Magnetic field oscillating at several frequencies for improving efficacy and/or reducing toxicity of magnetic hyperthermia

Abstract

Magnetic nanoparticles for use in a magnetic hyperthermia therapeutic treatment, prophylactic treatment or diagnosis method, wherein the magnetic nanoparticles are administered to a body part of an individual and the body part is exposed to a magnetic field oscillating at a high frequency and at a medium and/or low frequency, wherein the high frequency is 1 MHz at the most, the medium frequency is lower than the high frequency, and the low frequency is lower than the high frequency and lower than the medium frequency when it is present.

Claims

1. A method of therapeutic treatment, prophylactic treatment, cosmetic treatment, or diagnosis of an individual in which magnetic particles are exposed to an oscillating magnetic field, comprising: administering the magnetic nanoparticles to a body part of an individual in need thereof; and applying to the body part a magnetic field oscillating either: (a) at a high frequency, at a medium frequency, and at a low frequency, or (b) at a high frequency and at a low frequency, wherein the high frequency applied in (a) or (b) is 1 MHz at most, wherein the medium frequency applied in (a) is lower than the high frequency, wherein the low frequency applied in (a) or (b) is lower than the high frequency, and the low frequency applied in (a) is lower than the medium frequency, wherein applying the oscillating magnetic field at the low frequency in (a) or (b) is part of a repetition of at least one cycle of applying the oscillating magnetic field at the low frequency, and wherein the at least one cycle of applying the oscillating magnetic field at the low frequency has at least one property selected from the group consisting of: (i) the at least one cycle of applying the oscillating magnetic field at the low frequency comprises a heating step in which the oscillating magnetic field at the low frequency induces heating and a cooling step in which the oscillating magnetic field at the low frequency induces cooling, and (ii) the at least one cycle of applying the oscillating magnetic field at the low frequency comprises a step with increasing oscillating magnetic field strength at the low frequency and a step with decreasing the oscillating magnetic field strength at the low frequency, and wherein applying the oscillating magnetic field induces at least one of a temperature increase or a movement of the magnetic nanoparticles to cause internalization or externalization of the magnetic nanoparticles from cells or death of cells in the body part to provide the therapeutic treatment, prophylactic treatment, cosmetic treatment, or diagnosis.

2. The method according to claim 1, wherein the magnetic nanoparticles have a specific absorption rate (SAR) higher than 1 W/g.

3. The method according to claim 1, wherein the high frequency is between 1 and 1,000 kHz.

4. The method according to claim 3, wherein the high frequency heats the magnetic nanoparticles.

5. The method according to claim 1, wherein the applied magnetic field oscillates at the high frequency, at the medium frequency, and at the low frequency, and wherein the medium frequency is between 10.sup.−5 and 5×10.sup.5 Hz.

6. The method according to claim 1, wherein the applied magnetic field oscillates at the high frequency, at the medium frequency, and at the low frequency, and the medium frequency modulates the high frequency.

7. The method according to claim 1, wherein the applied magnetic field oscillates at the high frequency, at the medium frequency, and at the low frequency, and the medium frequency increases heating properties of the magnetic nanoparticles.

8. The method according to claim 1, wherein the low frequency is between 10.sup.−9 and 5×10.sup.5 Hz.

9. The method according to claim 1, wherein the heating step produces a temperature increase of more than 1° C. of the body part.

10. The method according to claim 1, wherein the cooling step induces a temperature decrease of more than 1° C. of the body part.

11. The method according to claim 1, wherein the applied magnetic field oscillates either: (a) at the high frequency, at the medium frequency, and at the low frequency, wherein both the medium and the low frequency increases a ratio between a maximum and an average amplitude of the applied magnetic field or (b) at the high frequency and at the low frequency, wherein the low frequency increases a ratio between a maximum and an average amplitude of the applied magnetic field.

12. The method according to claim 1, wherein the applied magnetic field oscillates either: (a) at the high frequency, at the medium frequency, and at the low frequency, wherein both the medium and the low frequency decreases diffusion of the magnetic nanoparticles outside of the body part, or (b) at the high frequency and at the low frequency, wherein the low frequency decreases diffusion of the magnetic nanoparticles outside of the body part.

13. The method according to claim 1, wherein a compound is bonded or linked to each of the magnetic nanoparticles before the oscillating magnetic field is applied, and the applied magnetic field oscillates either: (a) at the high frequency, at the medium frequency, and at the low frequency, wherein both the medium frequency and the low frequency causes release of the compound, or (b) at the high frequency and at the low frequency, wherein the low frequency causes release of the compound.

14. The method according to claim 1, wherein the at least one cycle of applying the oscillating magnetic field at the low frequency comprises the heating step and the cooling step, and wherein: i) a maximum temperature and a minimum temperature is reached during the heating step and the cooling step, respectively, and ii) at least one parameter of the magnetic field that modulates temperature is set at a first value to reach the maximum temperature during the heating step and then the at least one parameter of the magnetic field is set at a second value to reach the minimum temperature during the cooling step.

15. The method according to claim 14, wherein the at least one parameter is selected from the group consisting of: average or maximum magnetic field amplitude, magnetic field strength, magnetic field amplitude, magnetic field frequency, and spatial or temporal distribution of magnetic field lines.

16. The method according to claim 1, wherein the method of therapeutic treatment, prophylactic treatment, cosmetic treatment, or diagnosis is for therapeutic treatment, prophylactic treatment, cosmetic treatment, or diagnosis of a disease selected from the group consisting of a cancer, a tumor, and an infection.

17. The method according to claim 1, wherein applying to the body part a magnetic field oscillating either: (a) at the high frequency, at the medium frequency, and at the low frequency or (b) at the high frequency and at the low frequency is performed using a device generating the oscillating magnetic field located a distance of more than 50 cm from the body part.

18. The method according to claim 1, wherein the at least one cycle of applying the oscillating magnetic field at a low frequency comprises the heating step and the cooling step, and at least one of the heating step or the cooling step is performed for less than 1 day.

19. The method according to claim 1, wherein the at least one cycle of applying the oscillating magnetic field at a low frequency comprises the heating step and the cooling step, and the heating step is carried out for a duration that is short enough to limit Eddy or Foucault currents or toxic effects associated with applying the oscillating magnetic field to the body part.

20. A device suitable for magnetic hyperthermia comprising a generator of an oscillating magnetic field configured to oscillate at frequencies as high as 1 MHz and as low as 10.sup.−9 Hz.

21. The device according to claim 20, wherein the generator of the oscillating magnetic field is configured to generate an oscillating magnetic field that is able to reach a body part to be treated by magnetic hyperthermia that is located a distance of more than 1 cm from the generator.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: (a), for a magnetic field oscillating only at a high frequency, f.sub.h, variation of the magnitude of magnitude flux density with time, with indications of time t.sub.1 during which the magnetic field amplitude increases, time t.sub.2 during which the magnetic field amplitude decreases, time t.sub.3 during which the magnetic field is constant, with indication of H.sub.max corresponding to the maximum magnetic field amplitude among the different values of H.sub.max,i, where H.sub.max,i corresponds to the local maximum of magnetic field amplitude at each high frequency oscillation. f.sub.h may be equal or proportional to 1/[2(t.sub.1+t.sub.2+t.sub.3)]. (b), for a magnetic field oscillating at a high frequency, f.sub.h, and a medium frequency, f.sub.m, which may be equal or proportional to 1/[(t.sub.4+t.sub.5+t.sub.6)], variation of the magnitude of magnetic flux density with time, with indications of time t.sub.4 during which the magnetic field amplitude increases, time t.sub.5 during which the magnetic field amplitude decreases, time t.sub.6 during which the magnetic field is constant, with indication of H.sub.max corresponding to the maximum magnetic field amplitude among different values of H.sub.max,i, where H.sub.max,i corresponds to the local maximum of magnetic field amplitude at each high frequency oscillation and H.sub.av is equal or proportional to (Σ.sub.i=1.sup.i=n=H.sub.max,i)/n, where n is the number of high frequency oscillations.

