RU2633918C9 - Method for treatment of malignant new-formations by magnetic hyperthermia and pharmaceutical compositions for application in indicated method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: application of Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn_xMn_1-xFe₂O₄ (x=0-0.9), with average size of less than 40 nm, the Curie temperature ranges from 39 to 550°C, and the coercive force is 5 to 250 Oe. A pharmaceutical composition comprising these Zn-substituted magnetic nanoparticles and a pharmaceutically acceptable carrier is also provided. A method for malignant neoplasms treatment with magnetic hyperthermia, including the following steps (i) and (ii), is also proposed. Step (i) includes magnetic nanoparticles delivery to the tumour region in an effective concentration, selected from the group of magnetic metal oxides including Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn_xMn_1-xFe₂O₄ (x=0-0.9), with average size of less than 40 nm, the Curie temperature ranges from 39 to 550°C, and the average value of coercive force is 5 to 250 Oe. The step includes (ii) tumour region exposure to an alternating current (AC) magnetic field with an amplitude of 14 to 300 Oe and a frequency of 80 to 1000 kHz, where the exposure time of the said magnetic field at a temperature of 38 to 51°C is between 15 and 60 minutes. In addition, a method for experimental tumour cells damage by magnetic hyperthermia is proposed, comprising the following steps (i), (ii), (iii). At that, step (i) includes provision of magnetic nanoparticles selected from the group of magnetic metal oxides including Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn_xMn_1-xFe₂O₄ (x=0-0.9), with average size of less than 40 nm, the Curie temperature ranges from 39 to 550°C, and the average value of coercive force is from 5 to 250 Oe. Step (ii) includes addition of the said magnetic nanoparticles to the cell culture. Step (iii) comprises exposure of the said cell culture to a magnetic field of PT with an amplitude of 14 to 300°C and frequency of 80 to 1000 kHz, where the exposure time of the said magnetic field at a temperature of 38 to 51°C is between 15 and 60 minutes.

EFFECT: increased effectiveness of treatment due to uniform heating of the tumour tissue with reduced undesirable phenomena as a result of concomitant or subsequent selective damage or at least partial destruction of tumour cells in the tumour region without damaging the healthy surrounding tissue.

36 cl, 10 dwg, 4 tbl, 7 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to a method for the treatment of malignant neoplasms using magnetic hyperthermia and the use in this method of magnetic nanoparticles from the group of magnetic metal oxides, including ferrites, having increased heat production and an optimal value of the specific absorption coefficient (Specific Absorption Rate, SAR). The method provides selective heating of the tumor area and subsequent damage or destruction of cancer cells or malignant neoplasms without damaging healthy surrounding tissue. In addition, the invention relates to a pharmaceutical composition for magnetic hyperthermic treatment of malignant neoplasms containing magnetic nanoparticles.

BACKGROUND

Magnetic hyperthermia (MG) is a promising method of treating cancer, including exposing the entire body, its local area or tissue to elevated temperatures under the influence of an electromagnetic field to damage or destroy cancer cells or to destroy tumors. Two different approaches to hyperthermic heating include (a) hyperthermic methods using temperatures between 41 and 45 ° C and (b) methods of thermal ablation with increasing temperature above 45 ° C for local induction of tissue necrosis [1].

Various MG technologies and methods include local hyperthermia, regional hyperthermia, and whole body hyperthermia. Hyperthermia of the whole body can be caused by endogenous or exogenous sources of heat and, as a rule, provides heating of the whole body to temperatures of about 39-43 ° C [1, 2]. This method is usually used for the treatment of carcinomas with distant metastases [1, 3]. Regional hyperthermia affects the area of the human body or the whole organ. As a rule, the goal of such treatment is to weaken cancer cells, facilitating their destruction with the help of radiation and chemotherapeutic drugs [3]. Methods of local hyperthermia, as a rule, include the introduction of ferromagnetic or metal particles into the tumor area, followed by exposure to a magnetic or electromagnetic field: high frequency (HF), ultrahigh frequency (UHF), ultrahigh frequency (UHF), constant magnetic field, etc. . In some embodiments, with local MG, the administered particles are combined or attached to other anti-cancer drugs or cancer cell specific ligands. The main problems associated with the currently available methods of inducing hyperthermia include the need to localize the therapeutic effect in the target area, as well as to maximize the heating within the pathologically altered tissue without damaging normal tissue and reducing toxic side effects [2].

Magnetic nanoparticles (MNPs) have attracted attention as, for example, contrasting agents for magnetic resonance imaging (MRI), carriers of drug delivery systems and heat sources for hyperthermia for various methods of biomedical use [4]. They have unique properties, such as the ability to magnetic transportation, magnetic isolation and self-heating in a magnetic field of alternating current (PT) [5].

The use of MNPs as minimally invasive agents was first described by Gilchrist et al. (Gilchrist et al.) In 1957, which led to the emergence of MG methods [6]. When exposed to the magnetic field of a magnetic field, magnetic nanoparticles can exhibit pronounced thermal effects associated with losses during the magnetization reversal of particles [7]. In addition to providing the possibility of highly localized heat generation, the use of MNPs makes it possible to self-limit the temperature increase by using a magnetic material with a suitable Curie temperature [7]. One of the main problems associated with the use of magnetic nanoparticles in MG methods is the need for the maximum possible release of heat at the lowest dose of particles (due to their possible toxic effects on living organisms).

Nanoparticles can be divided based on their size into multi-domain MNPs, the size of which is more than about 40 nm depending on the parameters of the magnetic field, and single-domain nanoparticles, or “superparamagnetic nanoparticles,” with a diameter of less than 40 nm. Depending on the types of nanoparticles mentioned above, two different mechanisms are responsible for heat delivery. In the case of multi-domain magnetic nanoparticles, heat is delivered as a result of the displacement of the domain boundary (hysteresis losses) [1]. Single-domain particles cause heating as a result of losses due to reorientation of the magnetization vectors in a magnetic field, or losses due to friction, where this nanoparticle is able to rotate in the environment [1, 8].

