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Release of 5-fluorouracil from thermosensitive magnetoliposomes induced by an electromagnetic field
Journal of Controlled Release 46 (1997) 263–271
Release of 5-fluorouracil from thermosensitive magnetoliposomes induced by an electromagnetic field Ekapop Viroonchatapan a , Hitoshi Sato a , Masaharu Ueno b , Isao Adachi a , Kenji Tazawa c , a, Isamu Horikoshi * a
Department of Hospital Pharmacy, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930 -01, Japan Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930 -01, Japan c Second Department of Surgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930 -01, Japan Accepted 5 November 1996 b
Abstract Thermosensitive magnetoliposomes (TMs) have been proposed by the authors as a new drug carrier for magnetic targeting (Viroonchatapan et al., Pharm. Res. 12 (1995) 1176–1183; Viroonchatapan et al., Life Sci. 58 (1996) 2251–2261). The present study was carried out to investigate the properties of selective heating and thermosensitive drug release of TMs caused by an electromagnetic field. TMs containing 5-fluorouracil (5-FU) were prepared by reverse-phase evaporation. They were selectively heated by a 500-kHz electromagnetic field in a temperature-controlling unit kept at 378C, and the properties of single and multiple release of 5-FU from TMs were examined. The results showed that the temperature of TMs could be effectively elevated to 428C and maintained at this temperature against a cooling effect of the temperature-controlling unit, which mimics an in vivo situation where temperature rise in the site of TM delivery would be hindered by blood flow and surrounding tissues. The release of 5-FU from TMs in response to electromagnetic field-generated heat was accomplished. Moreover, the drug release could be executed several times by multiple exposure to the field. In conclusion, this paper presents for the first time a demonstration of both single and multiple thermosensitive drug release from TMs, induced by an electromagnetic field. It is suggested that TMs would be useful in future cancer treatment by magnetic targeting combined with drug release in response to electromagnetic induced hyperthermia. Keywords: Thermosensitive magnetoliposomes; Dextran magnetite; Electromagnetic induction heating; Drug release; 5-Fluorouracil
1. Introduction The use of thermosensitive liposomes in conjunction with local hyperthermia has been attempted for cancer treatment [1–7]. Besides earlier methods of heating (e.g. in a warm bath or with warmed *Corresponding author. Tel: 181 764 342281, Ext. 3250; Fax: 181 764 340297; WWW site: http: / / www.toyama-mpu.ac.jp.
perfusate), electromagnetic heating is now being investigated, especially for the local hyperthermia of deeper body structures [8–11]. Recently, dextran magnetite (DM) was reported to be a new agent for selective heating by electromagnetic induction [12– 15]. According to Jordan et al. [11], DM develops heat by the rotation of both a whole crystal magnetization vector and a subdomain particle, when coupled to an electromagnetic field.
0168-3659 / 97 / $17.00 Copyright 1997 Elsevier Science B.V. All rights reserved PII S0168-3659( 96 )01606-9
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We have proposed DM-incorporated thermosensitive liposomes (thermosensitive magnetoliposomes, TMs) as a new drug carrier from the viewpoint of magnetic targeting associated with drug release in response to electromagnetic induced hyperthermia. TMs formulated by the authors were shown to be efficiently held by a static magnetic field in an in vitro experiment [16] and in in situ perfused livers [17]. Local drug release was suggested to enhance the therapeutic effectiveness of drug-containing liposomes for local hyperthermia [18–20]. However, TMs have never been investigated with regard to drug release induced by an electromagnetic field. Therefore, the current study was designed to evaluate the properties of selective heating and drug release of TMs in a temperature-controlling unit kept at 378C, which mimics an in vivo situation, in an electromagnetic field. For this purpose, 5-fluorouracil (5FU), a powerful hydrophilic antitumor agent widely used in tumor treatment and dermatological applications, was entrapped in TMs. The prominent feature of this study compared with previous ones [1–7,18– 20] lies in the fact that the drug release is invoked by an external electromagnetic field.
2. Materials and methods
2.1. Materials Dextran magnetite (DM) was a gift from Meito Sangyo (Nagoya, Japan). Dipalmitoylphosphatidylcholine (DPPC) was supplied by Nippon Oil and Fats (Tokyo, Japan). TES (N-tris[hydroxymethyl]methyl-2-amino-ethanesulfonic acid) was purchased from Wako Pure Chemical Industries (Tokyo, Japan). 5-Fluorouracil (5-FU) was obtained from Nacalai Tesque (Kyoto, Japan). Calf serum was provided by Life Technologies (Grand Island, NY, USA). All other reagents used were commercially available and of analytical grade. The specifications of DM are as follows [16]: a hematite impurity was under the detection limit according to the results of X-ray diffraction; the average molecular weight of dextran in DM is 4000; the dextran / magnetite weight ratio is 0.43; its average core size is 8 nm ranging from 5 to 10 nm; its coercivity, magnetic susceptibility, saturation mag-
netization, and T 2 relaxivity are 240 A / m, 0.3 (g 21 2 Fe) , 0.1 Wb / m / g Fe, and 220 l / mmol per s, respectively.
