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Preparation of ferrimagnetic magnetite microspheres for in situ hyperthermic treatment of cancer
ARTICLE IN PRESS
Biomaterials 26 (2005) 2231–2238 www.elsevier.com/locate/biomaterials
Preparation of ferrimagnetic magnetite microspheres for in situ hyperthermic treatment of cancer Masakazu Kawashitaa,, Masashi Tanakab, Tadashi Kokuboc, Yoshiaki Inoued, Takeshi Yaoe, Sunao Hamadaf, Teruya Shinjof a
Ion Beam Engineering Experimental Laboratory, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan b Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan c Research Institute for Science and Technology, Chubu University, Kasugai-shi, Aichi 487-8501, Japan d Neturen Co. Ltd., Hiratsuka, Kanagawa 254-0013, Japan e Department of Fundamental Energy Science, Graduate School of Energy Science, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan f Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan Received 21 April 2004; accepted 12 July 2004 Available online 11 September 2004
Abstract Ferrimagnetic microspheres 20–30 mm in diameter are useful as thermoseeds for inducing hyperthermia in cancers, especially for tumors located deep inside the body. The microspheres are entrapped in the capillary bed of the tumors when they are implanted through blood vessels and heat cancers locally by their hysteresis loss when placed under an alternating magnetic field. In the present study, preparation of magnetite (Fe3O4) microspheres 20–30 mm in diameter was attempted by melting powders in high-frequency induction thermal plasma, and by precipitation from aqueous solution. The microspheres prepared by melting powders in highfrequency induction thermal plasma were composed of a large amount of Fe3O4 and a small amount of wustite (FeO), and those subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa were fully composed of Fe3O4 1 mm in size. The saturation magnetization and coercive force of the heat-treated microspheres were 92 emu g–1 and 50 Oe, respectively. The heat generation of the heat-treated microspheres was estimated to be 10 W g–1, under 300 Oe and 100 kHz. The microspheres prepared by precipitation from aqueous solution consisted of b-FeOOH, and those subsequently heat treated at 400 1C for 1 h in a 70% CO2+30% H2 atmosphere consisted of Fe3O4 crystals 50 nm in size. The saturation magnetization and coercive force of the heat-treated microspheres were 53 emu g–1 and 156 Oe, respectively. The heat generation of the heat-treated microspheres was estimated to be 41 W g–1, under 300 Oe and 100 kHz. The latter microspheres are believed to be promising thermoseeds for hyperthermic treatment of cancer. r 2004 Elsevier Ltd. All rights reserved. Keywords: Ferrimagnetism; Magnetite; Microsphere; Hysteresis loss; Hyperthermia
1. Introduction Cancer cells generally perish at around 43 1C because their oxygen supply via the blood vessels is not sufficient, whereas normal cells are not damaged at even higher temperatures [1]. In addition, tumors are Corresponding author. Tel.: +81-75-383-2330; fax: +81-75-3832343. E-mail address: [email protected] (M. Kawashita).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.07.014
more easily heated than the surrounding normal tissues, since the blood vessels and nervous systems are poorly developed in the tumor [2–4]. Therefore, hyperthermia is expected to be a very useful treatment of cancer with few side effects [1]. Various techniques for heating the tumors, such as treatments with hot water, infrared rays, ultrasound and microwaves have been attempted. However, deep-seated tumors cannot be heated effectively and locally using these techniques. Ferrimagnetic microspheres 20–30 mm in diameter are useful as
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thermoseeds for inducing hyperthermia in cancers, especially in those tumors located deep inside the body. These spheres are entrapped in the capillary bed of the tumors when they are implanted through blood vessels, and can heat cancers locally by their hysteresis loss when placed under an alternating magnetic field. So far, glass-ceramics containing lithium ferrite (LiFe5O8) in a matrix of hematite (a-Fe2O3) and a SiO2–P2O5 glassy phase [5–7]; magnetite (Fe3O4) in a matrix of b-wollastonite (b-CaSiO3) and a CaO–SiO2– B2O3–P2O5 glassy phase [8–15]; a-Fe [16]; Fe3O4 in a B2O3-free CaO–SiO2–P2O5 glassy phase [17]; or zinc–iron ferrite in a CaO–SiO2 glassy phase [18] have been developed for this purpose. However, none have been produced in the form of microspheres 20–30 mm in diameter, or have shown a high heat generating ability. In the present study, preparation of Fe3O4 microspheres was attempted by melting powders in a highfrequency induction thermal plasma, and by precipitation from an aqueous solution. The structure and magnetic properties of the microspheres obtained were investigated.
2. Materials and methods 2.1. Preparation of microspheres in high-frequency induction thermal plasma Pure Fe3O4 powders (purity: 99.5%, Kojundo Chemical Laboratory Co. Ltd., Saitama, Japan) were carried by argon gas at a flow rate of 5 l min–1 to be completely melted in a plasma flame of argon gas at a flow rate of 94 l min–1, which was produced by highfrequency induction heating, with a power of 19.0 kW at a frequency of 4 MHz. The temperature of the flame was estimated to be 12,000 to 13,000 1C. Argon gas at a flow rate of 20 l min–1 was introduced below the flame in order to decrease the temperature below the flame. The molten material fell into extra pure water, at room temperature, which was located at the base of a chamber 1 m in height. The solidified products were sieved using a nylon mesh in order to obtain particles of 20–30 mm in size. These particles were placed in an alumina boat, heated to various temperatures ranging from 400 to 600 1C at a rate of 5 1C min–1, kept at the respective temperature for 1 h, and allowed to cool under a reduced pressure of 5.1 103 Pa in an infrared gold image furnace (RHL-E-410P, ULVAC-RIKO Inc., Yokohama, Japan). 2.2. Preparation of microspheres in aqueous solution Fe3O4 powders (12 g) (Nacalai Tesque Inc., Kyoto, Japan) were added into 600 ml of 1 wt% aqueous solution of hydrofluoric acid (HF) (Nacalai Tesque