(2) FIG. 2: (a), Percentage of H.sub.max and H.sub.av μs a function of time following the switching on of coil 2 working with an alternating current I of 195 A. Similar curves were obtained for coil 1 at I equal to 50, 100, 150, 200, 250, 300, 350, 400 A, and for coil 2 at I equal to 50, 100, 150, 200, 250, 300, 350, 400, or 450 A, and for coil 3 at I equal to 25, 50, 100, 150, or 200 A, and for coil 4 at I equal to 25, 50, 100, 150, 200, or 250 A. (b), for a magnetic field oscillating at high, medium, and low frequency, variation of magnetic field amplitude as a function of time, showing the A.sub.7 low frequency sequence, taking place during t.sub.7 and corresponding to the heating step, followed by the A.sub.8 low frequency sequence, taking place during t.sub.8 and corresponding to the cooling step. f.sub.l may be equal or proportional to 1/(t.sub.7+t.sub.8). (c), for a magnetic field oscillating at high, and low frequency, variation of magnetic field amplitude as a function of time, showing the A.sub.9 low frequency sequence, taking place during t.sub.9 and corresponding to the heating step, followed by the A.sub.10 low frequency sequence, taking place during t.sub.10 and corresponding to the cooling step. f.sub.l may be equal or proportional to 1/(t.sub.9+t.sub.10).

(3) FIG. 3: (a), Variation of the magnetic flux density in axial direction as a function of time, showing both the high oscillation frequency, f.sub.h=195 kHz, and the medium oscillation frequency, f.sub.m=15 kHz. (b), Variation of the maximal flux density in the radial direction as a function of time, showing both the high oscillation frequency, f.sub.h=195 kHz, and the medium oscillation frequency, f.sub.m=15 kHz. (c), Magnitude of magnetic flux density as a function of time, showing both twice the high oscillation frequency, 2.Math.f.sub.h=390 kHz, and the medium oscillation frequency, f.sub.m=15 kHz. H.sub.max corresponds to the maximum value of the magnitude of magnetic flux density among the different values of H.sub.max,i, where H.sub.max,i is the maximum magnitude of magnetic flux density estimated for each high frequency oscillation. H.sub.av corresponds to the average value among all values of H.sub.max,i. The magnetic flux density, also designated as the strength of the magnetic field, is measured in the radial and axial direction of the cylindrical probe, where the probe is positioned so that its axial direction is parallel to the main magnetic field generated by coil 2. This is the reason why the strength of the magnetic field in the axial direction is much higher or larger than that in the radial direction. The magnitude of magnetic flux density is also designated as the amplitude of the magnetic field.

(4) FIG. 4: Positioning of the probe used to measure the strength or amplitude of the magnetic field in coil 2, as a function of the distance from the edge of coil 2, (0), measured in cm. (b), Measurement of H.sub.max and H.sub.av as a function of the distance, measured from the center of coil 2, using an alternating current of 500 A, where 1.5 cm corresponds to the center of the coil and 0 to the edge of the coil as indicated in (a).

(5) FIG. 5: Temperature variation as a function of time of a suspension of 100 μl of M-PLL, (a), or BNF-Starch, (b), at a concentration of 10 mg/mL, exposed to oscillating magnetic fields generated by coils 2 and 4 according to conditions (H.sub.av, H.sub.max, f.sub.m, f.sub.h) mentioned in table 1.

(6) FIG. 6: (a), Temperature variation as a function of time of confluent GL-261 cells brought into contact with 2 mg of BNF-Starch in 2 mL, exposed during 30 minutes to a magnetic field, oscillating at f.sub.h=196 kHz and f.sub.m=15 kHz, of fixed H.sub.av=61 mT and H.sub.max=85 mT, (-.circle-solid.-), of H.sub.av=49-61 mT and H.sub.max=68-85 mT to reach 45° C., (-.square-solid.-), of H.sub.av=53-61 mT and H.sub.max=75-85 mT to reach 50° C., (-.diamond-solid.-), of H.sub.av=55-61 mT and H.sub.max=77-85 mT to reach 55° C., (-.Math.-). In the control, confluent GL-261 cells were exposed to a magnetic field, oscillating at f.sub.h=196 kHz and f.sub.m=15 kHz, and of H.sub.av=61 mT and H.sub.max=85 mT. (b), Upper curve, temperature variation as a function of time of GL-261 cells brought into contact with 2 mg of BNF-Starch in 2 mL, exposed to sequences of application of the magnetic field, oscillating at f.sub.h=196 kHz and f.sub.m=15 kHz, H.sub.av=61 mT and H.sub.max=85 mT, corresponding to heating steps, followed by sequences of non-application of the magnetic field, corresponding to cooling steps. The heating times, t.sub.7, correspond to those necessary to reach 45° C., while the cooling times, t.sub.8, correspond to those necessary for the temperature to decrease from 45° C. to 35° C. In this case, the oscillating magnetic field oscillates at a low frequency of 1.6-2.5 10.sup.−3 Hz. Bottom curve, temperature variation as a function of time for GL-261 cells without BNF-Starch exposed to the same sequences of application and non-application of the magnetic field as for the upper curve. (c), Upper curve, temperature variation as a function of time of GL-261 cells brought into contact with 2 mg of BNF-Starch in 2 mL, exposed to sequences of application of the magnetic field, oscillating at f.sub.h=196 kHz, f.sub.m=15 kHz, H.sub.av=61 mT and H.sub.max=85 mT, corresponding to heating steps, followed by sequences of non-application of the magnetic field, corresponding to cooling steps. The heating times, t.sub.7, correspond to those necessary to reach 50° C., while the cooling times, t.sub.8, correspond to those necessary for the temperature to decrease from 50° C. to 37° C. In this case, the magnetic field oscillates at a low frequency of 1.2-1.7 10.sup.−3 Hz. Bottom curve, temperature variation as a function of time for GL-261 cells without BNF-Starch exposed to the same sequences of application and non-application of the magnetic field as for the upper curve. (d), Upper curve, temperature variation as a function of time for GL-261 cells brought into contact with 2 mg of BNF-Starch in 2 mL, exposed to sequences of application of the magnetic field, oscillating at f.sub.h=196 kHz and f.sub.m=15 kHz, H.sub.av=61 mT and H.sub.max=85 mT, corresponding to heating steps, followed by sequences of non-application of the magnetic field, corresponding to cooling steps. The heating times, t.sub.7, correspond to those necessary to reach 55° C., while the cooling times, t.sub.8, correspond to those necessary for the temperature to decrease from 55° C. to 37° C. In this case, the magnetic field oscillates at a low frequency of 0.9-1.1 10.sup.−3 Hz. Bottom curve, temperature variation as a function of time for GL-261 cells without BNF-Starch exposed to the same sequences of application and non-application of the magnetic field as for the upper curve.

(7) FIG. 7: (a), For the first hyperthermia session, temperature variation as a function of time of 25 μg in iron of BNF-Starch per mm.sup.3 of tumor introduced to GL-261 tumors and exposed continuously during 30 minutes to a magnetic field, oscillating at f.sub.h=202 kHz and f.sub.m=15 kHz, with H.sub.av=27 mT and H.sub.max=57 mT. 14 additional hyperthermia sessions during which a magnetic field, oscillating at f.sub.h=202 kHz and f.sub.m=15 kHz, with H.sub.av=24-31 mT and H.sub.max=54-67 mT, followed the first hyperthermia session. (b), For the first hyperthermia session, temperature variation as a function of time of 25 μg in iron of BNF-Starch per mm.sup.3 of tumor introduced to GL-261 tumors and exposed during 20 minutes to a magnetic field, oscillating at f.sub.h=202 kHz, f.sub.m=15 kHz, with H.sub.av=11 mT and H.sub.max=57 mT, applied during t.sub.7, followed by non-application of the magnetic field during t.sub.8. 20 hyperthermia sessions of 20 minutes each followed the first hyperthermia session, during which a magnetic field, oscillating at f.sub.h=202 kHz, f.sub.m=15 kHz and f.sub.l=19 10.sup.−3 Hz was applied. The times t.sub.7 and t.sub.8 associated to heating and cooling steps of the different cycles are indicated in table 8. (c) Heating times t.sub.7 μs a function of the hyperthermia session. (d), Cooling times t.sub.8 as a function of the hyperthermia session. (e), Low frequency of oscillation, f.sub.l=[1/(t.sub.7+t.sub.8)], as a function of the hyperthermia session.