2.2. Preparation of TMs The method of thermosensitive magnetoliposome (TM) preparation was reported in previous papers [16,17]. Briefly, DPPC (294 mg) was dissolved in 120 ml of an isopropyl ether and chloroform mixture (1:1, v / v). Then, it was emulsified with 20 ml of DM suspension (167 mM as magnetite) containing 192 mM 5-FU by 5-min sonication at 408C in a bath sonicator (Sonorex Super RK 156 BH, 150 / 300 W, Bandelin Electronic, Berlin, Germany) fixed at 35 kHz. The organic solvent was evaporated from the w / o emulsion in a rotary evaporator at 428C and low pressure (260–400 mmHg). TMs were separated from non-encapsulated DM and 5-FU by centrifugation (9603g) at 48C for 20 min and TMs were resuspended in 20 mM TES buffer solution containing 0.84% NaCl (pH 7.0). This step was repeated three times. The concentration of DPPC was adjusted to 8.21 mM. The average diameter of TMs was measured using a laser particle size analyzing system (Photal LPA-3000 / 3100, Otsuka Electronics, Osaka, Japan).
2.3. Determination of phosphorus, magnetite, and 5 -FU Total phosphorus was determined based on the method of Bartlett [21]. Magnetite was measured colorimetrically according to the method described by Kiwada et al. [22]. 5-FU was detected spectrophotometrically at 266 nm. Then, the contents of magnetite (mg / mmol DPPC) and 5-FU (mmol / mmol DPPC) of TM suspension were calculated. In order to quantify 5-FU without any effect of nonencapsulated DM, a new method to remove DM from the samples was developed and employed as described below.
2.4. High-gradient magnetic filtration apparatus Since DM cannot be well separated by centrifugation, a high-gradient magnetic filtration apparatus
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operated at the magnetic field of 15 000 Gauss, which was measured by a Gauss meter (Model HGM-8200, ADS Co., Tokyo, Japan). Then, a sample solution containing 5-FU and DM was pumped at 2.0 ml / min through the magnetic filtration column.
2.5. Thermal property of DM in an electromagnetic field
Fig. 1. Illustration of a high-gradient magnetic filtration apparatus.
was designed for removing DM as illustrated in Fig. 1. The apparatus was made up of a peristaltic pump (Eyela MP-3, Tokyo Rikakikai, Tokyo, Japan), a magnetic filtration column placed between magnetic pole pieces, and an electromagnet (Model MCD-1B, JASCO, Tokyo, Japan) connected with a power supply (Model 7020A, JASCO). The column was a 5-ml disposable syringe (I.D., 1.3 cm; O.D., 1.5 cm; Top, Tokyo, Japan) packed with 4 g of stainless steel wool (NAS 430, Nihonseisen, Osaka, Japan); the packed wool was 2-cm thick. The electromagnet was
A system for evaluating the thermal property of DM is illustrated in Fig. 2. The system consisted of an electromagnetic field-producing unit (Thermotron Model RF-IV, Yamamoto Vinyter, Osaka, Japan) with a 7-kW generator of a 500-kHz electromagnetic field and a four-turn pancake coil, a thermometry system (Fluoroptic Model 3000, Luxtron, Mountain View, CA, USA), a double-jacketed beaker, and a water circulator with Omron E5BX temperature controller (Cool Circulator CB15, Iuchi, Tokyo, Japan). The thermometry system, in which metal-free sensors (Luxtron Fluoroptic MPM, Luxtron) are implemented, provided a precise temperature determination without direct heating effect of the field on the sensors. DM suspension (50 m l) at various concentrations was introduced into a 1.5-ml microtube (12310MTB1.5, Iwaki Glass, Tokyo, Japan). Then,
Fig. 2. Diagram of an experimental system for evaluating the thermal properties of DM and TMs.
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the microtube was placed in the water (30 ml) inside the double-jacketed beaker located on the center of the pancake coil (see Fig. 2), the strongest fieldgenerated site. The temperature sensors of the thermometry system were placed in the DM suspension and in the water inside the double-jacketed beaker controlled at 378C by the water circulator. Then, DM suspension was inductively heated by an electromagnetic field and temperatures of the suspension and the water inside the beaker were monitored every 10 s with on-line data acquisition by a microcomputer (9801 NC, NEC, Tokyo, Japan).
2.6. Property of thermosensitive 5 -FU release from TMs induced by an electromagnetic field One milliliter of TMs with 8.21 mM DPPC was put into a microtube and centrifuged at 19203g for 20 min. The supernatant was decanted and the precipitate (50 m l of TMs) was redispersed with 950 ¨ m l of Sorensen buffer (53.4 mM Na 2 HPO 4 , 13.3 mM NaH 2 PO 4 , 75.2 mM NaCl; pH 7.4) or 50% calf ¨ serum in Sorensen buffer, followed by centrifugation at 19203g for 20 min to obtain concentrated TMs with 164 mM DPPC. The microtube was then exposed to an electromagnetic field using the same procedure as described for the thermal property of DM. The double-jacketed beaker was placed 5 cm out of the center of the pancake coil to keep the TM suspension near the phase transition temperature of DPPC (428C). After being exposed to the field for the given periods of time (5, 10, 20, 30, 60, and 120 min after reaching 428C), TMs were dispersed and centrifuged at 19203g for 20 min and the supernatant was collected. The supernatant obtained (500 m l) was mixed with 3.5 ml of methanol and filtered through the high-gradient magnetic filtration apparatus described above. 5-FU in the supernatant was determined by spectrophotometer (U-3410, Hitachi, Tokyo, Japan) at 266 nm. Then, the amount of 5-FU released was calculated against that of the control (0-min exposure to the field). Drug release experiments were also performed at 378C without an electromagnetic field. Moreover, the above procedure was repeated three times with 20-min field exposure and 20-min centrifugation (at 19203g) for the multiple thermosensitive release of 5-FU.