Inc., Kyoto, Japan), and stirred at 30 1C for 24 h. Excess Fe3O4 powder was filtered out in order to give a 1 wt% HF solution containing Fe3O4 at a saturated concentration (Fe–HF solution). Silica glass microspheres 12.4 mm in average diameter (0.75 g) (Admatechs Co. Ltd., Tokyo, Japan) were soaked in Fe–HF solution at 30 1C for various periods up to 24 days, under vigorous stirring using a Teflons propeller. The Fe–HF solution was renewed every 6 days. After the given period, the samples were filtered out, gently washed with distilled water, and then dried at room temperature for 1 day. The products thus obtained were placed in an alumina boat, heated to various temperatures ranging from 300 to 600 1C at a rate of 5 1C min–1, kept at the respective temperature for 1 h, and allowed to cool in an atmosphere of 70 vol% CO2 gas and 30 vol% H2 gas (70% CO2+30% H2 atmosphere). Some products were heated to 400 1C at a slow rate (0.5 1C min–1 from room temperature to 100 1C, 0.1 1C min–1 from 100 1C to 200 1C, 0.5 1C min–1 from 200 1C to 300 1C and 0.1 1C min–1 from 300 1C to 400 1C), kept at 400 1C for 1 h, and allowed to cool in the same atmosphere. 2.3. Structural analysis The structure of the products was analyzed using a powder X-ray diffractometer (XRD; RINT–1400, Rigaku Co., Tokyo, Japan). X-ray source, X-ray power, scanning rate and sampling angle were set at Ni-filtered CuKa radiation, 40 kV–200 mA, 2y=2 1min1 and 0.011, respectively. The crystalline phases precipitated in the specimens were identified by referring to data from the Joint Committee on Powder Diffraction Standards. The shape and microstructure of the products were observed using a field-emission scanning electron microscope (FE–SEM; S-4700, Hitachi Ltd., Tokyo, Japan). Products were pulverized into fine powders using a super hard alloy mortar, and suspended in 1 ml of ethanol. A few drops of the suspended solution were placed on a collodion film supported on a titanium metal microgrid mesh, and then observed by a transmission electron microscope (TEM; JEM2000FXIII, JEOL Co. Ltd., Tokyo, Japan) at 200 kV. 2.4. TG–DTA measurement Silica glass microspheres (5.95 mg) soaked in Fe–HF solution at 30 1C for 24 days were put into an aluminum holder, and subjected to thermogravimetry-differential thermal analysis (TG–DTA) (TG–DTA2000S, MAC Science Co. Ltd., Yokohama, Japan). The samples were heated to 400 1C at a rate of 3 1C min–1, and allowed to cool in a 70% CO2+30% H2 atmosphere.
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2.5. Measurement of magnetic properties The saturation magnetization and coercive force of the specimens were measured using a vibrating sample magnetometer (VSM-5–15, Toei Industry Co. Ltd., Tokyo, Japan) under a magnetic field of up to 10 kOe at 300 K. The heat generation of the specimens was calculated using the following equation: I P¼f H dB 107 ; (1) where f and H are the frequency and the magnetic field of the applied magnetic field and B is the magnetization of H the specimen under the given magnetic field. The term H dB; is the area of the hysteresis loss under the given magnetic field. The frequency and maximum strength of the magnetic field, generated using an apparatus developed by the present authors were 100 kHz and 300 Oe, respectively [8]. In the present calculation, therefore, 100 kHz was put into f, and the hysteresis H loop area measured under 300 Oe was put into H dB:
3. Results 3.1. Structure and magnetic properties of microspheres prepared in high-frequency induction thermal plasma Fig. 1 shows powder XRD patterns of the products prepared in high-frequency induction thermal plasma and then subjected to heat treatments at various : Fe3O4(Magnetite) : α-Fe2O3 (Hematite) : FeO (Wustite)
600°C
Intensity
temperatures at 5.1 103 Pa. Large peaks assigned to Fe3O4 were observed for all products examined. Besides Fe3O4, small peaks assigned to wustite (FeO) were observed in the product before the heat treatment. Peaks assigned to hematite (a-Fe2O3) were newly observed when the products were heat treated at 300, 400 and 500 1C. The products heat treated at 600 and 700 1C gave large peaks only, ascribed to Fe3O4. Fig. 2 shows FE–SEM photographs of the products prepared in high-frequency induction thermal plasma and then subjected to heat treatments at various temperatures under 5.1 103 Pa. All products examined took a form of microspheres with spherical shape and of 20–30 mm in diameter. The surface roughness of the microspheres was slightly increased by the heat treatment. Fig. 3(a) shows the magnetization curve, under magnetic fields of up to 10 kOe, of the products prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa. The saturation magnetization and the coercive force were 92 emu g–1 and 50 Oe, respectively. Fig. 4(a) shows the magnetization curve, under magnetic fields of up to 300 Oe, of the products prepared in highfrequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa. The heat generation of the heat-treated products was calculated to be 10 W g–1, at 300 Oe and 100 kHz. Table 1 summarizes the magnetic properties and heat generation of the products after heat treatment, in comparison with glass ceramics containing Fe3O4 in 36 wt% [9]. Fig. 5 shows an FE–SEM photograph of the surface of the product prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa. The products consisted of grains about 1 mm in size. 3.2. Structure and magnetic properties of microspheres prepared in aqueous solution
700°C
500°C 400°C
300°C Before treatment
10
2233
20
30
40
50
2 (CuKα) / degree Fig. 1. Powder XRD patterns of the products prepared in highfrequency induction thermal plasma and then subjected to heat treatments at various temperatures at 5.1 103 Pa.
Fig. 6 shows powder XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods. An extremely small peak ascribed to quartz was observed for silica glass microspheres before soaking. After the soaking in Fe–HF solution, peaks ascribed to b-FeOOH were observed, and their intensities increased with increasing soaking period. Fig. 7 shows FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods. Some precipitates were formed on the surface of the silica glass microspheres within 6 days, and the thickness of the precipitates increased with increasing soaking period. After soaking for 24 days, the diameter of the products reached approximately 25 mm. Fig. 8 shows powder XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatment at various
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Before Before treatment treatment
300° °C 300°C 300°C
20µm 20µm 500°C 500°C
400°C 400°C
20µm 20µm 600°C 600°C 500°C
20µm 20µm
20µm 20µm 700°C 700°C
20µm 20µm
20µm 20µm
Magnetization / emu . g-1
Fig. 2. FE–SEM photographs of the products prepared in high-frequency induction thermal plasma and then subjected to heat treatments at various temperatures at 5.1 103 Pa.
-10
-5
100 (a)
(b)
50
0
5 10 Magnetic field / kOe
-50
-100 Fig. 3. (a) Magnetization curve, under magnetic fields of up to 10 kOe, of the products prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa: and (b) those of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere.
temperatures in a 70% CO2+30% H2 atmosphere. Peaks ascribed to Fe3O4 were observed for the specimens heat treated at above 400 1C. Fig. 9 shows FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatment at various temperatures in a 70% CO2+30% H2 atmosphere. Large cracks occurred on the microspheres after the heat treatment, irrespective of the heat treatment temperature.