A wide range of currently synthesized magnetic nanoparticles includes, among others, MFs based on metals (Fe, Co, Ni), MFs based on ferrites (MgFe ₂ O ₄ , CoPt, MnAl, CoFe ₂ O ₄ , etc.) and nanoparticles such as iron oxide. Despite the relatively weak magnetic properties, iron oxide nanoparticles are widely used in biomedicine as hyperthermic agents due to their low toxicity and stable magnetic characteristics. For example, Fe ₃ O ₄ -based nanoparticles have been approved by the US Food and Drug Administration (FDA) for use in hyperthermic processes. However, Fe ₃ O ₄ -based nanoparticles are prone to phase transformation into α-Fe ₂ O ₃ , γ-Fe ₃ O ₄ or Fe ₃ O ₄ crystals depending on environmental conditions, while the heat generation characteristics and, accordingly, the magnetic characteristics of such nanoparticles change, which limits their clinical use [9]. In addition, despite the fact that nanoparticles based on Co, Ni, or Mg have also been studied, they cannot be used for the human body due to the low heat generation temperature (Co: 25 ° C, others: below 25 ° C). In particular, particles based on MgFe ₂ O _{4 are} difficult to synthesize, and they have a low heat generation temperature due to low magnetic anisotropy [9]. Thus, there is a need for nanoparticles useful as new highly functional hyperthermic agents.

When developing nanoparticles for magnetic hyperthermia, the following parameters should be considered:

- the size;

- the form;

- toxicity and biocompatibility;

- the ability to modify the surface;

- the complexity of the synthesis and reproducibility of the obtained properties;

- heat transfer (SAR);

- the required external magnetic field and the reaction rate to the action of the specified field;

- cost.

One of the most important parameters when creating magnetic nanoparticles for MG is SAR, or specific absorption coefficient [6, 10]. SAR quantifies the efficiency of the conversion of magnetic energy into heat by MNP colloids. The specified parameter is defined as the absorbed energy normalized to the mass of the MNP in an alternating magnetic field of a certain frequency and intensity H ₀ [6]:

SAR is a critical parameter that affects tissue temperature during hyperthermic treatment. Overheating of the tumor can lead to serious damage to surrounding healthy cells or uncontrolled necrosis. On the other hand, the desired therapeutic effect cannot be achieved with an insufficiently high temperature rise. SAR of MNPs strongly depends on the frequency and intensity of the applied magnetic field, as well as on the chemical, physical, and magnetic properties of the material [6].

The main disadvantages of the methods of hyperthermia based on nanoparticles include:

- possible overheating (the exact duration of exposure should be calculated);

- toxicity of dosage forms;

- the complexity of selective delivery to the field of application;

- uneven distribution of MNP in the tumor area;

- high manufacturing cost.

Thus, the latest developments in MG methods, including the use of MNPs, are aimed at ensuring fast and uniform heating of malignant tumors with a decrease in the risk of thermal damage to healthy tissue and an increase in the selectivity of hyperthermia treatment. Another significant problem is the creation of new types of magnetic particles and nanoparticles that provide increased biocompatibility and treatment efficiency and alleviate severe adverse events by increasing SAR values and lower doses of these particles, as well as the development of nanoparticles combined or attached to anticancer drugs, ligands or cancer cell specific antibodies.

According to the publication of US patent 4983159 [11] a method is described that provides necrosis of neoplasms in warm-blooded animals as a result of hyperthermia, including the injection of particles into the perineoplastic region of the body of warm-blooded animals. Particles are capable of hysteretic heating when exposed to an alternating magnetic field. In addition, the particle size is at least two microns or more to prevent their intracellular absorption in any viable or neoplastic tissue of the animal. After the particles are injected into the tumor tissue, the region of the tumor is exposed to an alternating magnetic field. The frequency of this field is higher than the frequency sufficient to induce any detectable neuromuscular reaction in response to an alternating magnetic field, but lower than the frequency that can cause any adverse heating of the viable healthy tissue of a warm-blooded animal caused by eddy current and / or dielectric heating . The tumor is within the field for a time sufficient to heat the particles and the tumor to which the particles are bound to a temperature of at least 42 ° C to induce necrosis of the tumor. However, relatively large particles cannot be injected into solid tumors, and the use of smaller ferromagnetic particles is ineffective due to the lack of heating in the RF field. In addition, this approach does not provide uniform heating of the tumor tissue, which causes a risk of subsequent stimulation of tumor growth.

According to another approach, the Eurasian patent ΕА 019412 [12] describes a method for local hyperthermia of malignant tumors based on the absorption of energy from an alternating magnetic field using ferromagnetic particles with high coercive force introduced into the tumor region. The specified method can be used for thermal destruction of tumors without damaging healthy tissue. In particular, the invention allows to solve the problem of increasing the efficiency of magnetic hyperthermia by controlling the field amplitude taking into account changes in the temperature of the tumor during hyperthermia, as well as the interconnected optimization of particle properties (size and coercive force) and parameters of an alternating magnetic field (frequency and amplitude range regulation). However, the temperature of the heated area is controlled using invasive thermocouple sensors, and there is no reliable data on the temperature distribution in the heated area, since the temperature of the tumor is measured in the immediate vicinity of the sensors.