(8) FIG. 8: (a), Variation of the tumor volume as a function of time (days) following day 0, the day of BNF-Starch administration, where BNF-Starch are either administered without application of a magnetic field, (.circle-solid.), BNF-Starch are administered followed by 15 hyperthermia session (3 per week) during which a magnetic field oscillating at f.sub.h=202 kHz and f.sub.m=15 kHz with H.sub.av=24-31 mT and H.sub.max=54-67 mT is applied during 30 minutes, (.box-tangle-solidup.), BNF-Starch are administered followed by 21 hyperthermia sessions (3 per week) during which a magnetic field oscillating at f.sub.h=202 kHz, f.sub.m=15 kHz, f.sub.l=4-25 mHz with H.sub.av=7-23 mT and H.sub.max=57 mT is applied during 20 minutes, (.Math.). (b), Mouse survival rate as a function of time (days) following day 0, the day of BNF-Starch administration, where BNF-Starch are either administered without application of a magnetic field, (.circle-solid.), BNF-Starch are administered followed by 15 hyperthermia session (3 per week) during which a magnetic field oscillating at f.sub.h=202 kHz and f.sub.m=15 kHz with H.sub.av=24-31 mT and H.sub.max=54-67 mT is applied during 30 minutes, (.box-tangle-solidup.), BNF-Starch are administered followed by 21 hyperthermia sessions (3 per week) during which a magnetic field oscillating at f.sub.h=202 kHz, f.sub.m=15 kHz, f.sub.l=4-25 mHz with H.sub.av=7-23 mT and H.sub.max=57 mT is applied during 20 minutes, (.Math.).

(9) FIG. 9: A schematic illustration of a device (1) that generates an oscillating magnetic field located at a distance (d) of more than 1 cm from a body part (2).

EXAMPLES

Example 1: Application of a Magnetic Field, Oscillating at High and Medium Frequencies, f.SUB.h .and f.SUB.m., to Heat Nanoparticles

(10) A volume of 100 μL of a suspension containing either nonpyrogenic magnetosomes coated with poly-L-lysine, also designated as central parts of magnetosomes coated with poly-L-lysine or magnetosome minerals coated with poly-L-lysine, (M-PLL) or BNF-Starch (Micromod, ref: 10-00-801) at a concentration of 10 mg/mL in iron was introduced into a 250 μL tube. The preparation, characterization and properties of M-PLL have been described in Patent PCT/FR2016/000095 which is incorporated herein by reference. The tube was placed at the center of each of the 5 induction coils, whose properties are summarized in table 1. Each coil was connected to a power source generating an alternating current of intensity varied between 73 and 682 A (Easy Heat 10 kW, Ambrell) to obtain the same H.sub.av, where the alternating current produced an oscillating magnetic field during 700 sec. The temperature variation inside the tube, following the application of the alternating magnetic field, was measured using a thermocouple (IT-18, Physitemp). The measurement of the alternating magnetic field was carried out using a magnetic probe placed at the center of each induction coil (Magnetic field probe, AMF life system), and an oscilloscope. The probe measured the variation with time of the axial voltage, U.sub.a, and of the radial voltage, U.sub.r. We deduced from U.sub.a and U.sub.r the variation of the magnetic flux density over time in the radial direction, H.sub.r=U.sub.r/[0.7f.sub.h], and in the axial direction, H.sub.a=U.sub.a/[0.6f.sub.h], where f.sub.h is the high frequency of oscillation. We also deduced the variation of the magnitude of magnetic flux density with time using the relation: H=[(H.sub.a).sup.2+(H.sub.r).sup.2].sup.1/2.

(11) With the probe, we measured the variation of the magnetic flux density in the axial and radial directions, as well as the magnitude of the magnetic flux density, during 650 10.sup.−6 seconds, with a magnetic field measurement carried out every 0.16 10.sup.−6 seconds. The average and maximum magnetic fields, H.sub.av and H.sub.max, were estimated, taking into accounts the maximum values of the amplitude of the magnetic field of each high frequency oscillation, H.sub.max,i, during a measurement time of 650 10.sup.−6 seconds.

(12) For a magnetic field oscillating only at high frequency, the variation with time of the magnetic field amplitude is shown in FIG. 1(a), while for a magnetic field oscillating at high and medium frequency, the variation with time of the magnetic field amplitude is shown in FIG. 1(b). For coils 1 to 4, the medium frequency of f.sub.m=15 kHz corresponds to a period T.sub.m=1/f.sub.m=67 10.sup.−6 seconds, which is much smaller than the measurement time and f.sub.m can hence be measured for coils 1 to 4. For coil 5, the medium frequency f.sub.m=2 kHz corresponds to a period T.sub.m=1/f.sub.m=500 10.sup.−6 seconds, which is also smaller than the measurement time and f.sub.m can hence be measured for coil 5.

(13) For coil 2, FIG. 2(a) shows the percentage of H.sub.max and H.sub.av reached as a function of time following the switching on of the device generating the oscillating magnetic field. It appears that 100% of H.sub.max and H.sub.av, corresponding to a stable magnetic field, is reached 30 seconds after the device generating the oscillating magnetic field has been switched on. For the other coils (1, 3, 4), a stabilized magnetic field was also reached after 30 seconds and stabilization time was always observed to be below 30 seconds.

(14) FIG. 2(b) shows the variation of the magnetic field amplitude as a function of time for a magnetic field oscillating at high, medium, and low frequency, with indications about A.sub.7 and A.sub.8 low frequency sequences. FIG. 2(c) shows the variation of the magnetic field amplitude as a function of time for a magnetic field oscillating at high and low frequency, with indications about A.sub.9 and A.sub.10 low frequency sequences.

(15) FIGS. 3(a), 3(b), and 3(c), show variations with time of the magnetic flux density in the axial direction (FIG. 3(a)), the variation with time of the magnetic flux density in the radial direction (FIG. 3(b)), the variation with time of the magnitude of magnetic flux density (FIG. 3(d)), measured for coil 2.

(16) For coils 1 to 5, magnetic fields oscillate at f.sub.h=202 kHz (coil 1), f.sub.h=195 kHz (coil 2), f.sub.h=231 kHz (coil 3), f.sub.h=329 kHz (coil 4), f.sub.h=91 kHz (coil 5), and f.sub.m=15 kHz (coils 1 to 4) or f.sub.m=2 kHz (coil 5), where these frequencies are measured as described in FIGS. 1(a) and 1(b).

(17) The maximum magnetic field, H.sub.max, corresponding to the maximum value of magnetic field amplitude among the different H.sub.max,i,

(18) $H_{\max }=\underset{i=1,n}{\mathrm{MAX}}{H}_{\max ,i},$
where H.sub.max,i is the maximum magnetic field amplitude of each high frequency oscillation and n is the number of oscillations considered in the measurement. H.sub.max is equal to H.sub.max=58 mT for an alternating current of intensity, I, of 190 A (coil 1), H.sub.max=34 mT for I=195 A (coil 2), H.sub.max=53 mT for I=73 A (coil 3), H.sub.max=56 mT for I=149 A (coil 4), H.sub.max=33 mT for 1=682 A (coil 5) (table 1).

(19) The average magnetic field, H.sub.av, which is estimated using the formula: H.sub.av=(Σ.sub.i=1.sup.i=nH.sub.max,i)/n, is H.sub.av=26 mT for I=190 A (coil 1), H.sub.av=25 mT for I=195 A (coil 2), H.sub.av=26 mT for I=73 A (coil 3), H.sub.av=24 mT for I=149 A (coil 4), H.sub.av=26 mT for 1=682 A (table 1).

(20) In this experiment, we have used a value of the alternating current for each coil that yields similar average magnetic fields.

(21) Coils 2 and 5 produce a ratio H.sub.max/H.sub.av=1.3-1.4, which is close to 1 (table 1).

(22) Coils 1, 3, and 4, produce a higher or larger ratio H.sub.max/H.sub.av of 2-2.3 (table 1).

(23) By using a double coil (coil 3) with a diameter about 2 times smaller than that of the single coil 1, the current required to reach a relatively similar high frequency f.sub.h (f.sub.h=231 kHz for coil 3 compared to f.sub.h=202 kHz for coil 1), similar maximum and average magnetic fields, is 2.6 times lower (table 1).

(24) For coil 4, the diameter and length are smaller than for coil 1, leading to a high oscillation frequency, which is higher or larger (329 kHz for coil 4 compared with 202 kHz for coil 1). Maximum and average fields are similar at 56-58 mT and 24-26 mT for coils 1 and 4 (table 1).