Fig. 10(a) shows FE–SEM photograph of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. The resultant microspheres had only small cracks on their surfaces. Fig. 3(b) shows the magnetization curve, under magnetic fields of up to 10 kOe, of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. It can be estimated from this figure that the saturation magnetization and coercive force of the specimen were 53 emu g–1 and 156 Oe, respectively. Fig. 4(b) shows the magnetization curve, under magnetic fields of up to 300 Oe, of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. The heat generation of the microspheres was calculated to be 41 W g–1, under 300 Oe and 100 kHz, which was four times as large as that of the magnetite microspheres prepared by high-frequency induction thermal plasma, as shown in Table 1. Fig. 10(b) shows a TEM photograph of the precipitates on the surface of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. The crystallite size of the precipitates was estimated to be approximately 50 nm.
4. Discussion It can be seen from Figs. 1 and 2 that the 20–30 mm microspheres prepared in high-frequency induction thermal plasma were mainly composed of Fe3O4,
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Table 1 Magnetic properties and heat generation of microspheres obtained in the present study, in comparison with those of Fe3O4-containing glass-ceramics [9] Coercive force (Oe)
Heat generation under 300 Oe, 100 kHz(W g1)
Sample
Saturation magnetization (emu g1)
Microspheres prepared in high-frequency induction thermal plasmaa Microspheres prepared in aqueous solutionb Fe3O4-containing glass-ceramics
92
50
10
53 32
156 120
41 10
a
Heat treated at 600 1C for 1 h under 5.1 103 Pa. Heated up to 400 1C at a slow rate, kept at 400 1C for 1 h in 70% CO2+30% H2 atmosphere.
-300
-200
: β-FeOOH
20
(b)
: SiO2 (quartz)
(a) 10 24 d
0
-100
100
200
300
Magnetic field / Oe
Intensity
Magnetization / emu . g-1
b
18 d
12 d
-10
6d -20 Before soaking Fig. 4. (a) Magnetization curve, under magnetic fields of up to 300 Oe, of the products prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa: and (b) those of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere.
10
20
30
40
50
2 / degree Fig. 6. XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods.
3µm 3µm Fig. 5. FE–SEM photograph of the surface of the product prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h at 5.1 103 Pa.
accompanied by a small amount of FeO, and those subsequently heat treated to above 600 1C under 5.1 103 Pa were almost completely composed of Fe3O4. The surfaces of the microsphere were smooth before the heat treatment, but became slightly rough after heat treatment above 400 1C. The increase in the surface roughness might be attributed to the formation of a-Fe2O3 and/or the crystal growth of Fe3O4. The saturation magnetization of the microspheres heat treated at 600 1C for 1 h under 5.1 103 Pa was 92 emu g–1, as shown in Fig. 3(a) and Table 1. It has been reported that stoichiometric Fe3O4 gives a saturation magnetization of 92 emu g–1 at room temperature
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Before soaking soaking Before
66 d d
5µm 5µm
12 12 d d
5µm 5µm
24 24 d d
18 18 d d
5µm 5µm
5µm 5µm
5µm 5µm
Fig. 7. FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods.
:
β-FeOOH
:
Fe3O4 (Magnetite)
small as 10 W g–1, under 300 Oe and 100 kHz (see Fig. 4(a) and Table 1). This might be attributed to the low coercive force (50 Oe) due to the large crystallite size of Fe3O4 (ffi1 mm), as shown in Fig. 5. It has been reported for Fe3O4 that the degree of ordering of the magnetic moment in the individual particles increases with increasing crystallite size, up to 40 nm, forming a wellordered single domain at 40 nm [20], and the number of magnetic domains in the individual particles increases with increasing crystallite size for larger sizes, to decrease the coercive force. It can be seen from Figs. 6 and 7 that b-FeOOH was precipitated on the surface of silica glass microspheres in Fe–HF solution within six days, and that the thickness of b-FeOOH increased with increasing soaking period. The mechanism of the precipitation and growth of bFeOOH is speculated as follows. According to the following (equilibrium (i)), a trivalent iron (Fe3+) fluoro complex and b-FeOOH are formed in the Fe–HF solution.
Intensity
600°C
500°C
400°C
300°C Before treatment
Fe3þ fluoro complex þ mH2 O2b-FeOOH þ nHF: (i)
10
20
30 2 / degree
40
50
Fig. 8. Powder XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatments at various temperatures in a 70% CO2+30% H2 atmosphere.
[19]. Thus, it was reveled that the heat-treated microspheres were fully composed of Fe3O4. However, the heat generation of the heat-treated microspheres was as
When silica glass microspheres are soaked in the Fe–HF solution, reaction (ii) occurs to form a chemically stable [SiF6]2 complex in the solution, and HF is consumed selectively at the surface of the silica glass microspheres. SiO2 þ 6HF ! ½SiF6 2 þ 2H2 O þ 2Hþ :
(ii)
As a result, equilibrium (i) is shifted to the right-hand side in order to compensate the decreased amount of HF in the solution and b-FeOOH is precipitated selectively on the surface of the silica glass microspheres. On the other hand, in the Fe–HF solution, dissolved divalent iron (Fe2+) ions are oxidized gradually to form Fe3+
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treatment Before Before treatment
2237
400° °C 400°C 400°C
300°C
(c) (c)
5µm 5µm
5µm 5µm
5µm 5µm
600°C 500°C 600°C
500°C
5µm 5µm
5µm 5µm
Fig. 9. FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatments at various temperatures in a 70% CO2+30% H2 atmosphere.
(a)
5µm 5µm
(b)
100 100 nm nm
Fig. 10. (a) FE–SEM photograph of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere and (b) TEM photograph of the precipitates on their surface.