The invention according to Japanese patent application JP 2013256405 [13] describes Ni-Zn-based ferrite magnetic nanoparticles for heat therapy having a core containing magnetic Ni _(1-i) Zn _i Fe ₂ O ₄ nanoparticles (0.2≤i≤0 , 6), and a shell containing amorphous SiO ₂ for coating the specified core. The average particle size is 10-20 nm. These nanoparticles provide a temperature rise of 4-15 ° C upon application of a magnetic field PT with a frequency of 15 kHz and an intensity of 210 E. However, these nanoparticles have a high manufacturing cost. In addition, it was previously described that Ni-based nanoparticles have a low heat-generating temperature, which limits their use in hyperthermia [9].

A magnetic system suitable for drug delivery using hyperthermia is described in Russian patent RU 2481125 [14]. The structure of this magnetic system includes magnetic particles of nanoscale formula M ^II M ₂ ^III O ₄ , where M ^II = Fe, Co, Ni, Ζn, Μn; M ^III = Fe, Cr, or maghemite functionalized by bifunctional compounds of the formula R ₁ - (CH ₂ ) _n -R ₂ (where n = 2-20, R _{1 is} selected from: CONHOH, CONHOR, PO (OH) ₂ , PO ( OH) (OR), COOH, COOR, SH, SR; R ₂ represents an external group and is selected from: OH, NH ₂ , COOH, COOR; R represents an alkyl group or an alkali metal selected from C _1-6 alkyl and K, Na or Li, respectively). The structure also contains a polymer, optionally containing a pharmacologically active molecule, which can be selected from anticancer agents, antimicrobial agents, anti-inflammatory agents, immunomodulators, and molecules that act on the central nervous system or are able to label cells to be identified using standard diagnostic detectors. The magnetic system is characterized as biocompatible and stable. However, these magnetic systems and, in particular, magnetic nanoparticles vary greatly in size (for example, the average diameter of the nanoparticles can be in the range from 4 to 200 nm). It is known that these small particles are in a single-domain state, and their ability to heat up at a specific amplitude and frequency of the magnetic field is determined by the magnetic susceptibility. An increase in particle size leads to the emergence of another heating mechanism based on hysteresis losses, and the heating value is determined by the region of the hesteresis loop, i.e. magnetization of saturation and coercive force. In addition, an increase in particle diameter leads to mechanisms mediated by viscous friction during rotation and particle motion in a magnetic field. Thus, different heating mechanisms are characteristic of particles of different sizes, which in turn can lead to certain difficulties in controlling the temperature and subsequent uneven heating of malignant tumors.

According to another approach, the Russian patent RU 2295933 [15] describes a method for magnetic treatment of malignant neoplasms, including the introduction of particles into the tumor and subsequent heating as a result of the magnetocaloric effect caused by the energy of the electromagnetic field. Particles are composed of a substance with a high value of the magnetocaloric effect, with a magnetic phase transition temperature close to the human body temperature, and are selected from the group including alkaline-earth, transition and noble metals, as well as their alloys and intermetallic compounds (for example, particles on based on an alloy of iron-rhodium, in particular, Fe _0.49 Rh _0.51 ). However, the applied magnetic field has a high value of strength (up to 60 Oe and more), which can cause a number of negative effects in living organisms. Thus, the use of such a magnetic field in hyperthermia methods cannot be considered absolutely safe.

According to the publication of US patent 7842281 [16] an improved composition of magnetic nanoparticles is described that provides an internal temperature controller for use in magnetic heating, in particular for use in medical treatment using magnetic hyperthermia. The composition contains magnetic nanoparticles, the Curie temperature of which is between 40 and 46 ° C, and may optionally contain a polymeric material and optionally a drug or radiosensitizing agent. The specified composition reduces or eliminates the problem of uneven heating and has the ability to self-regulate the maximum temperature reached, which makes the method of magnetic hyperthermia a more promising therapeutic opportunity. One embodiment of the invention relates to a composition containing MNPs whose Curie temperature is 47 ° C., wherein said nanoparticle composition contains an alloy of the formula Mn _0.5 Zn _0.5 Fe ₂ O ₄ , and an effective average diameter of the nanoparticles is between 10 nm and 400 nm.

The invention also provides methods for hyperthermic treatment of a patient in need of said treatment, comprising the steps of administering to the patient a composition comprising MNPs whose Curie temperature is between 40 and 46 ° C; and exposing the MNP in the patient’s body to an alternating magnetic field effective to induce hysteretic heating of the nanoparticles. However, the size of these magnetic nanoparticles varies significantly (effective average particle diameter is between 10 nm and 400 nm). As mentioned above, particles of different sizes are characterized by different heating mechanisms, which in turn can lead to certain difficulties in controlling the temperature, making it difficult, therefore, to uniformly heat malignant tumors. Moreover, this approach does not take into account the characteristics of the tumor to be treated, such as its location, density and hydrodynamic properties.

Thus, there is a need to develop improved methods of magnetic hyperthermic treatment with the elimination of one or more disadvantages according to the prior art. The authors believe that future success in the development of MG methods and their use for the treatment of cancer depends on the ability to create individual protocols for each type of cancer to be treated with hyperthermia in subjects, instead of developing a common universal method.

SUMMARY OF THE INVENTION

The aim of the present invention is an improved method of hyperthermic treatment of various diseases and disorders, in particular, malignant neoplasms, providing regulation of the number and local concentration of magnetic nanoparticles in the tumor area depending on the desired values predefined for each type of tumor and uniform heating and increased efficiency of hyperthermia while reducing adverse events. The inventors of the present invention unexpectedly found that a method of magnetic hyperthermic treatment of malignant neoplasms, comprising the steps of delivering magnetic nanoparticles selected from the group of magnetic metal oxides to the tumor region, including ferrites, the average size of which is less than 40 nm, and exposing the tumor region to a magnetic field of PT predefined parameters, provides concomitant and / or subsequent selective damage or at least partial destruction of the OLEV cells in the tumor without damaging surrounding healthy tissue.