3. Results
3.1. Magnetite and 5 -FU contents of TMs The physicochemical properties of TMs prepared in this study were essentially the same as previously reported [16,17]. The differential scanning calorimetry showed a thermal curve for TMs with a sharp transition peak at 428C, the phase transition temperature of DPPC [16], suggesting that DM does not interrupt the lipid packing within the liposomal bilayers. The percentage release of calcein from TMs was greatest at 428C [16]. Thus, it appears that DM does not qualitatively interfere with a temperaturedependent release characteristic of TMs. The electron micrographs revealed that TMs were completely filled with DM when they were prepared at high DM concentrations. In contrast, they were coated by or partially filled with DM when prepared at lower DM concentrations [16]. The average diameter of TMs used in the current study was determined to be 1.03 m m; 75% of total particles are distributed in the range of 1.3260.12 m m, and the other portion in the range of 0.1660.01 m m, expressed as mean6S.D. The contents of incorporated magnetite and 5-FU were 352 mg and 2.03 mmol per mmol DPPC, respectively.
3.2. Thermal property of DM in an electromagnetic field Fig. 3 shows thermal profiles of DM at various concentrations. Slight fluctuations of temperature were inevitably observed due to the high sensitivity of the thermometry system used and the discontinuous nature of the thermostat. The temperature of DM was sharply raised at the beginning of the exposure time and then it reached a plateau at different temperature depending on the DM concentration (15.6–250 mM magnetite). For the higher DM concentrations (500 and 1000 mM magnetite), temperature rise far exceeded the level required for hyperthermia. Increasing the DM concentration resulted in a higher rate and extent of heating. It was clearly shown that the DM concentration at approximately 250 mM magnetite was appropriate for heating close to 428C. Therefore, the concentration
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Fig. 3. Thermal profiles of DM exposed to an electromagnetic field. The numbers accompanying the lines correspond to the concentrations (mM as magnetite) of DM. Note that the water inside the double-jacketed beaker was not heated. See Fig. 2 for the experimental system.
of TMs was set at 250 mM magnetite for the subsequent experiment.
3.3. Property of thermosensitive release of 5 -FU from TMs induced by an electromagnetic field We employed a centrifugation method to concentrate TM suspension from 8.21 mM to 164 mM DPPC in order to achieve DM concentration at 250 mM magnetite because a high concentration of DM ($approximately 250 mM magnetite) is required for electromagnetic induction heating (see Fig. 3). Fig. 4 shows a thermal profile of TMs containing DM at the magnetite concentration of 250 mM. It was found that TMs could be heated approximately up to 428C even in a temperature-controlling unit kept at 378C. Fig. 5 depicts the time courses of 5-FU release from TMs during exposure to an electromagnetic field for 2 h under different conditions. The drug was released relatively rapidly at 428C for the initial 10 min, followed by a slower phase. In contrast, the drug release at 378C occurred to a lower extent. In both cases, the release of 5-FU from TMs in 50% calf ¨ serum was greater than that in Sorensen buffer. The releases of 5-FU from TMs in the serum and buffer after reaching 428C for 120 min were 2.54% and 2.28% of the total drug encapsulated in the liposomes, respectively. The amount of DM in the
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Fig. 4. Thermal profile of TMs exposed to an electromagnetic field. Note that the water inside the double-jacketed beaker was not heated. See Fig. 2 for the experimental system.
supernatant of TM suspension after exposure to the electromagnetic field was determined to be less than 0.2 mM as magnetite, indicating that DM did not leak from TMs. Fig. 6 illustrates thermal profiles of TM suspension and thermosensitive 5-FU release from TMs during multiple exposure to an electromagnetic field. The extents of drug release were almost the same for each medium. The release in ¨ 50% calf serum was higher than that in Sorensen buffer, which is similar to the results in Fig. 5.
Fig. 5. Time courses of thermosensitive 5-FU release from TMs in ¨ Sorensen buffer (pH 7.4) and 50% calf serum with (428C) and without (378C) exposure to an electromagnetic field. Drug releases in the buffer and serum after reaching 428C for 120 min were 2.28% and 2.54% of the total drug encapsulated in the liposomes, respectively. Each value is expressed as mean6S.D., n53. See text for details.
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Fig. 7. Schematic illustration of the thermosensitive magnetoliposome (TM). DM and drug molecules are incorporated in the TM.
Fig. 6. Thermal profiles of TM suspension (panel A) and ¨ thermosensitive release of 5-FU from TMs (panel B) in Sorensen buffer (pH 7.4) and 50% bovine serum at 428C, during multiple exposure to an electromagnetic field. Data in panel B are the mean6S.D. of triplicate experiments. See text for details.
