed in Fig.1g, the intact cell structures of the sample S2are lost due to ultrasonic treatment for 10 min. With ultrasonic treatment for 50 min, both of the cell structure and magnetosome chain of sample S3are completely destroyed, and the magnetosomes stick together (Fig.1h). The sample S1is not subjected to any ultrasonic treatment. Therefore, the S1remains its original state with complete cell morphology and magnetosome chains.

3.2 Rock magnetic properties

FORC diagrams show very weak magnetostatic interactions (Bu,1/2=1.0 mT) among magnetite particles of MTB cells because of separation by magnetosome membranes and cell structure (Fig.2a-b), which isconsistent with our previous reports (Zhang and Pan,2018). The coercivity value of MTBLA-2(Bc,FORC= 20.4 mT)is larger than that of MTBLA-1(Bc,FORC=17.4 mT),which is in agreement with the TEM results that the MTBLA-2owns larger magnetosomes and longer magnetosome chains (Fig.1a-d). On the contrary, the magnetostatic interactions among magnetite particles in Scmpare signif icantly stronger (Bu,1/2=9.6 mT)(Fig.2c), and the center of FORC diagram is much closer to theY-axis than that of MTBLA-1and MTBLA-2.This means that the coercivity of Scmpis smaller than MTB cells.

Table 1 Magnetic parameters of diff erent MTBs and S cmp

The coercivity (Bc), saturation magnetization (Ms),remanent coercivity (Bcr), and saturation remanent magnetization (Mrs) of MTBLA-1, MTBLA-2, and Scmpare determined by hysteresis loops and SIRM curves,and the results are shown in Fig.2d and Table 1 (Msis normalized by iron mass). It is noted that these samples have similar potbelly shape loop, which is characteristic of cubic type of magnetic anisotropy.The MTBLA-1, MTBLA-2, and Scmpsamples haveBcvalues of 12.7, 18.7, and 3.8 mT;Msvalues of 105.1,116.8, and 69.5 mAm2/g;Bcrvalues of 18.0, 21.6, and 9.8 mT; andMrs/Msvalues of 0.37, 0.48, and 0.11,respectively. As a result, the area surrounded by hysteresis loop, in descending order, are MTBLA-2,MTBLA-1, and Scmp, respectively.The changes in magnetosome chain structure can signif icantly aff ect the rock magnetism of MTB cells.The FORCs diagrams of the two ultrasound-treated samples, S2and S3, are clearly diff erent from that of intact cells, S1, (Fig.2e-g). In particular, there are two hot center spots in FORC diagrams of sample S3. This might have been caused by chain-structure destruction.As the results of hysteresis loops and SIRMs shown in Table 1;Bc,Bcr, andMrs/Msvalues of S1, S2, and S3are reduced in turn, and the corresponding areas of hysteresis loops also decreased in the same order(Fig.2h).

3.3 Heating effi ciency of MTB in AMF

Fig.1 TEM images (a, c, e, g, h) and statistics data of particle size (b, d, f) of MTB samples and commercial magnetic beads

Fig.2 Rock magnetic characterization of samples

Fig.3 Heating effi ciency of MTB cells in AMF

As shown in Fig.3a, the temperature rising curves of MTBLA-2under AMF (30 kA/m, 764 kHz) show that the same kind of MTB cells under the same cell concentration (OD=8 or 20) have the same rate of temperature rise in the overlapped curves. This means that the heat production stability of MTB in AMF is stable and repeatable in aqueous medium. The change in temperature (ΔT) values increase linearly with the concentration of MTB cells (Fig.3b). At the same iron concentration, the ΔTof MTBLA-2at 10 min is higher than that of MTBLA-1(Fig.3b insert), indicating that the magnetosomes of MTBLA-2have stronger heat conversion ability. This is consistent with the results of TEM (Fig.1a-d) and magnetic characterization(Fig.2a-b); therefore, the MTBLA-2cells with more and larger magnetosomes have superior magnetic properties and heat conversion effi ciency.