According to one embodiment of the invention, a method for treating malignant neoplasms using magnetic hyperthermia comprises the following steps:

(i) delivery to the tumor region of magnetic nanoparticles selected from the group of magnetic metal oxides, including Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), average size which is less than 40 nm, the Curie temperature is from 39 ° C to 550 ° C, and the average value of the coercive force is from 5 to 250 Oe, and

(ii) exposing the tumor region to a magnetic field of PT with an amplitude of from 14 O to 300 O and a frequency of from 80 kHz to 1000 kHz, where the exposure time to the specified effect at a temperature of 38 ° C to 51 ° C is from 15 to 60 minutes, providing:

(iii) concomitant and / or subsequent selective damage or at least partial destruction of the tumor cells selectively in the tumor area without damaging healthy surrounding tissue.

According to one embodiment of the invention, the strength and / or frequency of the electromagnetic field of the PT is selected based on a predetermined dependence of the effectiveness of hyperthermia on the type of tumor and the delivered concentration and / or type of nanoparticles to provide damage or partial destruction of at least 80 to 90%, preferably 95% tumor cells.

According to one embodiment of the invention, the magnetic nanoparticles are selected based on a predetermined dependence of the effectiveness of magnetic hyperthermia for a particular tumor type on at least one parameter selected from concentration, average particle size, particle size distribution, Curie temperature, average coercive force and particle coating , to provide damage or partial destruction of at least 80 to 90%, preferably 95% of tumor cells.

According to one embodiment of the invention, the duration and number of cycles of exposure to magnetic hyperthermia are selected based on a predetermined dependence of the effectiveness of hyperthermia on the type of tumor and / or particle characteristics to provide damage or partial destruction of at least 80 to 90%, preferably 95% of the tumor cells.

According to one embodiment of the invention, the magnetic nanoparticles are Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ , where x is 0-0.9. In some embodiments, the particles may comprise a protective shell or coating, wherein said protective shell or coating consists of a biocompatible polymeric material.

According to another embodiment of the invention, the method of magnetic hyperthermic treatment of malignant neoplasms includes the step of selecting magnetic field parameters (strength, frequency) and magnetic nanoparticle characteristics (size, shape) for each type of tumor, providing optimal SAR values, coercive force and heating temperature for damage or partial destruction of at least 50, preferably from 75 to 90%, more preferably 95% of tumor cells.

According to another embodiment of the invention, the invention relates to the use of Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ , where x is 0-0.9, for the treatment of malignant neoplasms using magnetic hyperthermia.

According to another embodiment of the invention, there is provided a pharmaceutical composition for treating malignant neoplasms using magnetic hyperthermia, containing Ζn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), pharmaceutically acceptable a carrier and optionally a biologically active molecule.

According to another embodiment of the invention, the use of Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9) for magnetic hyperthermia of malignant neoplasms is proposed. According to another embodiment of the invention, a method for experimental damage to tumor cells using magnetic hyperthermia, comprising the following stages:

(i) providing magnetic nanoparticles selected from the group of magnetic metal oxides, including Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), the average size of which is less than 40 nm, the Curie temperature is from 39 ° C to 550 ° C, and the average value of the coercive force is from 5 to 250 E.

(ii) the addition of magnetic nanoparticles to cell culture;

(iii) exposure of the indicated cell culture to a magnetic field of PT with an amplitude of from 14 O to 300 O and a frequency of from 80 kHz to 1000 kHz, where the exposure time to the specified exposure at a temperature of 38 ° C to 51 ° C is from 15 to 60 minutes, for providing:

(iv) concomitant or subsequent damage or at least partial destruction of the tumor cells.

These and other embodiments of the invention are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying graphic materials illustrate embodiments of the invention described in this application and, together with the description of the invention, disclose some of the non-limiting examples of the invention.

The Figure 1 shows images of samples of Zn _x Mn _1-x Fe ₂ O ₄ compounds with different Zn contents obtained by transmission electron microscopy: a - Zn _x Mn _1-x Fe ₂ O ₄ , x = 0; b - Zn _x Mn _1-x Fe ₂ O ₄ , x = 0.1; s - Zn _x Mn _1-x Fe ₂ O ₄ , x = 0.2; d is Zn _x Mn _1-x Fe ₂ O ₄ , x = 0.4; e is Zn _x Mn _1-x Fe ₂ O ₄ , x = 0.5; f is Zn _x Mn _1-x Fe ₂ O ₄ , x = 0.6; g - Zn _x Mn _1-x Fe ₂ O ₄ , x = 0.9.

Figure 2 shows the size distribution of Zn _x Mn _1-x Fe ₂ O ₄ nanoparticles depending on the degree of Zn substitution.

Figure 3 shows the design of an experimental device with an air cooling system for conducting in vitro experiments using MG ( a ) and its block diagram (b): 1 - generator, 2 - converter, 3 - cooling system, 4, 5, 6 - power source, L - coil.

Figure 4 shows the magnetization results for Ζn _x Μn _1-x Fe ₂ O ₄ with different contents of Ζn (x) [18].

The Figure 5 shows the dependence of coercivity on the concentration of Zn at room temperature.

Figure 6 shows the heating efficiency of the Zn _x Mn _1-x Fe ₂ O ₄ MNP samples as a graph of temperature versus time for various Zn (x) concentrations.

The Figure 7 shows the values of the specific absorption coefficient (SAR) depending on the size of the MNP.

Figure 8 shows the heating efficiency of Zn _0.2 Mn _0.8 Fe ₂ O ₄ particles as a graph of temperature versus time for various field strengths Η at a frequency f = 100 kHz.

The Figure 9 shows a graph of temperature versus time, including the ratio of the heating efficiency Zn _x Mn _1-x Fe ₂ O ₄ MNP (x = 0.1) to the heating efficiency of other compositions: polymer-coated and polymer-free magnetic particles (Fe ₃ O ₄ ) and LSMO particles (La _x Sr _1-x MnO ₃ , x = 0.75); Η = 95 E.