4. Discussion The inclusion of DM in the thermosensitive liposomes came from the idea that the combination of magnetoliposomes and thermosensitive liposomes for magnetic targeting can provide not only drug targeting by a static magnetic field but also drug release in response to hyperthermia, especially electromagnetic-induced selective hyperthermia. Transmission electron micrographs of TMs were published in a preceding paper [16] to show clearly the entrapment of DM and its distribution in the liposomes. The thermosensitive magnetoliposome (TM) containing drug is illustrated schematically in Fig. 7. TMs were shown to be efficiently targeted by a static magnetic field in the in vitro on-line flow system [16] and in situ perfused mouse liver [17]. Moreover, our preliminary study first reported the electromagnetic field-induced heating property of TMs [23]. In
that study, heat loss from TMs during the in vitro experiment was minimized by using an effective styrofoam insulation around the sample tube. This is why a lower DM concentration (10.4 mg Fe / ml or 62.5 mM magnetite) could cause a temperature rise up to 428C in vitro [23]. On the contrary, the present results indicated that a much higher concentration of magnetite (approximately 250 mM) is required for effectively elevating and maintaining the temperature of TMs at 428C against a cooling effect of a temperature-controlling unit (378C) which mimics an in vivo situation (i.e. the existence of cooling effect by blood flow and surrounding tissues). In this sense, the present in vitro study may be closer to an in vivo situation than those using styrofoam as a heat insulator for in vitro experiments of electromagnetic heating [11,23]. In a previous study, we reported that the temperature of phantom with magnetite particles was effectively elevated by an electromagnetic field of 500 kHz while leaving the temperature of phantom without the particles essentially unchanged [24]. The reasons for using the large DPPC liposomes without an incorporation of cholesterol in our study are as follows: (1) large unilamellar vesicles have the best temperature-controlled drug release properties as a result of their homogeneous size and lesser curvature compared with small unilamellar vesicles [6]; (2) incorporation of cholesterol into lipid bilayers has a detrimental effect on thermosensitive liposomes, i.e. it broadens the temperature-dependent release property of the liposomes by reducing the temperature of onset of drug release,
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resulting in a significant amount of drug release at 37–388C [25]; (3) as our intention, TMs were formulated for intra-arterial administration into a diseased organ in conjunction with magnetic targeting, so that the main portion of administered TMs will not systemically circulate over the whole body but be locally retained in the organ. Therefore, the stability of TMs may not be an unfavorable circumstance for this particular purpose. For the drug release experiments and determination of entrapped 5-FU in TMs, we developed a new method of removing DM from the samples using a high-gradient magnetic filtration apparatus (Fig. 1). We found that this is a rapid and effective means of avoiding interference with DM for an accurate spectrophotometric measurement of the drug. As shown in Fig. 5, the profile of drug release at 428C can be divided into two parts: a first rapid phase and a second slower phase. It is assumed that 5-FU release in the first phase is attributed to TMs residing near the interface between the TM suspension and releasing medium, while in the second phase it is due to 5-FU released and diffused from inside the suspension. This assumption is feasible because TM concentration (164 mM DPPC) was so high that the TM suspension used was relatively viscous. This also causes a cost problem in clinical use. The reason for the use of a high TM concentration was that it was not possible to detect substantial drug release at lower TM concentrations due to insufficient temperature rise. This limitation of the study should be solved when using a stronger electromagnetic field generator. The 5-FU release from TMs at 378C occurred to a lower extent than that at 428C and reached a plateau after 20 min (Fig. 5). It is indicated from this result that only a minor fraction of TMs located at the interface between the TM suspension and releasing medium released their content spontaneously. At both 378C and 428C, there is a tendency for the release of 5-FU in serum to be slightly higher than that in buffer, indicating a minor effect of serum on the drug release property of TMs. The releases of ¨ 5-FU from TMs in 50% calf serum and Sorensen buffer after reaching 428C for 120 min were 2.54% and 2.28% of the total drug encapsulated in the liposomes, respectively. From this result, in a case where 50 m l of TMs are injected into a tumor of
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1-ml size, the concentration of released 5-FU can be calculated as 55 m g / ml; this value was reported to be sufficient to produce at least 50% growth inhibition of several human tumor cell lines [26–29]. Moreover, the extent of 5-FU released can be increased when the larger volume of TMs is administered. Of clinical interest is the fact that multiple thermosensitive release of 5-FU from TMs, induced by an electromagnetic field, could be achieved (see Fig. 6). This finding indicates the possibility of using TMs to treat cancer by multiple hyperthermia and concomitant drug release, with which we expect more effective cancer therapy than single hyperthermia. In another experiment [30], moreover, a second hyperthermia using TMs was successfully carried out in vivo 48 h subsequent to the first hyperthermia while DM itself may be easily diffused out from the injected site due to its much smaller size and high dispersibility compared with TMs. Together with the observation of no DM leakage from TMs, TMs were shown to exist in the injected site for at least 2 days to such an extent that multiple hyperthermia is possible. Other than the electromagnetic induced heating and drug release properties, TMs have another important feature, i.e. drug targeting by a static magnetic field. With the use of a strong electromagnetic field generator, drug release from TMs after targeting to a specific organ may be possible. This would be helpful in treating a diseased organ by first targeting TMs and subsequently exposing the organ to an electromagnetic field. The possibility of targeting TMs to the liver was already provided [17] aiming to treat hepatocellular carcinoma (HCC), a hard healing type of cancer. Since powerful magnets are currently employed in clinical settings such as magnetic resonance imaging (MRI) systems, the clinical use of TMs with an extracorporeal magnet is feasible. Magnetic targeting may have a wide application because it is not organ-specific. In future, a combination of magnetic targeting with a specific targeting device (e.g. antibody and receptor) would provide a more sophisticated approach to the active delivery of drugs. In conclusion, this paper presents for the first time a demonstration of temperature-dependent drug release from TMs, both single and multiple release, induced by an electromagnetic field. We are now
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investigating in vivo drug release from TMs in a tumor by using a microdialysis technique.
Acknowledgments One of the authors (E.V.) thanks the Ministry of Education, Science, and Culture, Japan, for their scholarship to him. This work was supported in part by a grant from the Sagawa Foundation for the Promotion of Cancer Research, awarded to E.V., and a grant from the Magnetic Health Science Foundation to M.U.