Fig.4 Temperature rising curves under amplitude of 30 kA/m and frequency of 764 kHz

In order to evaluate the eff ect of magnetic f ield amplitude on the heat production effi ciency of MTB,we tested the temperature rising curves of MTBLA-2with iron concentration of 48.4 μg/mL using AMF frequency of 339 kHz at diff erent amplitudes of 10,15, 20, 25, 30, 35, 40, 45 kA/m. As AMF amplitude increased from 10 to 25 kA/m, the ΔTrises from 1.1 to 6.1 ℃ in 10 min, and the SARs of MTBLA-2are increased from 415.7 to 1 913.8 W/g (Fig.3c-d).However, the ΔTand SARs stop increasing with the amplitude when the amplitude exceeded 25 kA/m.To understand the eff ect of frequency on heat effi ciency of MTB cells, we also perform heating test using the sample MTBLA-2with iron concentration of 48.4 μg/mL as AMF frequency increased from 144 to 764 kHz under the constant amplitude of 30 kA/m.With the frequency increased, the ΔTgrows from 2.4 to 14.0 ℃ at 10 min (Fig.3e), and SARs grow linearly from 788.0 to 4 226.0 W/g (Fig.3f).

3.4 Comparison of heat production effi ciency between MTB and S cmp

The temperature rising curves of MTBLA-1,MTBLA-2, and Scmpwith same iron concentration of 49.2 μg/mL are tested under amplitude of 30 kA/m and frequency of 764 kHz. The ΔTvalues at 10 min are 11.6 ℃ for MTBLA-1, 16.6 ℃ for MTBLA-2, and 2.9 ℃ for Scmp, respectively (Fig.4a), and the corresponding SAR values are 3 440.2 W/g for MTBLA-1, 4 925.6 W/g for MTBLA-2, and 657.3 W/g for Scmp, respectively. As expected, sample MTBLA-2with more and larger magnetosomes possesses stronger ability of heat generation than sample MTBLA-1. Both of the two MTB samples produce more heat than commercial magnetic beads under the same conditions because of the advantages in magnetic properties, such asBc,Ms,Mrs/Ms.

3.5 Eff ect of magnetosome chains on heat production of MTB

To evaluate the eff ect of magnetosome chain integrity on heat production, the temperature rising curves of sample S1, S2, and S3with same iron concentration of 22.3 μg/mL are tested under amplitude of 30 kA/m and frequency of 764 kHz, and the corresponding SAR values were calculated according to the temperature rising curves. It is obvious that the sample S1owing to intact cell structure and magnetosome chain exhibit the strongest ability of heat production with ΔTvalue of 7.1 ℃ at 10 min (Fig.4b). By contrast, the ΔTvalues of S2and S3at 10 min are only 6.3 ℃ and 5.3 ℃, respectively.Likewise, the SAR of S1is 4 643.6 W/g, which is larger than that of S2(4 209.4 W/g) and S3(3 114.6 W/g). This highlights the important role of the intact magnetosome chain for heat generation during the magnetic hyperthermia process.

4 DISCUSSION

There are three physical principles involved in the heat transfer mechanisms involved in magnetic hyperthermia, namely: (i) resistance heating due to eddy currents, (ii) magnetic heating due to hysteresis loss, and (iii) magnetic heating due to Néel and Brownian relaxation processes (Ortega and Pankhurst,2013). In the case of single-domain particles,hysteresis losses are higher than any of the other losses (Hergt et al., 1998). When the magnetic f ield vary with time, the relationship between the area under AC hysteresis loop and the amount of heat generated per cycle (PFM) follows the next equation:

wherefis the frequency of the applied f ield,His the f ield amplitude andMis the magnetization. It is well known that magnetosomes belong to single-domain magnetic particles (Ding et al., 2010; Zhang and Pan,2018), and MTB shows typical rock magnetic characteristics of single-domain (Fig.2a-d), thus the magnetic hyperthermia mechanism of MTB mainly follows the principle of hysteresis loss. The experimental results accord with the prediction of the Eq.2: (i) ΔTand SARs linearly increase with f ield frequency; (ii) ΔTand SARs increase with f ield amplitude only within a certain range. When the magnetization of MTB is saturated and the area under the AC hysteresis loop reaches maximum, ΔTand SARs will no longer increase with the amplitude. In a word, the three main parameters of AC hysteresis loop,Ms,Mrs, andBc, determine the area under AC hysteresis loop, and thus determine the heat production effi ciency of single domain particles at the same frequency. Unfortunately, due to the limitation of equipment, we only test the low-frequency hysteresis loop, which could not be directly used in the calculation of heat production effi ciency, but could be used to evaluate the capacity of heat generation of the samples.