Figure 10 shows the images of control ( a ) and experimental (b) leukemia cells of the K562 line, which were incubated at 37 ° C ( a ) and 43 ° C (b) in the presence of Zn-substituted magnetic nanoparticles based on manganese ferrite (10 mg) . The initial increase in x250.

DETAILED DESCRIPTION OF THE INVENTION

In General, the present invention describes a method for the treatment of malignant neoplasms or target tissues using local hyperthermia using magnetic nanoparticles having improved heating characteristics to increase heat production and reduce adverse events.

I. DEFINITIONS

Throughout the present application and claims, the terms “includes” and “contains” and their variations, such as “comprising”, “including”, “has” and “having”, should be interpreted as inclusive. The use of items in the singular in the context of the described invention refers to both the singular and plural of these items, unless otherwise indicated in this application or the context clearly does not follow.

It should be understood that each range described in this application is a series of numbers belonging to the specified range. Thus, each range includes all and every value belonging to the specified range, as well as any of its subranges. Unless otherwise indicated, it is assumed that the boundary points of the range are part of the specified range.

When used in this application, the term "magnetic hyperthermia" (abbreviation "MG") refers to a method of medical treatment, including exposing the entire body, its local area or tissue to elevated temperatures under the influence of an electromagnetic field to damage or destroy cancer cells or to destroy tumors.

The term "magnetic nanoparticles" (abbreviation "MNP") when used in this application refers to a class of nanoparticles that can be controlled using magnetic field gradients. According to a preferred embodiment of the invention, the term refers to Zn-substituted magnetic manganese ferrite nanoparticles.

When used in this application, the terms "magnetic field of alternating current (PT)" or "alternating magnetic field" means a magnetic field caused by alternating current.

When used in this application, the terms "malignant neoplasm", "malignant neoplasm", "cancer cell", "tumor cell" or "tumor" refer to any neoplastic growth in a patient, including any metastases. Specific types of cancer that can be treated using the methods of the present invention are described herein.

In general, the term “heating efficiency” means the ratio of the heat generated during the process to the total energy expended or absorbed heat. When used in this application, this term defines the ability of magnetic nanoparticles to heat up to the optimum temperature in the described method of magnetic hyperthermia. The indicated optimal temperature provides fast and uniform heating of malignant neoplasms, reducing the risk of thermal damage to healthy tissue.

The term "Curie temperature" (or "T _s ") means the temperature at which specific materials lose the properties of a permanent magnet with the acquisition of induced magnetism. The particle size in the crystal lattice of the material changes the Curie temperature. Due to the small particle size, the electron spin oscillations become more significant, which thus leads to a decrease in T _c with a decrease in particle size, since the oscillations cause disorder [17].

When used in this application, the term "specific absorption coefficient" (or "SAR") is the amount of energy absorbed by the sample per unit mass. Particles must have high SAR values for rapid heating under the influence of the applied alternating magnetic field. SAR MNP is highly dependent on the frequency and intensity of the applied magnetic field, as well as on the chemical, physical and magnetic properties of the material. It is expected that particles having an optimal specific absorption coefficient (SAR) value do not cause overheating of the tumor tissue with an applied magnetic field and provide the desired therapeutic effect due to a sufficient rise in temperature.

More specifically, these particles provide heating of the tumor region to a desired temperature of from about 40 to about 46 ° C, using their low concentration and without overheating of regions with a high concentration of particles with minimal values of the frequency and amplitude of the applied field.

The terms "coercive force" or "coercivity" used in relation to a ferromagnetic material, mean the intensity of the applied magnetic field, necessary to reduce the magnetization of the specified material to zero after bringing the magnetization of the sample to saturation. In other words, this parameter measures the resistance of the ferromagnetic material to demagnetization.

The term "anisotropy constant" or "magnetic anisotropy constant" reflects the realization of the effect of magnetic anisotropy in a material where the location of nuclear magnetic moments in a certain direction of the crystal is energetically more effective than other directions. Under the influence of the applied magnetic field, the magnetic moment of a single-domain nanoparticle can rotate. In a solid, rotation occurs inside the particle (Neel relaxation). In a magnetic fluid, it can occur through Brownian diffusion. Important factors affecting the Neel relaxation time are the anisotropy constant K _eff and the particle volume V. When the particle diameter is less than 15 nm, fast Neel relaxation dominates. If the particle diameter is more than 15 nm, its slow Brownian relaxation affects its characteristics.

When used in this application, the terms "selectively" and "selectivity" (for example, "treatment selectivity") mean an effect with highly specific activity, providing a strictly localized effect. The term "cell damage coefficient" means the percentage of cancer cells undergoing visible morphological changes 24 hours after they are exposed to the magnetic field of the PT in the presence of magnetic nanoparticles.

When used in this application, the term "introduction" means the introduction of magnetic nanoparticles in accordance with the invention or the pharmaceutical composition containing them to a subject in need of the specified introduction. According to a preferred embodiment of the invention, the term refers to intravenous or intratumoral administration.

The terms “subject” and “patient” are used interchangeably herein and refer to an animal, such as a mammal, more preferably a human.

When used in this application, the terms "treat" and "treatment" refer to the suppression or reduction of the development of a pathological condition, disorder or disease or its clinical symptoms using specific therapy.

The term “biologically active molecule” is used to define a molecule of a substance that is administered to a subject capable of eliciting a specific cellular response. According to a preferred embodiment of the invention, the biologically active molecule is selected from an anticancer agent that recognizes tumor cells of an antibody or aptamer.

II. DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

According to one embodiment of the invention, a method for the treatment of malignant neoplasms using magnetic hyperthermia, comprising the following stages:

(i) delivery to the tumor site of magnetic nanoparticles selected from the group of magnetic metal oxides,

(ii) exposure of the tumor region to the magnetic field of the PT,

(iii) subsequent selective damage or destruction of the tumor cells in the area of the tumor without damaging healthy surrounding tissue.

According to one embodiment of the invention, the magnetic nanoparticles are Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ , where x is 0-0.9.

According to specific embodiments of the invention, the magnetic nanoparticles are Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _0.2 Mn _0.8 Fe ₂ O ₄ . The authors of the present invention unexpectedly discovered during the experiment that the introduced magnetic nanoparticles have increased heating efficiency and high specific absorption coefficient (SAR). The SAR of the MNP is highly dependent on the frequency and intensity of the applied magnetic field, along with the chemical, physical and magnetic properties of the material. As a result of a study conducted by the inventors, it was found that nanoparticles with an optimal SAR value described according to the invention provide the desired therapeutic effect due to a sufficient rise in temperature without overheating of the tumor tissue under the influence of the applied magnetic field. Based on an experimental study, the inventors determined the size of the nanoparticles for use according to the present invention in order to maximize the cell damage coefficient and select the composition of the MNP, for example, the degree of Zn substitution, and magnetic field parameters such as frequency and / or magnetic field strength. The size was measured as shown in Example 2.

According to one embodiment of the invention, a preferred average nanoparticle size is less than 40 nm. In some embodiments, the average nanoparticle size is in the range of 1 to 100 nm, 2 to 50 nm, 4 to 40 nm, or 5 to 30 nm. According to a preferred embodiment of the invention, the average nanoparticle size is from 5 to 25 nm. The authors found that the specified size range is a decisive factor for improving the heating efficiency of nanoparticles.

According to one embodiment of the invention, the Curie temperature of the MNPs for use in the described method can be from 39 ° C to 550 ° C. According to some embodiments of the invention, the Curie temperature of the nanoparticles is from 40 to 100 ° C, from 45 to 150 ° C, from 50 to 200 ° C, from 60 to 250 ° C, from 65 to 300 ° C, from 70 to 350 ° C , from 80 to 400 ° C, from 85 to 450 ° C, or from 90 to 550 ° C. According to a preferred embodiment of the invention, the Curie temperature of the nanoparticles is from 42 ° C to 45 ° C. The inventors have found that the specified temperature range provides the highest efficiency and cell damage rate of more than 50%.

According to one embodiment of the invention, magnetic nanoparticles for use in accordance with the present invention are characterized by an average coercive force of 5 to 250 Oe. According to some embodiments of the invention, the average coercive force is 5 to 40 Oe, 10 to 45 Oe, 17 to 35 Oe, from 20 to 50 Oe, from 24 to 48 Oe, from 30 to 90 Oe, from 45 to 120 Oe, from 60 to 110 Oe, from 78 to 190 Oe or from 95 to 250 Oe. According to a preferred embodiment of the invention the value of the coercive force is from 10 to 30 E. The authors showed that then the value of the coercive force strongly depends on the degree of Zn substitution (see Example 5).

The MNPs used in the method according to the present invention can be easily synthesized using production methods known to those skilled in the art. Methods suitable for producing MNPs include co-precipitation, hydrothermal synthesis, thermal decomposition, microemulsion methods, sol-gel synthesis, and flame spray synthesis. For example, magnetic nanoparticles for use in the described method can be synthesized using the co-precipitation method in an Ar atmosphere (Example 1, [18]).

According to one embodiment of the invention, magnetic nanoparticles for use in the described method may comprise a protective sheath or coating. The inventors have found that said protective sheath or coating provides an increase in the biocompatibility of MNPs. According to one embodiment of the invention, the protective sheath or coating consists of a biocompatible polymeric material. In some embodiments, the biocompatible polymer material is polyethylene glycol (PEG), polypropylene, dextran, polyvinyl difluoride (PVDF), polyvinyl pyrrolidone (PVP), or polyacrylic acid. In one embodiment, the biocompatible polymer material is polyethylene glycol (PEG) or dextran. The inventors have successfully found that non-covalent immobilization of PEG on the surface of nanoparticles increases the circulation time of nanoparticles in the bloodstream and the efficiency of their absorption by cells, and thus increases the coefficient of damage to cancer cells. Similarly, inventors have found that dextran-coated particles exhibit higher stability in a colloidal solution of nanoparticles and increased circulation time in the bloodstream. According to another embodiment of the invention, the biocompatible polymer material is polypropylene and / or polyvinyl difluoride (PVDF) (see Example 3).

According to an embodiment of the present invention, the magnetic field is an alternating magnetic field created by an external source and having an amplitude of from 14 Oe to 300 Oe and a frequency from 80 kHz to 1000 kHz. According to some embodiments of the invention, the amplitude of the magnetic field of the PT is from 14 to 90 Oe, from 30 Oe to 150 Oe, from 50 Oe to 200 Oe, from 60 Oe to 230 Oe, from 80 Oe to 250 Oe, from 40 to 200 Oe, from 70 to 290 Oe or from 90 Oe to 300 Oe. According to some embodiments of the invention, the magnetic field frequency of the PT is from 1 kHz to 100 kHz, from 50 kHz to 300 kHz, from 80 kHz to 500 kHz, from 100 kHz to 700 kHz , from 150 kHz to 900 kHz or from 90 kHz to 1000 kHz. The inventors found that the magnetic field of the PT with an amplitude of 100 Oe and a frequency of 100 kHz provides effective cell damage. Example 4 describes an experimental device for creating a magnetic field. A magnetic field is applied to a predetermined area and for a predetermined time to ensure that the magnetic nanoparticles are heated to a temperature sufficient to damage or destroy tumor cells without damaging healthy surrounding tissue.