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Release of 5-fluorouracil from thermosensitive magnetoliposomes induced by an electromagnetic field Ekapop Viroonchatapan a , Hitoshi Sato a , Masaharu Ueno b , Isao Adachi a , Kenji Tazawa c , a, Isamu Horikoshi * a
Department of Hospital Pharmacy, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930 -01, Japan Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930 -01, Japan c Second Department of Surgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930 -01, Japan Accepted 5 November 1996 b
Abstract Thermosensitive magnetoliposomes (TMs) have been proposed by the authors as a new drug carrier for magnetic targeting (Viroonchatapan et al., Pharm. Res. 12 (1995) 1176–1183; Viroonchatapan et al., Life Sci. 58 (1996) 2251–2261). The present study was carried out to investigate the properties of selective heating and thermosensitive drug release of TMs caused by an electromagnetic field. TMs containing 5-fluorouracil (5-FU) were prepared by reverse-phase evaporation. They were selectively heated by a 500-kHz electromagnetic field in a temperature-controlling unit kept at 378C, and the properties of single and multiple release of 5-FU from TMs were examined. The results showed that the temperature of TMs could be effectively elevated to 428C and maintained at this temperature against a cooling effect of the temperature-controlling unit, which mimics an in vivo situation where temperature rise in the site of TM delivery would be hindered by blood flow and surrounding tissues. The release of 5-FU from TMs in response to electromagnetic field-generated heat was accomplished. Moreover, the drug release could be executed several times by multiple exposure to the field. In conclusion, this paper presents for the first time a demonstration of both single and multiple thermosensitive drug release from TMs, induced by an electromagnetic field. It is suggested that TMs would be useful in future cancer treatment by magnetic targeting combined with drug release in response to electromagnetic induced hyperthermia. Keywords: Thermosensitive magnetoliposomes; Dextran magnetite; Electromagnetic induction heating; Drug release; 5-Fluorouracil
1. Introduction The use of thermosensitive liposomes in conjunction with local hyperthermia has been attempted for cancer treatment [1–7]. Besides earlier methods of heating (e.g. in a warm bath or with warmed *Corresponding author. Tel: 181 764 342281, Ext. 3250; Fax: 181 764 340297; WWW site: http: / / www.toyama-mpu.ac.jp.
perfusate), electromagnetic heating is now being investigated, especially for the local hyperthermia of deeper body structures [8–11]. Recently, dextran magnetite (DM) was reported to be a new agent for selective heating by electromagnetic induction [12– 15]. According to Jordan et al. [11], DM develops heat by the rotation of both a whole crystal magnetization vector and a subdomain particle, when coupled to an electromagnetic field.
0168-3659 / 97 / $17.00 Copyright 1997 Elsevier Science B.V. All rights reserved PII S0168-3659( 96 )01606-9
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We have proposed DM-incorporated thermosensitive liposomes (thermosensitive magnetoliposomes, TMs) as a new drug carrier from the viewpoint of magnetic targeting associated with drug release in response to electromagnetic induced hyperthermia. TMs formulated by the authors were shown to be efficiently held by a static magnetic field in an in vitro experiment [16] and in in situ perfused livers [17]. Local drug release was suggested to enhance the therapeutic effectiveness of drug-containing liposomes for local hyperthermia [18–20]. However, TMs have never been investigated with regard to drug release induced by an electromagnetic field. Therefore, the current study was designed to evaluate the properties of selective heating and drug release of TMs in a temperature-controlling unit kept at 378C, which mimics an in vivo situation, in an electromagnetic field. For this purpose, 5-fluorouracil (5FU), a powerful hydrophilic antitumor agent widely used in tumor treatment and dermatological applications, was entrapped in TMs. The prominent feature of this study compared with previous ones [1–7,18– 20] lies in the fact that the drug release is invoked by an external electromagnetic field.
2. Materials and methods
2.1. Materials Dextran magnetite (DM) was a gift from Meito Sangyo (Nagoya, Japan). Dipalmitoylphosphatidylcholine (DPPC) was supplied by Nippon Oil and Fats (Tokyo, Japan). TES (N-tris[hydroxymethyl]methyl-2-amino-ethanesulfonic acid) was purchased from Wako Pure Chemical Industries (Tokyo, Japan). 5-Fluorouracil (5-FU) was obtained from Nacalai Tesque (Kyoto, Japan). Calf serum was provided by Life Technologies (Grand Island, NY, USA). All other reagents used were commercially available and of analytical grade. The specifications of DM are as follows [16]: a hematite impurity was under the detection limit according to the results of X-ray diffraction; the average molecular weight of dextran in DM is 4000; the dextran / magnetite weight ratio is 0.43; its average core size is 8 nm ranging from 5 to 10 nm; its coercivity, magnetic susceptibility, saturation mag-
netization, and T 2 relaxivity are 240 A / m, 0.3 (g 21 2 Fe) , 0.1 Wb / m / g Fe, and 220 l / mmol per s, respectively.