ions, which have lower solubility than Fe2+ ions under acidic conditions [21]. Therefore, the increase in the amount of Fe3+ ions with low solubility in the Fe–HF solution might be responsible for the growth of bFeOOH on the surface of the silica glass microspheres. It was seen from Fig. 8 that the deposited b-FeOOH was transformed into Fe3O4 by heat treatment above 400 1C in a 70% CO2+30% H2 atmosphere. However, as shown in Fig. 9, large cracks occurred on the microspheres after the heat treatment. These cracks might be attributed to the shrinkage of the surface layer due to the dehydration of b-FeOOH and the formation of Fe3O4. Examining the TG–DTA curves shows that remarkable changes were observed between 100 to 200 1C and between 300 and 400 1C (data was not shown). Therefore, it is expected that the crack formation may be suppressed if the heat treatment temperature was increased at a slower rate from 100 to 200 1C and from 300 to 400 1C. As expected, the
formation of the large cracks was suppressed effectively when the silica glass microspheres were soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate (see Fig. 10(a)). The silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate showed ferrimagnetism, with saturation magnetization of 53 emu g–1 and coercive force of 156 Oe (see Fig. 3(b) and Table 1). The heattreated microspheres gave a magnetization curve with a large area (see Fig. 4(b)) and a heat generation of 41 W g–1, under 300 Oe and 100 kHz, which was four times as large as for pure Fe3O4 microspheres prepared in high-frequency induction thermal plasma, as described above (see Table 1). It can be seen from Fig. 10(b) that the heat-treated microspheres are composed of small crystals of Fe3O4 (ffi50 nm). Therefore, the larger heat generation of the microspheres prepared in aqueous solution than those prepared in high-frequency
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induction thermal plasma might be attributed to the higher coercive force due to the smaller crystallite size of Fe3O4 in the former.
5. Conclusions Pure Fe3O4 microspheres, 20–30 mm in diameter, were prepared by melting powders in high-frequency induction thermal plasma and subsequent heat treatment. These microspheres, however, showed heat generation as low as 10 W g–1, at 300 Oe and 100 kHz, as they were composed of Fe3O4 crystals 1 mm in size. Microspheres, 20–30 mm in diameter, composed of small crystals of Fe3O4 50 nm in size, were prepared by precipitation from aqueous solution and subsequent heat treatment. The heat-treated microspheres showed heat generation as high as 41 W g–1, under the same magnetic field. The latter microspheres are believed to be promising thermoseeds for hyperthermic treatment of cancer. Acknowledgments This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] Sugihara T. Gan-to-Tatakau-Hyperthermia (Japanese). Kyoto: Kinpoudou; 1986. pp. 37–48. [2] Cavaliere R, Ciocatto EC, Giovanella BC, Heidelberger C, Johnson RO, Margottini M, Mondovi B, Moricca G, RossiFanelli A. Selective heat sensitivity of cancer cells. Biochemical and clinical studies. Cancer 1967;20:1351–81. [3] Overgaard K, Overgaard J. Investigations on the possibility of a thermic tumour therapy. I. Short-wave treatment of a transplanted isologous mouse mammary carcinoma. Eur J Cancer 1972;8:65–78. [4] Overgaard J. Effect of hyperthermia on malignant cells in vivo. A review and a hypothesis. Cancer 1977;39:2637–46. [5] Luderer AA, Borrelli NF, Panzarino JN, Mansfield GR, Hess DM, Brown JL, Barnett EH, Hahn EW. Glass-ceramic-mediated, magnetic-field-induced localized hyperthermia: response of a murine mammary carcinoma. Radiat Res 1983;94:190–8.
[6] Borrelli NF, Luderer AA, Panzarino JN. Radio frequency induced hyperthermia for tumor therapy. US Patent. No.4 323 056, 1980. [7] Borrelli NF, Luderer AA, Panzarino JN. Hysteresis heating for the treatment of tumours. Phys Med Biol 1984;29:487–94. [8] Kokubo T, Ebisawa Y, Sugimoto Y, Kiyama M, Ohura K, Yamamuro T, Hiraoka M, Abe M. Preparation of bioactive and ferromagnetic glass-ceramic for hyperthermia. In: Hulbert JE, Hulbert SFeditors. Bioceramics, vol. 3. Terre Haute, Indiana: Rose-Hulman Institute of Technology; 1990. p. 213–23. [9] Ebisawa Y, Sugimoto Y, Hayashi T, Kokubo T, Ohura K, Yamamuro T. Crystallization of (FeO, Fe2O3)CaO–SiO2 glasses and magnetic properties of their crystallized products. J Ceram Soc Japan 1991;99:7–13. [10] Ebisawa Y, Kokubo T, Ohura K, Yamamuro T. Bioactivity of CaOSiO2-based glasses: in vitro evaluation. J Mater Sci: Mater. Med 1990;1:239–44. [11] Ebisawa Y, Kokubo T, Ohura K, Yamamuro T. Bioactivity of Fe2O3-containing CaOSiO2 glasses: in vitro evaluation. J Mater Sci: Mater Med 1992;4:225–32. [12] Ohura K, Ikenaga M, Nakamura T, Yamamuro T, Ebisawa Y, Kokubo T, Kotoura Y, Oka M. A heat-generating bioactive glass-ceramic for hyperthermia. J Appl Biomater 1991;2:153–9. [13] Ikenaga M, Ohura K, Yamamuro T, Kotoura Y, Oka M, Kokubo T. Localized hyperthermic treatment of experimental bone tumors with ferromagnetic ceramics. J Orthop Res 1993;11: 849–55. [14] Ebisawa Y, Miyaji F, Kokubo T, Ohura K, Nakamura T. Surface reaction of bioactive and ferromagnetic glass-ceramics in the system FeO–Fe2O3–CaO–SiO2. J Ceram Soc Japan 1997;105: 947–51. [15] Ebisawa Y, Miyaji F, Kokubo T, Ohura K, Nakamura T. Bioactivity of ferrimagnetic glass-ceramics in the system FeO– Fe2O3–CaO–SiO2. Biomaterials 1997;18:1277–84. [16] Konaka H, Miyaji F, Kokubo T. Preparation and magnetic properties of glass-ceramics containing a-Fe for hyperthermia. J Ceram Soc Japan 1997;105:833–6. [17] Kawashita M, Takaoka H, Kokubo T, Yao T, Hamada S, Shinjo T. Preparation of magnetite-containing glass-ceramics in controlled atmosphere for hyperthermia of cancer. J Ceram Soc Japan 2001;109:39–44. [18] Kawashita M, Iwahashi Y, Kokubo T, Yao T, Hamada S, Shinjo T. Preparation of glass-ceramics containing ferrimagnetic zinc– iron ferrite for the hyperthermal treatment of cancer. J Ceram Soc Japan 2004;112:373–9. [19] Kingery WD, Bowen HK, Uhlmann DR. Introduction to ceramics. New York: Wiley; 1976. p. 993. [20] Knoules JE. Coercivity and packing density in acicular particles. J Magn Magn Mater 1981;25:105–12. [21] Paul A, Zaman MS. The relative influences of Al2O3 and Fe2O3 on the chemical durability of silicate glasses at different pH values. J Mater Sci 1978;13:1499–502.