According to one embodiment of the invention, the magnetic field of the PT is created using an external field source with a field gradient of 3 to 100 T / m to accurately determine the movement and location of the nanoparticles at the site of the tumor during treatment, in particular after the stage of delivery of the magnetic nanoparticles to the tumor. According to some embodiments of the invention, an external field source generating a magnetic field of the PT is placed inside or outside the body of a patient in need of said treatment.

According to specific embodiments of the invention, the method described in this application may optionally include the stage of localization and regulation of the concentration of nanoparticles in the tumor region using an external field source based on a system of alkaline-earth permanent magnets using Halbach cylinders. Such an approach allows one to maximize the forces applied to magnetic nanoparticles directed to deeply located tissues, thus providing the possibility of treating a wider class of patients, for example, patients with deeper tumors [19]. The stage of localization and regulation of the concentration of nanoparticles at the site of the tumor is preferably carried out after the stage of delivery of magnetic nanoparticles to the tumor. The authors suggest that precise localization and regulation of the concentration of nanoparticles in the tumor can significantly increase the selectivity of treatment.

According to an embodiment of the present invention, the exposure time to the magnetic field of the PT can vary from 15 to 60 minutes. The specified time, as a rule, is sufficient to heat the tumor tissue to therapeutic temperatures and / or ablation temperatures (preferably 38-51 ° C). According to some embodiments of the invention, the time of exposure to the magnetic field of the PT is from 15 to 30 minutes, from 20 to 40 minutes, from 30 to 50 minutes, or from 40 to 60 minutes. According to a preferred embodiment of the invention, the exposure time to the magnetic field of the PT is from 30 to 45 minutes. The inventors have found that the specified time range is sufficient to effectively heat the tumor region to therapeutic temperatures of 42-45 ° C, with a reduced risk of thermal damage to healthy tissue.

According to one embodiment of the invention, the tumor region is heated to 38-51 ° C under the influence of an alternating magnetic field. Temperatures in excess of 45-50 ° C are usually used for local induction of tissue necrosis (thermal ablation methods), while temperatures in the range of 41 to 45 ° C can be used to damage cancer cells (which is preferred for small tumors) associated with defects membranes, conformational changes, inhibition of the synthesis of RNA, DNA and protein, etc. According to some embodiments of the invention, the tumor region is heated to a temperature of from 39 to 45 ° C, from 40 to 42 ° C, from 41 to 45 ° C, from 43 to 47 ° C, or from 45 to 51 ° C. According to a preferred embodiment of the invention, the tumor region is heated to a temperature of 42 ° C to 45 ° C. The authors showed that this temperature range provides effective damage to cancer cells with a damage coefficient of more than 50% (see Example 7).

In general, cell death is a cascade process that involves a number of stages, from early morphological changes to apoptosis, which have different durations. According to one embodiment of the invention, the step of selectively damaging or destroying the tumor cells in the region of the tumor can vary from 1 hour to 96 hours. The duration of this stage depends on specific factors, such as the type, size and location of the tumor, heating temperature, exposure time, etc. The inventors found that experimental damage or destruction of cancer cells with a cell damage coefficient of more than 50% takes about 24 hours (Example 7).

According to one embodiment of the invention, the method for treating malignant neoplasms using magnetic hyperthermia described herein may include the step of non-invasively measuring the temperature of the tumor. Possible ways to measure the temperature of a tumor in MG include, for example, invasive and non-invasive approaches, such as using a thermocouple probe, measuring temperature using magnetic resonance imaging or the temperature dependence of higher harmonics of magnetization. According to one embodiment of the invention, a temperature measurement using magnetic resonance imaging or the temperature dependence of the higher harmonics of magnetization is used at the stage of non-invasive measurement of tumor temperature. According to a preferred embodiment of the invention, the step of non-invasively measuring the temperature of the tumor includes measuring the temperature using magnetic resonance imaging. In some embodiments, the method optionally includes the step of invasively measuring the temperature of the tumor, for example using a thermocouple probe. The measurement of the temperature of the tumor is preferably carried out during the step of exposing the tumor region to the magnetic field of the PT.

In accordance with an embodiment of the present invention, a method for treating malignant neoplasms by magnetic hyperthermia may also include selecting electromagnetic field parameters (strength, frequency), characteristics of magnetic nanoparticles (concentration, average size, size distribution, Curie temperature, average coercive force, coating) , the duration and number of cycles of exposure to the magnetic field of the PT and the method of injection, localization and retention time of nanoparticles for each type of op aches. Optionally, this approach takes into account other parameters, such as the volume of nanoparticles, SAR, and the anisotropy constant; size, density and location of the tumor; the condition of the patient, etc., thus providing increased efficiency and selectivity of treatment.

In accordance with an embodiment of the present invention, the MNPs used in the described method can be administered by any effective convenient route, including intravenous, intramuscular, subcutaneous, intratumoral, oral, intranasal, inhaled, rectal, intravaginal or topical administration. According to a preferred embodiment of the invention, the magnetic nanoparticles are administered intravenously or into the tumor into specific areas of the body of a patient in need of said administration.

Dosage, regimen and duration of treatment depend on the type of tumor undergoing hyperthermia, as well as age, body weight and response to treatment of a particular patient and must be determined by a specialist individually for each clinical case. However, it should be noted that the concentration of nanoparticles, which is between 20 and 80 mg / cm ³ , can lead to an ablation temperature (> 50 ° C). Thus, homogeneous magnetic materials with a relatively low SAR value may be suitable for magnetic hyperthermia if the volume of the particle solution to be introduced is less than the size of the tumor.

