Nanohyperthermia of malignant tumors. II. In vivo tumor heating with manganese perovskite nanoparticles

In the previous publication it was shown the possibility of synthesized magnetic fluid based on lanthanum-strontium manganite nanoparticles to generate the heat in externally applied alternating magnetic field (AMF) in vitro as well as lack of side effects of magnetic fluid administered into both non-tumor and tumor-bearing mice and rats [1]. The proposition to use the nanoparticles of perovskite manganite in the tumor hyperthermia is based on the postulate that these nanoparticles are able to demonstrate the ferromagnetic effect in the range of x=0.25–0.5 with Curie point = 0–95 °C that allows to avoid the overheating of surrounding normal tissues [2–7]. Samples of proposed nanocomposites were received in some laboratories, and their ability to be heated in the AMF in the system in vitro was shown [8–14].

It has to be noted that mentioned publications have shown the ability of nanoparticles of perovskite manganite to be heated by AMF action in model experiment [4, 8, 10, 13–14] and only one study presented theoretical calculations of the possibility of effective autostabilization of the temperature in the vicinity of T °C= 42 °C for nanoparticles of perovskite manganite [7]. Unfortunately, studies which could demonstrate the possibility to heat the tumor tissue with nanoparticles of perovskite manganite combined with AMF have not to be found in the accessible literature.

Taking into account this circumstance the current study was aimed to evaluate the ability of nanoparticles of perovskite manganite, in particular in the form of magnetic fluid under action of external AMF, to heat the biological tissues, especially tumor in the system in vivo.

Materials and Methods

Magnetic fluid. Magnetic fluid based on the synthesized nanoparticles of perovskite manganite (La_1-xSr_xMnO₃) and water solution of agarose, has been used in current study. Characteristics of magnetic fluid were presented earlier [1].

Alternating magnetic field. The rat leg was treated by external AMF after injection into muscle or intramuscularly transplanted tumor (Guerin carcinoma). A high frequency generator worked out by Institute of Electrodynamics was used to induce an alternating current of 7.7 kA at a frequency of 300 kHz [1]. The current passes through a custom-made five-turn water-cooled coil of 30 mm in internal diameter and 30 mm in height to generate a magnetic field inside the coil.

Temperature measurement. Tumor temperature during heating was measured by means of cooper-constantan thermocouples (0.1 mm diameter; IF-Kyiv). The thermocouples were calibrated against a certified precision mercury-in-glass thermometer (Committee of State Standards, Ukraine) before and after each experiment. Temperature was recorded always with the power switched off.

Animal and tumor models. All studies were conducted with strain IEPOR bread rats (Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv, Ukraine) weighting 150–200 g and bearing Guerin carcinoma transplanted intramuscularly into the right flank. The principle and method of transplantation were conventional. Animal were kept in Makrolon cages bedded with dust-free wood granulate, and had free access to a standard diet and tap water. All experiments had been approved by the regional animal ethics committee.

All 35 female rats were taken into experiment 7–9 days after transplantation (tumor volume 1.0–1.5 cm³), anaesthetized with “calipsovet plus” at a dose of 0.1 ml/100g b.w., i.p. before the onset of experiment. Each animal was placed into a special prepared net-hammock suspended to make free leg. The net-hammock was fixed on the tripod so that the femur of animal was localized in the water-cooled magnetic induction coil with diameter of 3.0 cm (Fig. 1). Suspension of magnetic fluid of manganite perovskite was administered into the muscle (5 rats) or tumor (30 rats) at a dose of 0.5–0.6 ml (50 mg manganite per ml). Tissue temperature was measured when generator was switched off to avoid the influence of alternating magnetic field.

Fig. 1. Photograph of rat in the special created net-hammock (the femur is located in the coil of magnetic field generator)

In experiments in vitro temperature was measured every 5 min under the switched off generator but in experiments in vivo such procedure was repeated not so often to avoid the living tissue damage (every 15–20 min, in average).

Results and Discussion

It was shown earlier that synthesized nanoparticles of lanthanum-strontium manganite did not display the toxicity or provoked the side effects in experimental animals with transplanted tumors [1].