Biomaterials 26 (2005) 2231–2238 www.elsevier.com/locate/biomaterials
Preparation of ferrimagnetic magnetite microspheres for in situ hyperthermic treatment of cancer Masakazu Kawashitaa,, Masashi Tanakab, Tadashi Kokuboc, Yoshiaki Inoued, Takeshi Yaoe, Sunao Hamadaf, Teruya Shinjof a
Ion Beam Engineering Experimental Laboratory, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan b Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan c Research Institute for Science and Technology, Chubu University, Kasugai-shi, Aichi 487-8501, Japan d Neturen Co. Ltd., Hiratsuka, Kanagawa 254-0013, Japan e Department of Fundamental Energy Science, Graduate School of Energy Science, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan f Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan Received 21 April 2004; accepted 12 July 2004 Available online 11 September 2004
Abstract Ferrimagnetic microspheres 20–30 mm in diameter are useful as thermoseeds for inducing hyperthermia in cancers, especially for tumors located deep inside the body. The microspheres are entrapped in the capillary bed of the tumors when they are implanted through blood vessels and heat cancers locally by their hysteresis loss when placed under an alternating magnetic field. In the present study, preparation of magnetite (Fe3O4) microspheres 20–30 mm in diameter was attempted by melting powders in high-frequency induction thermal plasma, and by precipitation from aqueous solution. The microspheres prepared by melting powders in highfrequency induction thermal plasma were composed of a large amount of Fe3O4 and a small amount of wustite (FeO), and those subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa were fully composed of Fe3O4 1 mm in size. The saturation magnetization and coercive force of the heat-treated microspheres were 92 emu g–1 and 50 Oe, respectively. The heat generation of the heat-treated microspheres was estimated to be 10 W g–1, under 300 Oe and 100 kHz. The microspheres prepared by precipitation from aqueous solution consisted of b-FeOOH, and those subsequently heat treated at 400 1C for 1 h in a 70% CO2+30% H2 atmosphere consisted of Fe3O4 crystals 50 nm in size. The saturation magnetization and coercive force of the heat-treated microspheres were 53 emu g–1 and 156 Oe, respectively. The heat generation of the heat-treated microspheres was estimated to be 41 W g–1, under 300 Oe and 100 kHz. The latter microspheres are believed to be promising thermoseeds for hyperthermic treatment of cancer. r 2004 Elsevier Ltd. All rights reserved. Keywords: Ferrimagnetism; Magnetite; Microsphere; Hysteresis loss; Hyperthermia
1. Introduction Cancer cells generally perish at around 43 1C because their oxygen supply via the blood vessels is not sufficient, whereas normal cells are not damaged at even higher temperatures [1]. In addition, tumors are Corresponding author. Tel.: +81-75-383-2330; fax: +81-75-3832343. E-mail address: [email protected] (M. Kawashita).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.07.014
more easily heated than the surrounding normal tissues, since the blood vessels and nervous systems are poorly developed in the tumor [2–4]. Therefore, hyperthermia is expected to be a very useful treatment of cancer with few side effects [1]. Various techniques for heating the tumors, such as treatments with hot water, infrared rays, ultrasound and microwaves have been attempted. However, deep-seated tumors cannot be heated effectively and locally using these techniques. Ferrimagnetic microspheres 20–30 mm in diameter are useful as
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thermoseeds for inducing hyperthermia in cancers, especially in those tumors located deep inside the body. These spheres are entrapped in the capillary bed of the tumors when they are implanted through blood vessels, and can heat cancers locally by their hysteresis loss when placed under an alternating magnetic field. So far, glass-ceramics containing lithium ferrite (LiFe5O8) in a matrix of hematite (a-Fe2O3) and a SiO2–P2O5 glassy phase [5–7]; magnetite (Fe3O4) in a matrix of b-wollastonite (b-CaSiO3) and a CaO–SiO2– B2O3–P2O5 glassy phase [8–15]; a-Fe [16]; Fe3O4 in a B2O3-free CaO–SiO2–P2O5 glassy phase [17]; or zinc–iron ferrite in a CaO–SiO2 glassy phase [18] have been developed for this purpose. However, none have been produced in the form of microspheres 20–30 mm in diameter, or have shown a high heat generating ability. In the present study, preparation of Fe3O4 microspheres was attempted by melting powders in a highfrequency induction thermal plasma, and by precipitation from an aqueous solution. The structure and magnetic properties of the microspheres obtained were investigated.
2. Materials and methods 2.1. Preparation of microspheres in high-frequency induction thermal plasma Pure Fe3O4 powders (purity: 99.5%, Kojundo Chemical Laboratory Co. Ltd., Saitama, Japan) were carried by argon gas at a flow rate of 5 l min–1 to be completely melted in a plasma flame of argon gas at a flow rate of 94 l min–1, which was produced by highfrequency induction heating, with a power of 19.0 kW at a frequency of 4 MHz. The temperature of the flame was estimated to be 12,000 to 13,000 1C. Argon gas at a flow rate of 20 l min–1 was introduced below the flame in order to decrease the temperature below the flame. The molten material fell into extra pure water, at room temperature, which was located at the base of a chamber 1 m in height. The solidified products were sieved using a nylon mesh in order to obtain particles of 20–30 mm in size. These particles were placed in an alumina boat, heated to various temperatures ranging from 400 to 600 1C at a rate of 5 1C min–1, kept at the respective temperature for 1 h, and allowed to cool under a reduced pressure of 5.1 103 Pa in an infrared gold image furnace (RHL-E-410P, ULVAC-RIKO Inc., Yokohama, Japan). 2.2. Preparation of microspheres in aqueous solution Fe3O4 powders (12 g) (Nacalai Tesque Inc., Kyoto, Japan) were added into 600 ml of 1 wt% aqueous solution of hydrofluoric acid (HF) (Nacalai Tesque
Inc., Kyoto, Japan), and stirred at 30 1C for 24 h. Excess Fe3O4 powder was filtered out in order to give a 1 wt% HF solution containing Fe3O4 at a saturated concentration (Fe–HF solution). Silica glass microspheres 12.4 mm in average diameter (0.75 g) (Admatechs Co. Ltd., Tokyo, Japan) were soaked in Fe–HF solution at 30 1C for various periods up to 24 days, under vigorous stirring using a Teflons propeller. The Fe–HF solution was renewed every 6 days. After the given period, the samples were filtered out, gently washed with distilled water, and then dried at room temperature for 1 day. The products thus obtained were placed in an alumina boat, heated to various temperatures ranging from 300 to 600 1C at a rate of 5 1C min–1, kept at the respective temperature for 1 h, and allowed to cool in an atmosphere of 70 vol% CO2 gas and 30 vol% H2 gas (70% CO2+30% H2 atmosphere). Some products were heated to 400 1C at a slow rate (0.5 1C min–1 from room temperature to 100 1C, 0.1 1C min–1 from 100 1C to 200 1C, 0.5 1C min–1 from 200 1C to 300 1C and 0.1 1C min–1 from 300 1C to 400 1C), kept at 400 1C for 1 h, and allowed to cool in the same atmosphere. 2.3. Structural analysis The structure of the products was analyzed using a powder X-ray diffractometer (XRD; RINT–1400, Rigaku Co., Tokyo, Japan). X-ray source, X-ray power, scanning rate and sampling angle were set at Ni-filtered CuKa radiation, 40 kV–200 mA, 2y=2 1min1 and 0.011, respectively. The crystalline phases precipitated in the specimens were identified by referring to data from the Joint Committee on Powder Diffraction Standards. The shape and microstructure of the products were observed using a field-emission scanning electron microscope (FE–SEM; S-4700, Hitachi Ltd., Tokyo, Japan). Products were pulverized into fine powders using a super hard alloy mortar, and suspended in 1 ml of ethanol. A few drops of the suspended solution were placed on a collodion film supported on a titanium metal microgrid mesh, and then observed by a transmission electron microscope (TEM; JEM2000FXIII, JEOL Co. Ltd., Tokyo, Japan) at 200 kV. 2.4. TG–DTA measurement Silica glass microspheres (5.95 mg) soaked in Fe–HF solution at 30 1C for 24 days were put into an aluminum holder, and subjected to thermogravimetry-differential thermal analysis (TG–DTA) (TG–DTA2000S, MAC Science Co. Ltd., Yokohama, Japan). The samples were heated to 400 1C at a rate of 3 1C min–1, and allowed to cool in a 70% CO2+30% H2 atmosphere.