2.2. Preparation of TMs The method of thermosensitive magnetoliposome (TM) preparation was reported in previous papers [16,17]. Briefly, DPPC (294 mg) was dissolved in 120 ml of an isopropyl ether and chloroform mixture (1:1, v / v). Then, it was emulsified with 20 ml of DM suspension (167 mM as magnetite) containing 192 mM 5-FU by 5-min sonication at 408C in a bath sonicator (Sonorex Super RK 156 BH, 150 / 300 W, Bandelin Electronic, Berlin, Germany) fixed at 35 kHz. The organic solvent was evaporated from the w / o emulsion in a rotary evaporator at 428C and low pressure (260–400 mmHg). TMs were separated from non-encapsulated DM and 5-FU by centrifugation (9603g) at 48C for 20 min and TMs were resuspended in 20 mM TES buffer solution containing 0.84% NaCl (pH 7.0). This step was repeated three times. The concentration of DPPC was adjusted to 8.21 mM. The average diameter of TMs was measured using a laser particle size analyzing system (Photal LPA-3000 / 3100, Otsuka Electronics, Osaka, Japan).
2.3. Determination of phosphorus, magnetite, and 5 -FU Total phosphorus was determined based on the method of Bartlett [21]. Magnetite was measured colorimetrically according to the method described by Kiwada et al. [22]. 5-FU was detected spectrophotometrically at 266 nm. Then, the contents of magnetite (mg / mmol DPPC) and 5-FU (mmol / mmol DPPC) of TM suspension were calculated. In order to quantify 5-FU without any effect of nonencapsulated DM, a new method to remove DM from the samples was developed and employed as described below.
2.4. High-gradient magnetic filtration apparatus Since DM cannot be well separated by centrifugation, a high-gradient magnetic filtration apparatus
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operated at the magnetic field of 15 000 Gauss, which was measured by a Gauss meter (Model HGM-8200, ADS Co., Tokyo, Japan). Then, a sample solution containing 5-FU and DM was pumped at 2.0 ml / min through the magnetic filtration column.
2.5. Thermal property of DM in an electromagnetic field
Fig. 1. Illustration of a high-gradient magnetic filtration apparatus.
was designed for removing DM as illustrated in Fig. 1. The apparatus was made up of a peristaltic pump (Eyela MP-3, Tokyo Rikakikai, Tokyo, Japan), a magnetic filtration column placed between magnetic pole pieces, and an electromagnet (Model MCD-1B, JASCO, Tokyo, Japan) connected with a power supply (Model 7020A, JASCO). The column was a 5-ml disposable syringe (I.D., 1.3 cm; O.D., 1.5 cm; Top, Tokyo, Japan) packed with 4 g of stainless steel wool (NAS 430, Nihonseisen, Osaka, Japan); the packed wool was 2-cm thick. The electromagnet was
A system for evaluating the thermal property of DM is illustrated in Fig. 2. The system consisted of an electromagnetic field-producing unit (Thermotron Model RF-IV, Yamamoto Vinyter, Osaka, Japan) with a 7-kW generator of a 500-kHz electromagnetic field and a four-turn pancake coil, a thermometry system (Fluoroptic Model 3000, Luxtron, Mountain View, CA, USA), a double-jacketed beaker, and a water circulator with Omron E5BX temperature controller (Cool Circulator CB15, Iuchi, Tokyo, Japan). The thermometry system, in which metal-free sensors (Luxtron Fluoroptic MPM, Luxtron) are implemented, provided a precise temperature determination without direct heating effect of the field on the sensors. DM suspension (50 m l) at various concentrations was introduced into a 1.5-ml microtube (12310MTB1.5, Iwaki Glass, Tokyo, Japan). Then,
Fig. 2. Diagram of an experimental system for evaluating the thermal properties of DM and TMs.
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the microtube was placed in the water (30 ml) inside the double-jacketed beaker located on the center of the pancake coil (see Fig. 2), the strongest fieldgenerated site. The temperature sensors of the thermometry system were placed in the DM suspension and in the water inside the double-jacketed beaker controlled at 378C by the water circulator. Then, DM suspension was inductively heated by an electromagnetic field and temperatures of the suspension and the water inside the beaker were monitored every 10 s with on-line data acquisition by a microcomputer (9801 NC, NEC, Tokyo, Japan).
2.6. Property of thermosensitive 5 -FU release from TMs induced by an electromagnetic field One milliliter of TMs with 8.21 mM DPPC was put into a microtube and centrifuged at 19203g for 20 min. The supernatant was decanted and the precipitate (50 m l of TMs) was redispersed with 950 ¨ m l of Sorensen buffer (53.4 mM Na 2 HPO 4 , 13.3 mM NaH 2 PO 4 , 75.2 mM NaCl; pH 7.4) or 50% calf ¨ serum in Sorensen buffer, followed by centrifugation at 19203g for 20 min to obtain concentrated TMs with 164 mM DPPC. The microtube was then exposed to an electromagnetic field using the same procedure as described for the thermal property of DM. The double-jacketed beaker was placed 5 cm out of the center of the pancake coil to keep the TM suspension near the phase transition temperature of DPPC (428C). After being exposed to the field for the given periods of time (5, 10, 20, 30, 60, and 120 min after reaching 428C), TMs were dispersed and centrifuged at 19203g for 20 min and the supernatant was collected. The supernatant obtained (500 m l) was mixed with 3.5 ml of methanol and filtered through the high-gradient magnetic filtration apparatus described above. 5-FU in the supernatant was determined by spectrophotometer (U-3410, Hitachi, Tokyo, Japan) at 266 nm. Then, the amount of 5-FU released was calculated against that of the control (0-min exposure to the field). Drug release experiments were also performed at 378C without an electromagnetic field. Moreover, the above procedure was repeated three times with 20-min field exposure and 20-min centrifugation (at 19203g) for the multiple thermosensitive release of 5-FU.