(25) For coil 5, the diameter and length are significantly larger than for the other coils at 28 and 15 cm, respectively, and the medium and high oscillation frequencies are smaller at f.sub.m=2 kHz and f.sub.h=91 kHz (table 1).

(26) Heating properties of M-PLL suspensions and BNF-Starch exposed to the oscillating magnetic fields generated by coils 1 to 5 for M-PLL and by coils 2 and 4 for BNF-Starch have also been studied. Variations of temperature with time of 100 μL of a M-PLL or BNF-Starch suspension at 10 mg/mL are presented in FIGS. 5(a) and 5(b) when these two suspensions are exposed to the alternating magnetic field generated by coil 2 (I=195 A, H.sub.max=34 mT, H.sub.av=25 mT) and coil 4 (1=149 A, H.sub.max=56 mT, H.sub.av=24 mT). The specific absorption rate, SAR, of M-PLL and BNF-Starch suspensions was measured, using the relationship: SAR=(C.sub.eau/X.sub.Fe) (ΔT/δt), where the SAR is measured in Watt per gram of iron, C.sub.eau=4.2 J.Math.g.sup.−1.Math.K.sup.−1 is the calorific capacity of water, X.sub.Fe=0.01 g/mL is the iron concentration of the M-PLL or BNF-Starch suspension and ΔT/δt is the initial slope of the temperature variation with time, measured in ° C./sec. Temperature variation, indicated by ΔT, is calculated by subtracting: 1) the temperature reached by the M-PLL or BNF-Starch suspension after 650 seconds of application of the oscillating field by 2) the temperature measured before application of the oscillating magnetic field.

(27) For M-PLL, the results of table 2 show that the induction coils which produce the highest SAR of 192-244 W/g.sub.Fe and ΔT of 68-75° C., are coils 1, 3 and 4, which generate the oscillating magnetic field with the highest values of maximum magnetic field of 53-58 mT and highest value of H.sub.max/H.sub.av of 2-2.3. In contrast, the induction coils, which produce the lowest SAR of 6-84 W/g.sub.Fe and ΔT of 5-57° C., are coils 2 and 5, which produce the lowest values of maximum magnetic field of 33-34 mT and H.sub.max/H.sub.av of 1.3-1.4. Furthermore, coil 5 which generates maximum and average magnetic field of 33 mT and 26 mT, respectively, similar to that of coil 2 of 34 mT and 35 mT, respectively, but has lower f.sub.h and f.sub.m values (f.sub.h=91 kHz and f.sub.m=2 kHz for coil 5 compared with f.sub.h=195 kHz and f.sub.m=15 kHz for coil 2), leading to SAR and ΔT, which are more than 10 to 14 times lower for coil 5 than those of coil 2 (table 2).

(28) For BNF-Starch, the results of table 2 show that the induction coils which produce the highest SAR of 13 W/g.sub.Fe and ΔT of 12° C., is coil 4, which generate the oscillating magnetic field with the highest values of maximum magnetic field of 56 mT and highest value of H.sub.max/H.sub.av of 2.3. In contrast, the induction coil, which produces the lowest SAR of 8 W/g.sub.Fe and ΔT of 7° C. is coil 2, which produce the lowest values of maximum magnetic field of 34 mT and H.sub.max/H.sub.av of 1.4. Moreover, BNF-Starch produce much lower SAR and ΔT than the M-PLL, both for coils 2 and 4, which can be explained by their lower coercivity, H.sub.c are 10 mT to BNF-starch and 6 mT for M-PLL, and lower ratio between remanent and saturating magnetization, M.sub.r/M.sub.s are equal to 0.19 for M-PLL and 0.15 for BNF starch.

(29) We can Conclude from this Example that:

(30) i), Best heating properties, i.e. highest values of SAR and ΔT, are obtained for coils 1, 3 and 4, with the highest maximum magnetic field of 55±3 mT and the highest ratio H.sub.max/H.sub.av of 2-2.3, suggesting that the maximum magnetic field and/or H.sub.max/H.sub.av should be maximized in order to reach best nanoparticle heating properties under the application of an oscillating magnetic field.

(31) ii), It is possible to obtain similar heating properties, i.e. similar values of SAR and ΔT, with similar values of H.sub.max=55±3 mT and the highest ratio H.sub.max/H.sub.av of 2-2.3, (coils 1, 3, 4), suggesting that H.sub.max=55±3 mT and H.sub.max/H.sub.av could be modified or adjusted to yield the desired heating properties.

(32) iii), It is possible to obtain similar heating properties, i.e. similar values of SAR and ΔT, with coils of different diameters, coil number, and coil length (coils 1, 3, 4), suggesting that coil diameter, coil number, and coil length, could be modified or adjusted without necessarily modifying heating properties.

(33) iv), For coils generating similar values of H.sub.max and H.sub.max/H.sub.av (coils 2 and 5), better heating properties, i.e. higher or larger values of SAR and ΔT, are obtained for the coil with the highest f.sub.m and f.sub.h values (coil 2), suggesting that f.sub.m and/or f.sub.h should be maximized in order to reach best nanoparticle heating properties under the application of an oscillating magnetic field.

(34) v) M-PLL lead to better heating properties than BNF-Starch both for coils 2 and 4, indicating that nanoparticle magnetic properties such as H.sub.c and M.sub.r/M.sub.s should be maximized to reach best heating properties under the application of a magnetic field oscillating at high and medium frequency.

(35) vi) The maximum and/or average magnetic field applied to heat M-PLL or BNF-Starch is larger than the coercivity of the nanoparticles (H.sub.c=10 mT for BNF-Starch and H.sub.c=6 mT for M-PLL at room temperature), which should enable rotation of the magnetic moment of the nanoparticles by application of the alternating magnetic field.

Example 2: Application of an Oscillating Magnetic Field to Heat Nanoparticle Suspensions Outside of the Coil

(36) 100 μL of a suspension containing uncoated iron oxide nanoparticles (SIGMA-ALDRICH, reference 544884) at different concentrations (422, 194, 87 and 57 mg/mL in iron) were introduced into a tube of 250 μL. The tube was then positioned at 5 cm and 8 cm from the edge of coil 2. The positions of the tube are indicated by −5 and −8 in the schematic diagram (FIG. 4(a). The tube was exposed to a magnetic field using the Ambrell Easy Heat LI 10 KW that generates an alternating current of 500 A for 1500 sec. The variation of temperature of the tube containing the nanoparticles was measured using a thermocouple (IT-18, Physitemp) while the properties of the magnetic field were measured using a magnetic probe (AMF life system) and an oscilloscope.

(37) We measured the variation of the average, H.sub.av, and maximum, H.sub.max, magnetic field as a function of the distance from the edge of coil 2, designated as 0 in FIG. 4(a). FIG. 4(b) shows similar variations of H.sub.max and H.sub.av as a function of the distance from the center, 1.5 cm, of the coil. As can be seen in FIG. 4(b), the average and maximum magnetic fields decrease exponentially outside of coil 2, but are non-negligible up to 6 cm outside of the coils and could hence potentially be used to heat nanoparticles. Outside of the center of coil 2, the magnetic field appears to be “evanescent”, or is characterized by a magnitude or frequency of oscillation, preferably lower than those measured at the center or inside the coil (table 3).

(38) Table 3 shows that the magnetic field oscillates at f.sub.h=192 kHz at 5 cm from the edge of coil 2 and at f.sub.h=189 kHz at 8 cm from the edge of coil 2. A medium frequency could not be detected for a measurement time of 650 10.sup.−6 seconds. In addition, the average and maximum magnetic fields are about two times lower at 8 cm from the edge of the coil (H.sub.av=H.sub.max=5 mT) than at 5 cm from the edge of the coil (H.sub.av=H.sub.max=12-13 mT), table 3.

(39) The SAR and ΔT of chemical nanoparticles (SIGMA-ALDRICH, reference 544884) were estimated for a tube containing 100 μL of these nanoparticles, positioned at 8 cm and 5 cm from the edge of coil 2, and exposed to the magnetic field, oscillating at f.sub.h=189-192 kHz with H.sub.max and H.sub.av indicated in table 3. When the tube containing the nanoparticles suspension was positioned at 8 cm from the edge of coil 2, table 4 indicates that SAR and ΔT values are low at 0.4-0.6 W/g.sub.Fe and 1-8° C., respectively, which can be explained by the fact that the average and maximum magnetic fields are small at 5 mT (table 3). When the tube containing the nanoparticles suspension was positioned 5 cm from the edge of coil 2, SAR and ΔT values were higher or larger at 2-3 W/g.sub.Fe and 13-45° C., respectively, explained by the fact that the maximum and average magnetic fields are larger at 12-13 mT, respectively (table 3).