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2.5. Measurement of magnetic properties The saturation magnetization and coercive force of the specimens were measured using a vibrating sample magnetometer (VSM-5–15, Toei Industry Co. Ltd., Tokyo, Japan) under a magnetic field of up to 10 kOe at 300 K. The heat generation of the specimens was calculated using the following equation: I P¼f H dB 107 ; (1) where f and H are the frequency and the magnetic field of the applied magnetic field and B is the magnetization of H the specimen under the given magnetic field. The term H dB; is the area of the hysteresis loss under the given magnetic field. The frequency and maximum strength of the magnetic field, generated using an apparatus developed by the present authors were 100 kHz and 300 Oe, respectively [8]. In the present calculation, therefore, 100 kHz was put into f, and the hysteresis H loop area measured under 300 Oe was put into H dB:
3. Results 3.1. Structure and magnetic properties of microspheres prepared in high-frequency induction thermal plasma Fig. 1 shows powder XRD patterns of the products prepared in high-frequency induction thermal plasma and then subjected to heat treatments at various : Fe3O4(Magnetite) : α-Fe2O3 (Hematite) : FeO (Wustite)
600°C
Intensity
temperatures at 5.1 103 Pa. Large peaks assigned to Fe3O4 were observed for all products examined. Besides Fe3O4, small peaks assigned to wustite (FeO) were observed in the product before the heat treatment. Peaks assigned to hematite (a-Fe2O3) were newly observed when the products were heat treated at 300, 400 and 500 1C. The products heat treated at 600 and 700 1C gave large peaks only, ascribed to Fe3O4. Fig. 2 shows FE–SEM photographs of the products prepared in high-frequency induction thermal plasma and then subjected to heat treatments at various temperatures under 5.1 103 Pa. All products examined took a form of microspheres with spherical shape and of 20–30 mm in diameter. The surface roughness of the microspheres was slightly increased by the heat treatment. Fig. 3(a) shows the magnetization curve, under magnetic fields of up to 10 kOe, of the products prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa. The saturation magnetization and the coercive force were 92 emu g–1 and 50 Oe, respectively. Fig. 4(a) shows the magnetization curve, under magnetic fields of up to 300 Oe, of the products prepared in highfrequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa. The heat generation of the heat-treated products was calculated to be 10 W g–1, at 300 Oe and 100 kHz. Table 1 summarizes the magnetic properties and heat generation of the products after heat treatment, in comparison with glass ceramics containing Fe3O4 in 36 wt% [9]. Fig. 5 shows an FE–SEM photograph of the surface of the product prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa. The products consisted of grains about 1 mm in size. 3.2. Structure and magnetic properties of microspheres prepared in aqueous solution
700°C
500°C 400°C
300°C Before treatment
10
2233
20
30
40
50
2 (CuKα) / degree Fig. 1. Powder XRD patterns of the products prepared in highfrequency induction thermal plasma and then subjected to heat treatments at various temperatures at 5.1 103 Pa.
Fig. 6 shows powder XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods. An extremely small peak ascribed to quartz was observed for silica glass microspheres before soaking. After the soaking in Fe–HF solution, peaks ascribed to b-FeOOH were observed, and their intensities increased with increasing soaking period. Fig. 7 shows FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods. Some precipitates were formed on the surface of the silica glass microspheres within 6 days, and the thickness of the precipitates increased with increasing soaking period. After soaking for 24 days, the diameter of the products reached approximately 25 mm. Fig. 8 shows powder XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatment at various
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Before Before treatment treatment
300° °C 300°C 300°C
20µm 20µm 500°C 500°C
400°C 400°C
20µm 20µm 600°C 600°C 500°C
20µm 20µm
20µm 20µm 700°C 700°C
20µm 20µm
20µm 20µm
Magnetization / emu . g-1
Fig. 2. FE–SEM photographs of the products prepared in high-frequency induction thermal plasma and then subjected to heat treatments at various temperatures at 5.1 103 Pa.
-10
-5
100 (a)
(b)
50
0
5 10 Magnetic field / kOe
-50
-100 Fig. 3. (a) Magnetization curve, under magnetic fields of up to 10 kOe, of the products prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa: and (b) those of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere.
temperatures in a 70% CO2+30% H2 atmosphere. Peaks ascribed to Fe3O4 were observed for the specimens heat treated at above 400 1C. Fig. 9 shows FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatment at various temperatures in a 70% CO2+30% H2 atmosphere. Large cracks occurred on the microspheres after the heat treatment, irrespective of the heat treatment temperature.