3. Results
3.1. Magnetite and 5 -FU contents of TMs The physicochemical properties of TMs prepared in this study were essentially the same as previously reported [16,17]. The differential scanning calorimetry showed a thermal curve for TMs with a sharp transition peak at 428C, the phase transition temperature of DPPC [16], suggesting that DM does not interrupt the lipid packing within the liposomal bilayers. The percentage release of calcein from TMs was greatest at 428C [16]. Thus, it appears that DM does not qualitatively interfere with a temperaturedependent release characteristic of TMs. The electron micrographs revealed that TMs were completely filled with DM when they were prepared at high DM concentrations. In contrast, they were coated by or partially filled with DM when prepared at lower DM concentrations [16]. The average diameter of TMs used in the current study was determined to be 1.03 m m; 75% of total particles are distributed in the range of 1.3260.12 m m, and the other portion in the range of 0.1660.01 m m, expressed as mean6S.D. The contents of incorporated magnetite and 5-FU were 352 mg and 2.03 mmol per mmol DPPC, respectively.
3.2. Thermal property of DM in an electromagnetic field Fig. 3 shows thermal profiles of DM at various concentrations. Slight fluctuations of temperature were inevitably observed due to the high sensitivity of the thermometry system used and the discontinuous nature of the thermostat. The temperature of DM was sharply raised at the beginning of the exposure time and then it reached a plateau at different temperature depending on the DM concentration (15.6–250 mM magnetite). For the higher DM concentrations (500 and 1000 mM magnetite), temperature rise far exceeded the level required for hyperthermia. Increasing the DM concentration resulted in a higher rate and extent of heating. It was clearly shown that the DM concentration at approximately 250 mM magnetite was appropriate for heating close to 428C. Therefore, the concentration
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Fig. 3. Thermal profiles of DM exposed to an electromagnetic field. The numbers accompanying the lines correspond to the concentrations (mM as magnetite) of DM. Note that the water inside the double-jacketed beaker was not heated. See Fig. 2 for the experimental system.
of TMs was set at 250 mM magnetite for the subsequent experiment.
3.3. Property of thermosensitive release of 5 -FU from TMs induced by an electromagnetic field We employed a centrifugation method to concentrate TM suspension from 8.21 mM to 164 mM DPPC in order to achieve DM concentration at 250 mM magnetite because a high concentration of DM ($approximately 250 mM magnetite) is required for electromagnetic induction heating (see Fig. 3). Fig. 4 shows a thermal profile of TMs containing DM at the magnetite concentration of 250 mM. It was found that TMs could be heated approximately up to 428C even in a temperature-controlling unit kept at 378C. Fig. 5 depicts the time courses of 5-FU release from TMs during exposure to an electromagnetic field for 2 h under different conditions. The drug was released relatively rapidly at 428C for the initial 10 min, followed by a slower phase. In contrast, the drug release at 378C occurred to a lower extent. In both cases, the release of 5-FU from TMs in 50% calf ¨ serum was greater than that in Sorensen buffer. The releases of 5-FU from TMs in the serum and buffer after reaching 428C for 120 min were 2.54% and 2.28% of the total drug encapsulated in the liposomes, respectively. The amount of DM in the
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Fig. 4. Thermal profile of TMs exposed to an electromagnetic field. Note that the water inside the double-jacketed beaker was not heated. See Fig. 2 for the experimental system.
supernatant of TM suspension after exposure to the electromagnetic field was determined to be less than 0.2 mM as magnetite, indicating that DM did not leak from TMs. Fig. 6 illustrates thermal profiles of TM suspension and thermosensitive 5-FU release from TMs during multiple exposure to an electromagnetic field. The extents of drug release were almost the same for each medium. The release in ¨ 50% calf serum was higher than that in Sorensen buffer, which is similar to the results in Fig. 5.
Fig. 5. Time courses of thermosensitive 5-FU release from TMs in ¨ Sorensen buffer (pH 7.4) and 50% calf serum with (428C) and without (378C) exposure to an electromagnetic field. Drug releases in the buffer and serum after reaching 428C for 120 min were 2.28% and 2.54% of the total drug encapsulated in the liposomes, respectively. Each value is expressed as mean6S.D., n53. See text for details.
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Fig. 7. Schematic illustration of the thermosensitive magnetoliposome (TM). DM and drug molecules are incorporated in the TM.
Fig. 6. Thermal profiles of TM suspension (panel A) and ¨ thermosensitive release of 5-FU from TMs (panel B) in Sorensen buffer (pH 7.4) and 50% bovine serum at 428C, during multiple exposure to an electromagnetic field. Data in panel B are the mean6S.D. of triplicate experiments. See text for details.