It is obvious that the MTB whole cells have huge advantages in heat production compared with commercial magnetic particles under the conditions of nearly similar particle size, same iron concentration and magnetic f ield. Under the experimental conditions described above, the ΔTvalues of MTBLA-1and MTBLA-2at 10 min are 3 times and 4.7 times more than that of sample Scmp, respectively (Fig.4a). As the values ofBc,Ms, andMrs/Msof MTB are signif icantly greater than those of sample Scmp, it means the lager value of AC hysteresis loop area and higher capacity of heat generation. Comparing with the sample Scmp,the magnetic parameters and heat production effi ciency of MTB whole cells greatly benef it from the inherent chain-like arrangement of magnetosomes.

Combined the rock magnetic and hyperthermia measurements, it indicates that the chain alignment of magnetosomes plays important roles in the magnetic parameters and heating effi ciency of MTB intact cells. The destruction of the magnetosome chain is directly proportional to the decrease in magnetic parameters, such asBc,Bcr, andMrs/Ms(Li et al.,2012). Magnetic hyperthermia also follows the same law (Fig.4b). The MTB intact cell organizes magnetosomes like necklaces through magnetosome membrane and cytoskeletal proteins, which reduces the interaction between magnetic particles and increases magnetic anisotropy and coercivity. The excellent magnetic properties make MTB whole cells have great heat production effi ciency and promising medical application prospects.

The MTB whole cells used in this study shows excellent property in heat production; however, there are still some limitations. For safety and patient tolerance reasons, the applied magnetic f ield is limited in clinical magnetic hyperthermia therapy by“Brezovich criterion” (Brezovich, 1988). In this case,achieving the “therapeutic window” of tumor would requires 109magnitude of bacteria, equivalent to a dose of approximately 1-mL OD565=20 bacteria cells with iron concentration of about 49.2 μg/mL, because one single MSR-1 cell contains only limited number of magnetosomes, averaging of 17.7 per cell in our study. Large number of foreign bacteria entering the body is bound to increase the safety risk. One strategy for reducing the dose of MTB is to continue to improve the heat production effi ciency of unit MTB cells. Our study and previous literatures show that using sodium nitrate instead of ammonium chloride as medium nitrogen source could signif icantly improve the parameters of magnetosome (Heyen and Schüler, 2003), and then improve the heat production effi ciency. Li et al. (2016) proves that cobaltcontaining magnetosomes with higher coercivity could be synthesized by adding cobalt to the culture medium. It is well known that MSR-1 cultured in fermenter have higherCmagvalue than that cultured in shaking table (Zhang et al., 2011), indicating higher coercivity and greater effi ciency in heat generation.All these indicate that the MSR-1 cells with higher heat effi ciency can be obtained by optimizing medium and culture mode. In addition, as more and more species of MTB have been discovered, we are pleasantly surprised to f ind a kind of giant rod-shaped MTB containing hundreds of magnetosomes per cell(Lin et al., 2009; Li et al., 2020), which means huge heat effi ciency and potential for clinical application.However, the vast majority of MTB, including giant rod-shaped MTB, have not achieved pure culture at present. Therefore, it is very signif icant to study the culture of MTB in future.

5 CONCLUSION

MTB whole cells with single-domain magnetosome exhibit excellent magnetic hyperthermia ability, and the heat generation principle mainly follows the hysteresis loss mechanism. So the heat production effi ciency of MTB cells is closely related to the parameters of hysteresis loop, such asBc,Ms, andMrs.Benef itting from the chain structures of magnetosomes,MTB cells have more superior magnetic properties than commercial magnetic beads with similar size,and thus have higher effi ciency in heat production. As expected, MTB cells with more and larger magnetosomes possess greater effi ciency in heat production. Therefore, improving the magnetic properties of MTB cells by optimizing the medium and culture method may further improve the heat conversion effi ciency.

6 DATA AVAILABILITY STATEMENT

The data that support the f indings of the current study are available on reasonable request from the corresponding author.

7 ACKNOWLEDGMENT

We thank Tang XU from the Institute of Geology and Geophysics, CAS for his help in TEM analysis.