(40) It is therefore possible to heat a suspension of nanoparticles by positioning the tube containing a suspension of nanoparticles outside of coil 2, at 5 cm from the edge of this coil, by applying an oscillating magnetic field with maximum magnetic field larger than 10 mT.

Example 3: Application of a Magnetic Field Oscillating at a Low Frequency, f.SUB.l., a Medium Frequency, f.SUB.m., and a High Frequency, f.SUB.h., for the Destruction of Tumor Cells In Vitro

(41) Description of the Various Treatments:

(42) 500,000 GL-261 cells were seeded in petri dishes containing a culture medium composed of 1.6 ml of RPMI and 0.4 ml of calf serum. After incubation for 12 hours at 37° C. in the presence of 5% CO.sub.2, the cells adhered to the surface of the Petri dishes and were confluent. For the application of the oscillating magnetic field, coil 2 was used.

(43) The Following 11 Conditions of Treatment were Tested:

(44) Condition 1:

(45) Confluent GL-261 cells were brought into contact with the culture medium and incubated at 37° C. for 12 hours.

(46) Condition 2:

(47) Confluent GL-261 cells were brought into contact with the culture medium, exposed to a magnetic field, oscillating at high frequency f.sub.h=196 kHz and medium frequency f.sub.m=15 kHz, with H.sub.av=61 mT and H.sub.max=85 mT, applied continuously during 30 minutes.

(48) Condition 3:

(49) Confluent GL-261 cells were brought into contact with culture medium and 2 mg of BNF-Starch in iron and were then exposed to a magnetic field, oscillating at f.sub.h=196 kHz and f.sub.m=15 kHz, with average magnetic field varied between 49 and 61 mT and maximum magnetic field varied between 68 and 85 mT, to reach an average temperature of 45° C. during 30 minutes continuously.

(50) Condition 4:

(51) Confluent GL-261 cells were brought into contact with culture medium and 2 mg of BNF-Starch in iron and were then subjected to 10 cycles. During each cycle, a magnetic field, oscillating at a high frequency of 196 kHz, medium frequency 15 kHz, with an average magnetic field of 61 mT and maximum magnetic field of 85 mT, was applied, leading to a heating step up to 45° C., followed by the non-application of an alternating magnetic field, leading to a cooling step during which the temperature decreases from 45° C. down to 34-35° C. The times of heating and cooling steps, t.sub.7 and t.sub.8, as well as the low oscillation frequency, average and maximum magnetic fields deduced from t.sub.7 and t.sub.8 are indicated in table 5. The variations of temperatures with time during these low frequency cycles are indicated in FIG. 6(b), upper curve.

(52) Condition 5:

(53) Confluent GL-261 cells were brought into contact with culture medium without BNF-Starch, were then exposed to the same 10 cycles as in condition 4 with the same magnetic field, t.sub.7 and t.sub.8 values. The variations of temperatures with time during the low frequency cycles are shown in FIG. 6(b), bottom curve.

(54) Condition 6:

(55) Confluent GL-261 cells were brought into contact with culture medium and 2 mg of BNF-Starch in iron, exposed to a magnetic field, oscillating at a high frequency of 196 kHz and medium frequency of 15 kHz, with an average magnetic field varied between 53 and 61 mT and a maximum magnetic field varied between 68 and 85 mT, to reach an average temperature of 50° C. continuously during 30 minutes.

(56) Condition 7:

(57) Confluent GL-261 cells were brought into contact with culture medium and 2 mg in iron of BNF-Starch, were then exposed to 6 cycles. During each cycle, a magnetic field, oscillating at a high frequency of 196 kHz and medium frequency 15 kHz, with an average magnetic field of 61 mT and maximum magnetic field of 85 mT was applied, leading to a heating step up to 50° C., followed by the non-application of an alternating magnetic field to decrease the temperature from 50° C. down to 37° C. during a cooling step. The times of heating and cooling steps, t.sub.7 and t.sub.8, as well as the low oscillation frequency, average and maximum magnetic fields deduced from t.sub.7 and t.sub.8 are indicated in table 6. The variations of temperatures with time during these low frequency cycles are indicated in FIG. 6(c), upper curve.

(58) Condition 8:

(59) Confluent GL-261 cells were brought into contact with culture medium without BNF-Starch and exposed to the same 6 cycles as in condition 7 with the same magnetic field and same time t.sub.7 and t.sub.8 μs in condition 7. The variations of temperature with time during these low frequency cycles are shown in FIG. 6(c), bottom curve.

(60) Condition 9:

(61) Confluent GL-261 cells were brought into contact with culture medium and 2 mg of BNF-Starch in iron, exposed to a magnetic field oscillating at a high frequency of 196 kHz and medium frequency of 15 kHz, with an average magnetic field varied between 55 and 61 mT and a maximum magnetic field varied between 77 and 85 mT, to reach an average temperature of 55° C. continuously during 30 minutes.

(62) Condition 10:

(63) Confluent GL-261 cells were brought into contact with culture medium and 2 mg of BNF-Starch in iron, and were then exposed to 4 cycles. During each cycle, a magnetic field, oscillating at a high frequency of 196 kHz and medium frequency 15 kHz, with an average magnetic field of 61 mT and maximum magnetic field of 85 mT was applied, leading to a heating step at 55° C., followed by the non-application of an alternating magnetic field to decrease the temperature from 55° C. down to 37° C. during a cooling step. The times of heating and cooling steps, t.sub.7 and t.sub.8, as well as the low oscillation frequency, average and maximum magnetic fields deduced from t.sub.7 and t.sub.8 are indicated in table 7. The variations of temperatures with time during these low frequency cycles are indicated in FIG. 6(d), upper curve.

(64) Condition 11:

(65) Confluent GL-261 cells were brought into contact without the BNF-Starch, and were then exposed to the same 4 cycles as in condition 10 with same magnetic field and same time t.sub.7 and t.sub.8 μs in condition 10. The variations of temperature with time during these low frequency cycles are shown in FIG. 6(d), bottom curve.

(66) Condition 12:

(67) Confluent GL-261 cells were brought into contact with culture medium and 2 mg of BNF-Starch in iron and incubated at 37° C. for 30 min.

(68) After treatments, the culture medium was removed, cells were rinsed with PBS, PBS was replaced with culture medium and the cells were then incubated at 37° C. in the presence of 5% CO.sub.2 for 12 hours. For each condition 1 to 12 of treatment, the percentage of dead cells, living cells, and the percentage of necrotic and apoptotic cells were measured. For that, the Petri dishes were rinsed with PBS, trypsin was added to detach cells, one milliliter of culture medium was added to the cells and the mixture was centrifuged at 1000 rpm for 10 minutes. The supernatant was removed and then replaced with 200 μl of PBS in order to obtain approximately 2.10.sup.6 cells per ml. 5 μl of Annexin V Alexa Fluoride and 1 μl of propidium iodide at 1 mg per ml were added to the cells. After 15 minutes, 800 μl of an Annexin 1× binding buffer solution were added to the cells and the fluorescence of the mixture was measured using a flow cytometer, which makes it possible to deduce the percentage of living cells, necrotic and apoptotic cells.

(69) For condition 2, the temperature increased by 11° C. during the 30 minutes of continuous application of the oscillating magnetic field (FIG. 6(a)). For this condition, the percentage of living cells is 92%, similar to that of 97% obtained with the untreated cells (condition 1), indicating that continuous magnetic field application leading to continuous temperature increase of 11° C. does not induce significant cell death.

(70) For conditions 5, 8, and 11, the temperature increased, due to Eddy or Foucault currents, by 5° C. on average from 25° C. to 30° C. (condition 5, FIG. 6(b), bottom curve), by 4° C. on average from 27° C. to 31° C. (condition 8, FIG. 6(c), bottom curve), or 7° C. on average from 27° C. to 34° C. (condition 11, FIG. 6(d), bottom curve) during sequences of magnetic field application, and the temperature decreased by a relatively similar amount during sequences of non-application of the magnetic field. For these conditions, the percentage of living cells is 94%, similar to that of 97% obtained with the untreated cells (condition 1), indicating that Eddy or Foucault currents did not induce significant cell death.