4. Discussion The inclusion of DM in the thermosensitive liposomes came from the idea that the combination of magnetoliposomes and thermosensitive liposomes for magnetic targeting can provide not only drug targeting by a static magnetic field but also drug release in response to hyperthermia, especially electromagnetic-induced selective hyperthermia. Transmission electron micrographs of TMs were published in a preceding paper [16] to show clearly the entrapment of DM and its distribution in the liposomes. The thermosensitive magnetoliposome (TM) containing drug is illustrated schematically in Fig. 7. TMs were shown to be efficiently targeted by a static magnetic field in the in vitro on-line flow system [16] and in situ perfused mouse liver [17]. Moreover, our preliminary study first reported the electromagnetic field-induced heating property of TMs [23]. In
that study, heat loss from TMs during the in vitro experiment was minimized by using an effective styrofoam insulation around the sample tube. This is why a lower DM concentration (10.4 mg Fe / ml or 62.5 mM magnetite) could cause a temperature rise up to 428C in vitro [23]. On the contrary, the present results indicated that a much higher concentration of magnetite (approximately 250 mM) is required for effectively elevating and maintaining the temperature of TMs at 428C against a cooling effect of a temperature-controlling unit (378C) which mimics an in vivo situation (i.e. the existence of cooling effect by blood flow and surrounding tissues). In this sense, the present in vitro study may be closer to an in vivo situation than those using styrofoam as a heat insulator for in vitro experiments of electromagnetic heating [11,23]. In a previous study, we reported that the temperature of phantom with magnetite particles was effectively elevated by an electromagnetic field of 500 kHz while leaving the temperature of phantom without the particles essentially unchanged [24]. The reasons for using the large DPPC liposomes without an incorporation of cholesterol in our study are as follows: (1) large unilamellar vesicles have the best temperature-controlled drug release properties as a result of their homogeneous size and lesser curvature compared with small unilamellar vesicles [6]; (2) incorporation of cholesterol into lipid bilayers has a detrimental effect on thermosensitive liposomes, i.e. it broadens the temperature-dependent release property of the liposomes by reducing the temperature of onset of drug release,
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resulting in a significant amount of drug release at 37–388C [25]; (3) as our intention, TMs were formulated for intra-arterial administration into a diseased organ in conjunction with magnetic targeting, so that the main portion of administered TMs will not systemically circulate over the whole body but be locally retained in the organ. Therefore, the stability of TMs may not be an unfavorable circumstance for this particular purpose. For the drug release experiments and determination of entrapped 5-FU in TMs, we developed a new method of removing DM from the samples using a high-gradient magnetic filtration apparatus (Fig. 1). We found that this is a rapid and effective means of avoiding interference with DM for an accurate spectrophotometric measurement of the drug. As shown in Fig. 5, the profile of drug release at 428C can be divided into two parts: a first rapid phase and a second slower phase. It is assumed that 5-FU release in the first phase is attributed to TMs residing near the interface between the TM suspension and releasing medium, while in the second phase it is due to 5-FU released and diffused from inside the suspension. This assumption is feasible because TM concentration (164 mM DPPC) was so high that the TM suspension used was relatively viscous. This also causes a cost problem in clinical use. The reason for the use of a high TM concentration was that it was not possible to detect substantial drug release at lower TM concentrations due to insufficient temperature rise. This limitation of the study should be solved when using a stronger electromagnetic field generator. The 5-FU release from TMs at 378C occurred to a lower extent than that at 428C and reached a plateau after 20 min (Fig. 5). It is indicated from this result that only a minor fraction of TMs located at the interface between the TM suspension and releasing medium released their content spontaneously. At both 378C and 428C, there is a tendency for the release of 5-FU in serum to be slightly higher than that in buffer, indicating a minor effect of serum on the drug release property of TMs. The releases of ¨ 5-FU from TMs in 50% calf serum and Sorensen buffer after reaching 428C for 120 min were 2.54% and 2.28% of the total drug encapsulated in the liposomes, respectively. From this result, in a case where 50 m l of TMs are injected into a tumor of
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1-ml size, the concentration of released 5-FU can be calculated as 55 m g / ml; this value was reported to be sufficient to produce at least 50% growth inhibition of several human tumor cell lines [26–29]. Moreover, the extent of 5-FU released can be increased when the larger volume of TMs is administered. Of clinical interest is the fact that multiple thermosensitive release of 5-FU from TMs, induced by an electromagnetic field, could be achieved (see Fig. 6). This finding indicates the possibility of using TMs to treat cancer by multiple hyperthermia and concomitant drug release, with which we expect more effective cancer therapy than single hyperthermia. In another experiment [30], moreover, a second hyperthermia using TMs was successfully carried out in vivo 48 h subsequent to the first hyperthermia while DM itself may be easily diffused out from the injected site due to its much smaller size and high dispersibility compared with TMs. Together with the observation of no DM leakage from TMs, TMs were shown to exist in the injected site for at least 2 days to such an extent that multiple hyperthermia is possible. Other than the electromagnetic induced heating and drug release properties, TMs have another important feature, i.e. drug targeting by a static magnetic field. With the use of a strong electromagnetic field generator, drug release from TMs after targeting to a specific organ may be possible. This would be helpful in treating a diseased organ by first targeting TMs and subsequently exposing the organ to an electromagnetic field. The possibility of targeting TMs to the liver was already provided [17] aiming to treat hepatocellular carcinoma (HCC), a hard healing type of cancer. Since powerful magnets are currently employed in clinical settings such as magnetic resonance imaging (MRI) systems, the clinical use of TMs with an extracorporeal magnet is feasible. Magnetic targeting may have a wide application because it is not organ-specific. In future, a combination of magnetic targeting with a specific targeting device (e.g. antibody and receptor) would provide a more sophisticated approach to the active delivery of drugs. In conclusion, this paper presents for the first time a demonstration of temperature-dependent drug release from TMs, both single and multiple release, induced by an electromagnetic field. We are now
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investigating in vivo drug release from TMs in a tumor by using a microdialysis technique.
Acknowledgments One of the authors (E.V.) thanks the Ministry of Education, Science, and Culture, Japan, for their scholarship to him. This work was supported in part by a grant from the Sagawa Foundation for the Promotion of Cancer Research, awarded to E.V., and a grant from the Magnetic Health Science Foundation to M.U.
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