Fig. 10(a) shows FE–SEM photograph of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. The resultant microspheres had only small cracks on their surfaces. Fig. 3(b) shows the magnetization curve, under magnetic fields of up to 10 kOe, of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. It can be estimated from this figure that the saturation magnetization and coercive force of the specimen were 53 emu g–1 and 156 Oe, respectively. Fig. 4(b) shows the magnetization curve, under magnetic fields of up to 300 Oe, of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. The heat generation of the microspheres was calculated to be 41 W g–1, under 300 Oe and 100 kHz, which was four times as large as that of the magnetite microspheres prepared by high-frequency induction thermal plasma, as shown in Table 1. Fig. 10(b) shows a TEM photograph of the precipitates on the surface of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere. The crystallite size of the precipitates was estimated to be approximately 50 nm.
4. Discussion It can be seen from Figs. 1 and 2 that the 20–30 mm microspheres prepared in high-frequency induction thermal plasma were mainly composed of Fe3O4,
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Table 1 Magnetic properties and heat generation of microspheres obtained in the present study, in comparison with those of Fe3O4-containing glass-ceramics [9] Coercive force (Oe)
Heat generation under 300 Oe, 100 kHz(W g1)
Sample
Saturation magnetization (emu g1)
Microspheres prepared in high-frequency induction thermal plasmaa Microspheres prepared in aqueous solutionb Fe3O4-containing glass-ceramics
92
50
10
53 32
156 120
41 10
a
Heat treated at 600 1C for 1 h under 5.1 103 Pa. Heated up to 400 1C at a slow rate, kept at 400 1C for 1 h in 70% CO2+30% H2 atmosphere.
-300
-200
: β-FeOOH
20
(b)
: SiO2 (quartz)
(a) 10 24 d
0
-100
100
200
300
Magnetic field / Oe
Intensity
Magnetization / emu . g-1
b
18 d
12 d
-10
6d -20 Before soaking Fig. 4. (a) Magnetization curve, under magnetic fields of up to 300 Oe, of the products prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h under 5.1 103 Pa: and (b) those of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere.
10
20
30
40
50
2 / degree Fig. 6. XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods.
3µm 3µm Fig. 5. FE–SEM photograph of the surface of the product prepared in high-frequency induction thermal plasma and subsequently heat treated at 600 1C for 1 h at 5.1 103 Pa.
accompanied by a small amount of FeO, and those subsequently heat treated to above 600 1C under 5.1 103 Pa were almost completely composed of Fe3O4. The surfaces of the microsphere were smooth before the heat treatment, but became slightly rough after heat treatment above 400 1C. The increase in the surface roughness might be attributed to the formation of a-Fe2O3 and/or the crystal growth of Fe3O4. The saturation magnetization of the microspheres heat treated at 600 1C for 1 h under 5.1 103 Pa was 92 emu g–1, as shown in Fig. 3(a) and Table 1. It has been reported that stoichiometric Fe3O4 gives a saturation magnetization of 92 emu g–1 at room temperature
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Before soaking soaking Before
66 d d
5µm 5µm
12 12 d d
5µm 5µm
24 24 d d
18 18 d d
5µm 5µm
5µm 5µm
5µm 5µm
Fig. 7. FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for various periods.
:
β-FeOOH
:
Fe3O4 (Magnetite)
small as 10 W g–1, under 300 Oe and 100 kHz (see Fig. 4(a) and Table 1). This might be attributed to the low coercive force (50 Oe) due to the large crystallite size of Fe3O4 (ffi1 mm), as shown in Fig. 5. It has been reported for Fe3O4 that the degree of ordering of the magnetic moment in the individual particles increases with increasing crystallite size, up to 40 nm, forming a wellordered single domain at 40 nm [20], and the number of magnetic domains in the individual particles increases with increasing crystallite size for larger sizes, to decrease the coercive force. It can be seen from Figs. 6 and 7 that b-FeOOH was precipitated on the surface of silica glass microspheres in Fe–HF solution within six days, and that the thickness of b-FeOOH increased with increasing soaking period. The mechanism of the precipitation and growth of bFeOOH is speculated as follows. According to the following (equilibrium (i)), a trivalent iron (Fe3+) fluoro complex and b-FeOOH are formed in the Fe–HF solution.
Intensity
600°C
500°C
400°C
300°C Before treatment
Fe3þ fluoro complex þ mH2 O2b-FeOOH þ nHF: (i)
10
20
30 2 / degree
40
50
Fig. 8. Powder XRD patterns of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatments at various temperatures in a 70% CO2+30% H2 atmosphere.
[19]. Thus, it was reveled that the heat-treated microspheres were fully composed of Fe3O4. However, the heat generation of the heat-treated microspheres was as
When silica glass microspheres are soaked in the Fe–HF solution, reaction (ii) occurs to form a chemically stable [SiF6]2 complex in the solution, and HF is consumed selectively at the surface of the silica glass microspheres. SiO2 þ 6HF ! ½SiF6 2 þ 2H2 O þ 2Hþ :
(ii)
As a result, equilibrium (i) is shifted to the right-hand side in order to compensate the decreased amount of HF in the solution and b-FeOOH is precipitated selectively on the surface of the silica glass microspheres. On the other hand, in the Fe–HF solution, dissolved divalent iron (Fe2+) ions are oxidized gradually to form Fe3+
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treatment Before Before treatment
2237
400° °C 400°C 400°C
300°C
(c) (c)
5µm 5µm
5µm 5µm
5µm 5µm
600°C 500°C 600°C
500°C
5µm 5µm
5µm 5µm
Fig. 9. FE–SEM photographs of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and then subjected to heat treatments at various temperatures in a 70% CO2+30% H2 atmosphere.
(a)
5µm 5µm
(b)
100 100 nm nm
Fig. 10. (a) FE–SEM photograph of silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate in a 70% CO2+30% H2 atmosphere and (b) TEM photograph of the precipitates on their surface.