(71) For conditions 3 and 4, temperature either increased continuously during 30 minutes by 18° C. (condition 3, FIG. 6(a)) or increased by 20° C. and decreased by 10° C. during the first cycle, and increased by 10° C. and decreased by 10° C. during the remaining 9 cycles (condition 4, FIG. 6(b), upper curve). The percentage of living cells is 95% after continuous heating (condition 3) and 56% after sequential heating (condition 4), indicating that at this heating temperature of 45° C., the application of a magnetic field oscillating at a high, medium, and low frequency (condition 4) leads to enhanced toxicity towards cancer cells compared with a magnetic field oscillating at the high and medium frequency (condition 3).

(72) For conditions 6 and 7, the temperature either increased continuously during 30 minutes by 23° C. (condition 6, FIG. 6(a)), or increased by 24° C. and decreased by 9° C. during a first cycle, and increased by 15° C. and decreased by 15° C. during the remaining 5 cycles (condition 7, FIG. 6(c), upper curve). The percentage of living cells is 91% after continuous heating (condition 6) and 0.4% after sequential conditions (condition 7), indicating that at this heating temperature of 50° C., the application of a magnetic field oscillating at high, medium, and low frequency (condition 7) leads to enhanced toxicity towards cancer cells compared with a magnetic field oscillating at the high and medium frequency (condition 6).

(73) For conditions 9 and 10, the temperature increased continuously during 30 minutes by 28° C. (condition 9, FIG. 6(a)), or increased by 29° C. and decreased by 18° C. during the first cycle, and increased by 18° C. and decreased by 18° C. during the remaining 3 cycles (condition 10, FIG. 6(d), upper curve). The percentage of living cells is 2% after continuous heating (condition 9) and 1.6% under sequential conditions (condition 10), indicating that at this highest heating temperature of 55° C., application of magnetic fields oscillating at high, medium and low frequencies or at high and medium frequency, both lead to the destruction of most cancer cells.

(74) For condition 12, temperature did not increase. The percentage of living cells is then 99%, indicating that the cytotoxic effect observed when nanoparticles are exposed to the oscillating magnetic field is due to the application of the magnetic field and not to nanoparticle toxicity.

(75) We can Conclude from this Example that:

(76) The application of a magnetic field oscillating at high, medium, and low frequency, enables to strengthen treatment safety, i) to v), to enhance treatment efficacy, vi), and to use a method that does not necessitate to vary the magnetic field strength or frequency to reach a desired temperature during the heating steps.

(77) i) In the absence of nanoparticles, cytotoxicity induced by the alternating magnetic field, in the presence of heat generated by Eddy or Foucault currents, is not observed.

(78) ii) In the absence of nanoparticles, the application of a magnetic field oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz, and f.sub.l=0.9-2.5 mHz, enables to heat sequentially, limiting the increase in temperature compared with the application of a field oscillating at f.sub.h=195 kHz and f.sub.m=15 kHz. Increase in temperature, due to Eddy or Foucault currents, is 11° C. under the application of a magnetic field oscillating at f.sub.h=195 kHz and f.sub.m=15 kHz compared to 6-8° C. under the application of a magnetic field oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz and f.sub.l=0.9-2.5 mHz.

(79) iii) For a magnetic field, oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz and f.sub.l=0.9-2.5 mHz, the average magnetic field is 31-44 mT, compared with 61 mT for a magnetic field oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz. It is therefore possible to decrease the average magnetic field by a factor of ˜1.4-2 by adding a low frequency and therefore decrease potential toxicity associated with the application of a too high average magnetic field.

(80) iv) Temperatures of 44-45° C. are reached during 33 seconds using a magnetic field, oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz, and f.sub.l=1.6-2.5 mHz (FIG. 6(b)), whereas these temperatures are reached during 24 minutes using a magnetic field oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz (FIG. 6(a)). Temperatures of 49-50° C. are reached during 47 seconds using a magnetic field, oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz, and f.sub.l=1.2-1.7 mHz (FIG. 6(c)), whereas these temperatures are reached during 23 minutes using a magnetic field oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz (FIG. 6(a)). Temperatures of 52.5-53.5° C. are reached during 55 seconds using a magnetic field, oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz, and f.sub.l=0.9-1.1 mHz (FIG. 6(d)), whereas these temperatures are reached during 16 minutes using a magnetic field oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz (FIG. 6(a)). Using a low oscillation frequency enables to decrease the time during which maximum temperature is achieved by a factor of 17 to 44, compared with the application of an oscillating magnetic field without the low frequency. This should enhance treatment safety and does not reduce the efficacy of cell destruction by the magnetic field.

(81) v) The application of a magnetic field, oscillating at high, medium, and low frequency, enables to obtain a series of temperature gradient increase (+ΔT) followed by temperature gradient decrease (−ΔT). Temperatures of 44-45° C. were reached 10 times following ΔT of 8-20° C. followed by −ΔT of 8-10° C. by applying a magnetic field, oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz, and f.sub.l=1.6-2.5 mHz (FIG. 6(b)), whereas temperatures of 44-45° C. were reached only once following ΔT of 17-20° C. by applying a magnetic field, oscillating at f.sub.h=195 kHz, f.sub.m=15 kHz. Enhanced efficacy observed using the magnetic field oscillating at f.sub.h, f.sub.m, and f.sub.l, can be explained by the larger number of (+ΔT) and (−ΔT), compared with the magnetic field oscillating only at f.sub.h and f.sub.m.

(82) vi) When cancer cells are brought into contact with nanoparticles and heated to a temperature, which is below 55° C., the application of a magnetic field, oscillating at a high, medium, and low frequency leads to enhanced cytotoxicity compared with the application of a magnetic field, oscillating at high and medium frequencies.

(83) vii) The application of a magnetic field enables a treatment where the number of sequences of magnetic field application, the number of sequences of non-application of the magnetic field, the average and maximum magnetic fields, the frequency of the applied magnetic field, the times of application and non-application of the magnetic field, are fixed at the beginning of the treatment depending on the temperature that one desires to reach. With this method, it is not necessary to vary the magnetic field strength or amplitude to reach a desired temperature during a sequence of magnetic field application. Moreover, once the cycles with associated heating and cooling times have been estimated, it is not necessary to measure the temperature during treatments. Cycles with defined heating and cooling times can be used for the treatment.

Example 4: Application of a Magnetic Field, Oscillating at a High Frequency of 196 kHz, Medium Frequency of 15 Hz, and Low Frequency of 4-25 mHz, for Efficient In Vivo Destruction of Tumors

(84) Using a 1 mL 25 g syringe, a volume of 100 μl containing 10.sup.7 GL-261 murine glioblastoma cells was administered subcutaneously on the left flank, between the paw and the back of female mice black 6 C57BL/6J. The tumors grew during 10 to 15 days until they reached a size of 60 to 90 mm.sup.3. When the tumors reached this size, the mice were anesthetized with isoflurane gas and maintained at 37° C. by using heating plates. Using the same syringe, 50 μl of a suspension of BNF-starch nanoparticles at an iron concentration of 50 mg/mL were administered at the center of the tumors. The suspension of BNF-Starch was administered at a quantity of 25.Math.t, measured in μg of iron comprised in nanoparticles, where t is the size of the treated tumors in mm.sup.3. Three different groups of mice were treated as follows:

(85) A first group of 4 mice was exposed to 21 hyperthermia sessions, lasting 7 weeks with 3 sessions per week. Each session of hyperthermia consisted in 4 to 86 cycles (table 8). At the beginning of each cycle, to initiate the heating step, a magnetic field, oscillating at high and medium frequency, with f.sub.h=195 kHz, f.sub.m=15 kHz, H.sub.av=27 mT and H.sub.max=57 mT was switched on during a time t.sub.7. As soon as the intra-tumor temperature reached 39.3-47.4° C., the oscillating magnetic field was stopped and the cooling step started to allow the intratumor temperature to decrease to 35-37.9° C. Cycles were repeated until a total exposure time of about 20 minutes was obtained for each hyperthermia session. The heating and cooling times, t.sub.7 and t.sub.8, measured during the different cycles of each hyperthermia session, as well as H.sub.av, H.sub.max, and f.sub.l, deduced from the values of t.sub.7 and t.sub.8 are indicated in table 8 and are average values among the 4 mice.