At the first step of current study it was examined the ability of magnetic fluid of manganite perovskite to be heated in model experiment. Magnetic fluid was taken at a volume of 1.5 ml into the plastic tube which was located then in the coil of the generator of AMF in such way the tube setting up in the center of the field configuration. During the experiment temperature of magnetic fluid was measured when the generator was switched off. Obtained results were presented in Fig. 2. It was registered the increase of magnetic fluid temperature up to 72–75 °С within 10 min of AMF action. Fig. 3 represents the plot of temperature versus time after the onset of AMF action, and clearly demonstrates the increase of magnetic fluid temperature in comparison with the initial one. It has to be noted that specimens of magnetic fluid synthesized in diffe­rent periods of time and used in this study displayed almost the same changes of temperature under AMF in experiment in vitro (Fig. 4).

Fig. 2. Magnetic heating of manganite perovskite magnetic fluid under applied AMF (300 kHz, 7.7 kA/m). Ordinate — temperature (Т °С), abscisse — duration of AMF action (min)

Fig. 3. Plot of temperature (t/t₀) versus time of the magnetic heating of manganite perovskite magnetic fluid under applied AMF (300 kHz, 7.7 kA/m). Ordinate — temperature (t/t₀), abscisse — duration of AMF action (min)

Fig. 4. Magnetic heating of manganite perovskite magnetic fluid under applied AMF (300 kHz, 7.7 kA/m). Specimens of magnetic fluid synthesized in the different period of study were examined. Ordinate — temperature (Т °С), abscisse — duration of AMF action (min)

At the second step it was provided the experiments in vivo. The heating of magnetic fluid was evaluated after it was injected into the muscle of intact animal leg or into the tumor in the femur as well as into the muscle of opposite leg of tumor-bearing rats. It was determined that the temperature of femur muscle of intact rat (magnetic fluid was administered into the muscle) was increased by 8 °С within 20–30 min of AMF action (Fig. 5). The temperature of tumor (administration into the tumor, Guerin carcinoma) was increased by 8.3 °С within 10 min, by 10.8 °С within 20 min, by 13.6 °С within 30 min of AMF (Fig. 6, 7). These results have distinctly shown that magnetic fluid on the basis of lanthanum-strontium manganite is able to increase the tumor temperature, i.e., “to heat” the tumor under influence of external AMF and can be considered as an effective inductor of tumor local hyperthermia.

Obtained results are the first demonstration of the possibility to heat the tumor up to therapeutically relevant temperature, i.e., 43–44 ⁰ with nanoparticles of manganite perovskite in the system in vivo. The temperature in muscle tissue surrounding the tumor node was not increased over 37–39 °C indicating the minimal possibility of the normal tissue overheating (Fig. 8). This observation confirms the idea about the self-controlled heating by exploitation of manganite substances [4, 12].

Fig. 6. Magnetic heating of Guerin carcinoma after intratumoral administration of manganite perovskite magnetic fluid under applied AMF (300 kHz, 7.7 kA/m). It is given individual curves of rat tumor heating; experiment on 13.12.11: 1 and 2 — Guerin carcinoma was transplanted into both femurs of the same rat. Оrdinate — temperature (T °С), abscisse — duration of AMF action (min)

Fig. 7. Plot of temperature (t/t₀) versus time of the magnetic heating of Guerin carcinoma after intratumoral administration of manganite perovskite magnetic fluid under applied AMF (300 kHz, 7.7 kA/m). It is given individual curves of rat tumor heating. Оrdinate — temperature (t/t₀), abscisse — duration of AMF (min)

It should to be also noted that the analysis of obtained results has indicated that there is necessity to discuss the problems of coating, agglomeration of nanoparticles as well as their size that may influence the efficacy of heating, the distribution of nanoparticles in the tumor tissue as well as the manifestation of their magnetic properties in the specific tumor microenvironment.

The therapeutic relevance of nanohyperthermia method using manganite perovskite magnetic fluid and external AMF as single mean as well as in combination with chemotherapy is under evaluation now in experiment with transplanted tumors in vivo. Moreover, the advantage of nanohyperthermia in comparison with conventional radiofrequency hyperthermia has to be assessed obligatory, as we suppose.

Fig. 8. Plot of temperature (t/t₀) versus time of the magnetic heating of muscle and Guerin carcinoma after intramuscular or intratumoral administration of manganite perovskite magnetic fluid under applied AMF (300 kHz, 7.7 kA/m). Ordinate — temperature (t/t₀), abscisse — duration of AMF action (min)

Acknowledgements

This work was supported by STCU (grant #5213).

References

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