ions, which have lower solubility than Fe2+ ions under acidic conditions [21]. Therefore, the increase in the amount of Fe3+ ions with low solubility in the Fe–HF solution might be responsible for the growth of bFeOOH on the surface of the silica glass microspheres. It was seen from Fig. 8 that the deposited b-FeOOH was transformed into Fe3O4 by heat treatment above 400 1C in a 70% CO2+30% H2 atmosphere. However, as shown in Fig. 9, large cracks occurred on the microspheres after the heat treatment. These cracks might be attributed to the shrinkage of the surface layer due to the dehydration of b-FeOOH and the formation of Fe3O4. Examining the TG–DTA curves shows that remarkable changes were observed between 100 to 200 1C and between 300 and 400 1C (data was not shown). Therefore, it is expected that the crack formation may be suppressed if the heat treatment temperature was increased at a slower rate from 100 to 200 1C and from 300 to 400 1C. As expected, the
formation of the large cracks was suppressed effectively when the silica glass microspheres were soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate (see Fig. 10(a)). The silica glass microspheres soaked in Fe–HF solution at 30 1C for 24 days and subsequently heated to 400 1C at a slow rate showed ferrimagnetism, with saturation magnetization of 53 emu g–1 and coercive force of 156 Oe (see Fig. 3(b) and Table 1). The heattreated microspheres gave a magnetization curve with a large area (see Fig. 4(b)) and a heat generation of 41 W g–1, under 300 Oe and 100 kHz, which was four times as large as for pure Fe3O4 microspheres prepared in high-frequency induction thermal plasma, as described above (see Table 1). It can be seen from Fig. 10(b) that the heat-treated microspheres are composed of small crystals of Fe3O4 (ffi50 nm). Therefore, the larger heat generation of the microspheres prepared in aqueous solution than those prepared in high-frequency
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induction thermal plasma might be attributed to the higher coercive force due to the smaller crystallite size of Fe3O4 in the former.
5. Conclusions Pure Fe3O4 microspheres, 20–30 mm in diameter, were prepared by melting powders in high-frequency induction thermal plasma and subsequent heat treatment. These microspheres, however, showed heat generation as low as 10 W g–1, at 300 Oe and 100 kHz, as they were composed of Fe3O4 crystals 1 mm in size. Microspheres, 20–30 mm in diameter, composed of small crystals of Fe3O4 50 nm in size, were prepared by precipitation from aqueous solution and subsequent heat treatment. The heat-treated microspheres showed heat generation as high as 41 W g–1, under the same magnetic field. The latter microspheres are believed to be promising thermoseeds for hyperthermic treatment of cancer. Acknowledgments This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] Sugihara T. Gan-to-Tatakau-Hyperthermia (Japanese). Kyoto: Kinpoudou; 1986. pp. 37–48. [2] Cavaliere R, Ciocatto EC, Giovanella BC, Heidelberger C, Johnson RO, Margottini M, Mondovi B, Moricca G, RossiFanelli A. Selective heat sensitivity of cancer cells. Biochemical and clinical studies. Cancer 1967;20:1351–81. [3] Overgaard K, Overgaard J. Investigations on the possibility of a thermic tumour therapy. I. Short-wave treatment of a transplanted isologous mouse mammary carcinoma. Eur J Cancer 1972;8:65–78. [4] Overgaard J. Effect of hyperthermia on malignant cells in vivo. A review and a hypothesis. Cancer 1977;39:2637–46. [5] Luderer AA, Borrelli NF, Panzarino JN, Mansfield GR, Hess DM, Brown JL, Barnett EH, Hahn EW. Glass-ceramic-mediated, magnetic-field-induced localized hyperthermia: response of a murine mammary carcinoma. Radiat Res 1983;94:190–8.
[6] Borrelli NF, Luderer AA, Panzarino JN. Radio frequency induced hyperthermia for tumor therapy. US Patent. No.4 323 056, 1980. [7] Borrelli NF, Luderer AA, Panzarino JN. Hysteresis heating for the treatment of tumours. Phys Med Biol 1984;29:487–94. [8] Kokubo T, Ebisawa Y, Sugimoto Y, Kiyama M, Ohura K, Yamamuro T, Hiraoka M, Abe M. Preparation of bioactive and ferromagnetic glass-ceramic for hyperthermia. In: Hulbert JE, Hulbert SFeditors. Bioceramics, vol. 3. Terre Haute, Indiana: Rose-Hulman Institute of Technology; 1990. p. 213–23. [9] Ebisawa Y, Sugimoto Y, Hayashi T, Kokubo T, Ohura K, Yamamuro T. Crystallization of (FeO, Fe2O3)CaO–SiO2 glasses and magnetic properties of their crystallized products. J Ceram Soc Japan 1991;99:7–13. [10] Ebisawa Y, Kokubo T, Ohura K, Yamamuro T. Bioactivity of CaOSiO2-based glasses: in vitro evaluation. J Mater Sci: Mater. Med 1990;1:239–44. [11] Ebisawa Y, Kokubo T, Ohura K, Yamamuro T. Bioactivity of Fe2O3-containing CaOSiO2 glasses: in vitro evaluation. J Mater Sci: Mater Med 1992;4:225–32. [12] Ohura K, Ikenaga M, Nakamura T, Yamamuro T, Ebisawa Y, Kokubo T, Kotoura Y, Oka M. A heat-generating bioactive glass-ceramic for hyperthermia. J Appl Biomater 1991;2:153–9. [13] Ikenaga M, Ohura K, Yamamuro T, Kotoura Y, Oka M, Kokubo T. Localized hyperthermic treatment of experimental bone tumors with ferromagnetic ceramics. J Orthop Res 1993;11: 849–55. [14] Ebisawa Y, Miyaji F, Kokubo T, Ohura K, Nakamura T. Surface reaction of bioactive and ferromagnetic glass-ceramics in the system FeO–Fe2O3–CaO–SiO2. J Ceram Soc Japan 1997;105: 947–51. [15] Ebisawa Y, Miyaji F, Kokubo T, Ohura K, Nakamura T. Bioactivity of ferrimagnetic glass-ceramics in the system FeO– Fe2O3–CaO–SiO2. Biomaterials 1997;18:1277–84. [16] Konaka H, Miyaji F, Kokubo T. Preparation and magnetic properties of glass-ceramics containing a-Fe for hyperthermia. J Ceram Soc Japan 1997;105:833–6. [17] Kawashita M, Takaoka H, Kokubo T, Yao T, Hamada S, Shinjo T. Preparation of magnetite-containing glass-ceramics in controlled atmosphere for hyperthermia of cancer. J Ceram Soc Japan 2001;109:39–44. [18] Kawashita M, Iwahashi Y, Kokubo T, Yao T, Hamada S, Shinjo T. Preparation of glass-ceramics containing ferrimagnetic zinc– iron ferrite for the hyperthermal treatment of cancer. J Ceram Soc Japan 2004;112:373–9. [19] Kingery WD, Bowen HK, Uhlmann DR. Introduction to ceramics. New York: Wiley; 1976. p. 993. [20] Knoules JE. Coercivity and packing density in acicular particles. J Magn Magn Mater 1981;25:105–12. [21] Paul A, Zaman MS. The relative influences of Al2O3 and Fe2O3 on the chemical durability of silicate glasses at different pH values. J Mater Sci 1978;13:1499–502.