(86) A second group of 10 mice was exposed to 15 hyperthermia sessions, lasting 5 weeks, with 3 sessions per week. Each session of hyperthermia consisted in 30 minutes of application of a magnetic field, oscillating at f.sub.h=202 kHz, f.sub.m=15 kHz, H.sub.av=24-31 mT and H.sub.max=54-67 mT to target an intratumor temperature of 37-48° C. For the first hyperthermia session, the targeted intratumor temperature was always reached while for subsequent hyperthermia sessions, it was not always possible to reach the targeted intratumor temperature and the average and maximum magnetic fields were then set at H.sub.av=31 mT and H.sub.max=67 mT, with f.sub.h=202 kHz and f.sub.m=15 kHz. For mice in which tumor volumes exceeded 150% of initial tumor sizes and targeted temperature of 43-46° C. was not reached, mice received a second intratumor nanoparticle administration of BNF-Starch at 25 μg in iron of nanoparticles per mm.sup.3 of tumor.

(87) A third group of 10 mice was not treated further following BNF-Starch administration.

(88) In groups 1 and 2, the intra-tumor temperature was measured using an optical fiber positioned at the center of the tumors (Luxtron, LumaSense Technologies). In the groups, mice were euthanized when/if mouse weight had decreased by more than 20%.

(89) For group 1, the heating and cooling times, t.sub.7 and t.sub.8, are presented as a function of the number of hyperthermia session in FIGS. 7(c) and 7(d). The heating time is observed to be relatively low and constant at 20 seconds during the 13 first hyperthermia sessions and then it strongly increases from 20 seconds to 320 seconds between the 13.sup.th and 22.sup.nd hyperthermia session, which may be due to BNF-Starch progressively leaving the tumor and/or being degraded following the 13.sup.th hyperthermia session. A lower quantity of nanoparticles in the tumor would indeed require longer heating times to reach similar temperatures. By contrast, the cooling time remains relatively constant at 40 seconds, during the various hyperthermia sessions, suggesting that the cooling time does not depend on nanoparticle distribution and/or degradation. Instead, it may indeed depend on the environment of the nanoparticles such as type of tissue, organ, or blood irrigation of the tumor. The low frequency of oscillation is plotted as a function of number of hyperthermia session in FIG. 7(d). It is relatively constant and high at 16 10.sup.−3 Hz during the first 10 hyperthermia sessions and decreases down to 4 10.sup.−3 Hz between the 10.sup.th and 22.sup.nd hyperthermia session. This seems to indicate that to reach sufficiently high low frequency of oscillation, which may be necessary to reach antitumor activity, nanoparticle concentration should be sufficiently high and/or nanoparticles should not be degraded.

(90) Length and width of the tumors, L and l, were measured in the different mice using a caliper every 2 days and the tumor volume was estimated using the formula; V.sub.tumoral=0.5(L.Math.l.sup.2). Average tumor volumes of the three groups of mice are plotted as a function of time following day 0 (the day of BNF-Starch administration) in FIG. 8(a). Average tumor volumes strongly increase for group 3 and mice are euthanized at day 16, suggesting the absence of antitumor activity in group 3 (FIGS. 8(a) and 8(b)). Average tumor volumes increase less significantly in group 2 than in group 3 (FIG. 8(a)) and 20% of mice are still alive 245 days following BNF-Starch administration in group 2 (FIG. 8(b)), suggesting partial antitumor activity in group 2. Average tumor volumes decrease to zero at day 44 for group 1 and 100% of mice of group 1 are still alive 245 days following BNF-Starch administration (FIG. 8(b)), showing strong antitumor activity for group 1.

(91) We can Conclude from this Example that:

(92) i) By heating magnetic nanoparticles comprised in tumors using a magnetic field oscillating at three frequencies (f.sub.h=202 kHz, f.sub.m=15 kHz, and f.sub.l=4-25 mHz), it was possible to reach stronger antitumor efficacy than by using a magnetic field oscillating at two frequencies (f.sub.h=202 kHz and f.sub.m=15 kHz).

(93) ii) When we used a magnetic field oscillating at three frequencies (f.sub.h=202 kHz, f.sub.m=15 kHz, and f.sub.l=4-25 mHz), it was possible to reach strong antitumor efficacy without nanoparticle re-administration, whereas when we used a magnetic field oscillating at two frequencies (f.sub.h=202 kHz and f.sub.m=15 kHz), partial antitumor activity could only be reached when nanoparticles were re-administered. This suggests that the application of a magnetic field oscillating at three frequencies (f.sub.h=202 kHz, f.sub.m=15 kHz, and f.sub.l=4-25 mHz) leads to nanoparticles being less degraded and/or leaving less rapidly the tumor than the application of a magnetic field oscillating at two frequencies (f.sub.h=202 kHz and f.sub.m=15 kHz).

(94) iii) The heating time increases with the number of hyperthermia session and therefore seems to depend on nanoparticle concentration, whereas the cooling time remains relatively constant during the various hyperthermia sessions and therefore seems to be independent from nanoparticle concentration.

(95) iv) The low frequency of oscillation is higher or larger during the first to 11.sup.th hyperthermia sessions at 16 10.sup.−3 Hz than between the 16.sup.th and 22.sup.nd hyperthermia session, where f.sub.l is 4 10.sup.−3 Hz. This suggests that as the nanoparticle progressively leave the tumor and/or are degraded, f.sub.l decreases.

(96) TABLE-US-00001 TABLE 1 Properties of the different coils when magnetic field stabilized Measurement of f.sub.m, f.sub.b, H.sub.max and H.sub.av at the center of each coil Length Diameter Number of Coil f.sub.oc (kHz) f.sub.x (kHz) (cm) (cm) I (A) H.sub.coxt (mT) H.sub.av (mT) H.sub.coxt/H.sub.av turns 1 15 202 3.5 7 190 58 26 2.2 4 2 15 195 4 7 195 34 25 1.4 4 3 15 231 3.5 3 73 53 26 2.0 2 coils of four tunrs one in the other 4 15 329 2 3.5 149 56 24 2.3 3 5 2 91 15 28 682 33 26 1.3 3

(97) TABLE-US-00002 TABLE 2 Magnetic heating properties (f.sub.h, f.sub.m, H.sub.max, H.sub.av as indicated in table 1) M-PLL BNF Coil ΔT (° C.) S AR (W/g.sub.Fe) ΔT (° C.) S AR (W/g.sub.Fe) 1 71 244 2 57 84 7 8 3 68 202 4 75 192 12 13 5 5 6

(98) TABLE-US-00003 TABLE 3 Conditions of magnetic field application when magnetic field stabilized Measurement of f.sub.m, f.sub.b, H.sub.max and H.sub.av at 5 cm and 8 cm from the edge of coil 2 Distance Current from the intensity edge of the Coil (Å) f.sub.k (kHz) f.sub.m (kHz) coil (cm) H.sub.coxt (mT) H.sub.av (mT) H.sub.max/H.sub.av 2 550 192 None 5 13 12 1.1 189 None 8 5 5 1.1

(99) TABLE-US-00004 TABLE 4 SIGMA nanoparticles heating properties as a function of the distance from the edge of the coil 2 (f.sub.h, f.sub.m, H.sub.max, H.sub.av as indicated in table 3) Distance from the Concentration in edge of the coil iron (mg/mL) (cm) ΔT (° C.) SAR (W/g.sub.Fe) 422 5 45 2 8 8 0.4 194 5 29 2 8 5 0.5 87 5 15 3 8 1 0.4 57 5 13 3 8 1 0.6

(100) TABLE-US-00005 TABLE 5 Treatments of GL-261 cells brought into contact with 2 mg of chemical nanoparticles (BNF-Starch), exposed during t.sub.7 to a magnetic field oscillating at f.sub.h = 196 kHz and f.sub.m = 15 kHz with H.sub.max = 85 mT and H.sub.av = 61 mT to reach 45° C., followed by the non application of the magnetic field during t.sub.8. (Coil 2) H.sub.av, H.sub.max, f.sub.l deduced from low Time of application of the Time of non-application of the frequency sequences (t.sub.7 and t.sub.8) Cycle magnetic field, t.sub.7 (heating steps) magnetic field, t.sub.8 (cooling steps) H.sub.av (mT) H.sub.max (mT) f.sub.l (mHz) 1 7 minutes 23 secondes 2 minutes 57 secondes 44 85 1.61 2 3 minutes 31 secondes 3 minutes 23 secondes 31 85 2.42 3 3 minutes 21 secondes 3 minutes 17 secondes 31 85 2.51 4 3 minutes 30 secondes 3 minutes 9 secondes 32 85 2.51 5 3 minutes 27 secondes 3 minutes 25 secondes 31 85 2.43 6 3 minutes 34 secondes 3 minutes 10 secondes 32 85 2.48 7 3 minutes 35 secondes 3 minutes 6 secondes 33 85 2.49 8 3 minutes 40 secondes 3 minutes 30 secondes 31 85 2.33 9 3 minutes 45 secondes 3 minutes 36 secondes 31 85 2.27 10 3 minutes 42 secondes 3 minutes 36 secondes 31 85 2.28

(101) TABLE-US-00006 TABLE 6 Treatments of GL-261 cells brought into contact with 2 mg of chemical nanoparticles (BNF-Starch), exposed during t.sub.7 to a magnetic field oscillating at f.sub.h = 196 kHz and f.sub.m = 15 kHz with H.sub.max = 85 mT and H.sub.av = 61 mT to reach 50° C., followed by the non application of the magnetic field during t.sub.8. (Coil 2) H.sub.av, H.sub.max, f.sub.l deduced from low Time of application of the Time of non-application of the frequency sequences (t.sub.7 and t.sub.8) Cycle magnetic field, t.sub.7 (heating step) magnetic field, t.sub.8 (cooling step) H.sub.av (mT) H.sub.max (mT) f.sub.l (mHz) 1 9 minutes 07 secondes 4 minutes 12 secondes 42 85 1.25 2 5 minutes 09 secondes 4 minutes 25 secondes 33 85 1.74 3 5 minutes 42 secondes 4 minutes 32 secondes 34 85 1.63 4 5 minutes 36 secondes 4 minutes 40 secondes 33 85 1.62 5 5 minutes 34 secondes 4 minutes 37 secondes 33 85 1.64 6 5 minutes 34 secondes 4 minutes 37 secondes 33 85 1.64

(102) TABLE-US-00007 TABLE 7 Treatments of GL-261 cells brought into contact with 2 mg of chemical nanoparticles (BNF-Starch), exposed during t.sub.7 to a magnetic field oscillating at f.sub.h = 196 kHz and f.sub.m = 15 kHz with H.sub.max = 85 mT and H.sub.av = 61 mT to reach 55° C., followed by the non application of the magnetic field during t.sub.8. (Coil 2) H.sub.av, H.sub.max, f.sub.l deduced from low Time of application of the Time of non-application of the frequency sequences (t.sub.7 and t.sub.8) Cycle magnetic field, t.sub.7 (heating step) magnetic field, t.sub.8 (cooling step) H.sub.av (mT) H.sub.max (mT) f.sub.l (mHz) 1 11 minutes 56 secondes 5 minutes 54 secondes 41 85 0.93 2 8 minutes 49 secondes 5 minutes 49 secondes 37 85 1.14 3 8 minutes 40 secondes 5 minutes 50 secondes 36 85 1.15 4 9 minutes 15 secondes 5 minutes 23 secondes 39 85 1.14

(103) TABLE-US-00008 TABLE 8 Treatments of GL-261 tumors of 60-80 mm.sup.3 by administration of 25 μg/mm.sup.3 in iron of BNF-Starch, followed by application of a magnetic field, oscillating at f.sub.k = 202 kHz and f.sub.m = 15 kHz with H.sub.max = 57 mT and H.sub.av = 27 mT during t.sub.7 to a magnetic field oscillating to reach 45° C., following by the non application of the magnetic field during t.sub.8. (Coil 1) Maximum Minimum Time of temperature Time of non- temperature H.sub.av, H.sub.max, f.sub.t deduced from application of reached application of reached low frequency sequences the magnetic during the magnetic during (t.sub.7 and t.sub.8) field, t.sub.7 heating steps field, t.sub.8 cooling H.sub.max Hyperthermia Cycle (heating step) (° C.) (cooling step) steps (° C.) H.sub.av (mT) (mT) f.sub.t (mHz) 1 1 20 sec. 46.4-47.4 41 sec. 36.5-37.5 9 57 16 2 to 86 14 sec.   45-46.4 42 sec.   36-37.5 7 57 18 2 1 16 sec. 44.4-46 37 sec. 36.4-37.9 8 57 19 2 to 71 16 sec. 45.2-46.9 36 sec. 36.7-37.5 8 57 19 3 1 26 sec. 43.8-45.7 47 sec.   36-38.3 10 57 14 2 to 64 18 sec. 44.3-45.6 47 sec. 36.3-37.3 7 57 15 4 1 26 sec. 43.8-47.2 39 sec. 36.7-37.1 11 57 15 2 to 32 37 sec.   45-45.4 35 sec. 36.2-37.4 14 57 14 5 1 19 sec. 44.5-45.4 29 sec. 36.6-37.7 11 57 21 2 to 34 35 sec. 44.6-45.4 33 sec. 37.3-37.6 14 57 15 6 1 28 sec. 44.7-46 33 sec.   37-37.5 12 57 16 2 to 35 32 sec. 44.6-45.9 39 sec. 37.3-37.6 12 57 14 7 1 33 sec.   44-44.8 41 sec.   34-37.6 12 57 14 2 to 34 26 sec. 44.4-45.2 38 sec.   37-37.4 11 57 16 8 1 14 sec. 45.2-46.4 26 sec. 36.6-37.6 9 57 25 2 to 84 18 sec. 44.9-45.5 25 sec. 35.9-36.8 11 57 23 9 1 28 sec. 44.5-45.1 43 sec. 36.8-37.3 11 57 14 2 to 57 21 sec. 44.4-45.1 36 sec. 36.7-37.1 10 57 18 10 1 34 sec. 44.9-47 37 sec.   36-37.6 13 57 14 2 to 49 24 sec. 44.6-46.3 49 sec. 36.3-37 9 57 14 11 1 28 sec. 45.1-46 47 sec. 36.3-37.4 10 57 13 2 to 60 19 sec.   44-45.3 47 sec. 36.3-37.2 8 57 15 12 1 28 sec. 44.7-45.8 51 sec. 36.4-37.3 10 57 13 2 to 58 20 sec. 44.4-45.1 47 sec. 36.6-37.4 8 57 15 13 1 38 sec. 44.8-47 76 sec.   36-37.6 9 57 9 2 to 34 34 sec. 44.6-45.2 104 sec.  36.4-37.1 7 57 7 14 1 38 sec. 44.3-46.4 58 sec. 35.7-36.8 11 57 10 2 to 36 33 sec. 44.3-45 56 sec. 36.2-36.9 10 57 11 15 1 43 sec. 44.5-47 72 sec. 36.8-37.1 10 57 9 2 to 28 41 sec. 44.6-45 65 sec. 36.5-36.8 10 57 9 16 1 143 sec.  39.6-44.9 83 sec.   35-36.9 17 57 4 2 to 4 210 sec.  39.3-44.6 32 sec. 35.6-36.8 23 57 4 17 Same as cycle 16 18 19 20 21

(104) TABLE-US-00009 TABLE 9 Treatments of GL-261 tumors of 60-80 mm.sup.3 by administration of 25 μg/mm.sup.3 in iron of BNF-Starch, followed by application of a magnetic field, oscillating at f.sub.h = 202 kHz and f.sub.m = 15 kHz. (Coil 1) Maximum temperature reached during heating steps Nanoparticules H.sub.av H.sub.max Hyperthermia (° C.) admininistration (mT) (mT) 1 40-46.sup.  yes 24-31 54-67 2 32-46.sup.  no 25-27 54-57 3 31-48.sup.  no 25-27 54-57 4 31-47.8 yes 24-31 54-67 5 37-47.5 no 25-27 54-57 6 37-47.6 no 25-27 54-57 7 37-47.7 yes 24-31 54-67 8 37-47.8 no 25-27 54-57 9 37-47.9 no 25-27 54-57 10 37-47.1 no 25-27 54-57 11 37-47.1 no 25-27 54-57 12 37-47.1 no 25-27 54-57 13 37-47.1 no 25-27 54-57 14 37-47.1 no 25-27 54-57 15 37-47.1 no 25-27 54-57