According to another aspect of the invention, the disease or disorder to be treated in a subject using the method described herein may include, but is not limited to, breast cancer, lung cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, stomach cancer, liver cancer , pancreatic cancer, colon cancer, uterine cancer, cervical cancer, ovarian cancer, bladder cancer, testicular cancer, prostate cancer, bone cancer and brain cancer.

According to another embodiment of the invention, the invention relates to the use of Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ , where x is 0-0.9, for the treatment of malignant neoplasms using magnetic hyperthermia. According to specific embodiments of the invention, the invention relates to the use in the described method of Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _0.2 Mn _0.8 Fe ₂ O ₄ .

According to one embodiment of the invention, there is provided a pharmaceutical composition for treating malignant neoplasms using magnetic hyperthermia, comprising Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9) and a pharmaceutically acceptable carrier. In some embodiments, the magnetic nanoparticles are Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _0.2 Mn _0.8 Fe ₂ O ₄ . A pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material or carrier, such as a liquid or solid excipient, diluent, excipient or solvent, and may include sugars (lactose, glucose, sucrose, etc.), starch (corn starch or potato starch) , cellulose and its derivatives, malt, gelatin, glycols (e.g. propylene glycol), polyols (glycerin, sorbitol, mannitol, etc.), buffering agents, isotonic saline, ethyl alcohol and other non-toxic biocompatible substances.

Said pharmaceutical composition may optionally comprise a biologically active molecule, wherein said biologically active molecule is selected from an anticancer agent that recognizes tumor cells of an antibody or aptamer. Examples of biologically active molecules suitable for use in the pharmaceutical composition of the invention include, but are not limited to, anti-cancer agents such as carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, methotrexate, paclitaxel, tamoxifen, vincristine; antibodies, such as alemtuzumab, bevacizumab, cetuximab, matuzumab, obinutuzumab, ofatumumab, panitumumab, pertuzumab, rituximab, trastuzumab; aptamers, such as toll-like receptor (TLR) agonists or GCC receptor ligands. The authors suggest that the use of MNP in combination with biologically active substances in the described method can significantly increase the effectiveness and selectivity of treatment.

The pharmaceutical composition according to the invention for the treatment of malignant neoplasms using MG can be presented in the form of a solution for infusion, a solution for injection, powder or lyophilisate. The specialist in the field of medicine should determine the dosage, regimen and duration of treatment individually for each clinical case, depending on the characteristics of the patient to be treated. The pharmaceutical composition described herein can be administered to a subject using various methods, including intravascular, intramuscular, subcutaneous, intratumoral, oral, intranasal, inhalation, rectal, intravaginal or topical administration.

The pharmaceutical composition according to the invention is suitable for treating with MG a disease or disorder, such as a malignant neoplasm or cancer, in a patient in need of said treatment. Examples include, but are not limited to, breast cancer, lung cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, colon cancer, uterine cancer, cervical cancer, ovarian cancer, urinary cancer bladder cancer, testicular cancer, prostate cancer, bone cancer and brain cancer.

The following are non-limiting examples illustrating the present invention.

EXAMPLES

EXAMPLE 1. Obtaining nanoparticles

The magnetic nanoparticles used in the described method can be easily obtained using methods known to a person skilled in the art. MNPs of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9) for use in the magnetic hyperthermia method were synthesized using the co-precipitation method in an Ar atmosphere [18]. To synthesize a sample with 50% Zn substitution, solutions of 0.5 M MnCl ₂ , ZnNO ₃ and 1 M FeCl _{3 were} mixed in a 1: 1: 2 ratio and heated to 85 ° C, stirred, then a 0.64 M NaOH solution was added as a catalyst. The mixture was heated to 95 ° C for one hour, then washed and characterized. The remaining samples with Zn substitution of 10%, 20%, 90% were obtained in a similar way. The following formula describes the specified process:

The resulting nanoparticles (x = 0.5) have a size of approximately 10 nm and correspond to a narrow peak in the size distribution diagram, and are also characterized by high monodispersity.

EXAMPLE 2. ANALYSIS OF SAMPLES USING PHOTOCORRELATION SPECTROSCOPY AND TRANSMISSION ELECTRON MICROSCOPY

The obtained samples of nanoparticles were analyzed using the following methods. Photocorrelation spectroscopy (FCC) is a method for determining the particle size in a solution using a diffusion coefficient. The Brownian motion of particles in solution and the subsequent local heterogeneity of their concentration leads to a correlation of the refractive index of a particular medium and fluctuations in the intensity of scattered light when passing through the medium. Thus, it is possible to determine the diffusion coefficient, since it is proportional to the time constant of the exponentially decaying attenuation of the intensity of the scattered light. The hydrodynamic radius of a nanoparticle is calculated using the Stokes-Einstein equation, which relates the diffusion coefficient and viscosity of a liquid to particle size.

Transmission electron microscopy (TEM) is based on the principle of transmission / reflection of an electron beam through a thin (approximately 0.1 μm) surface, followed by image enlargement. Samples were applied to the grid by diluting the solution to a very low concentration of particles. A drop (about 1-2 μl) was applied to a substrate grid (copper grid coated with formar) and dried at room temperature in the absence of magnetic field sources. To analyze the samples, a Hitachi H-7000 transmission electron microscope (100 kW, 0.5 T) was used.

The results are presented in Figures 1 and 2. Figure 1 shows images of samples with different Zn contents obtained using TEM. Figure 2 shows the size distribution of nanoparticles depending on the degree of Zn substitution.

EXAMPLE 3. METHOD OF LAYERED APPLICATION OF MNP ON POLYPROPYLENE AND POLYVINYL DIFFLUORIDE COATING

Magnetic nanoparticles for use in the described method may contain a protective shell or coating consisting of a biocompatible polymer material, which allows to increase the biocompatibility of MNPs. In some embodiments, the biocompatible polymer material is polypropylene and / or polyvinyl difluoride (PVDF). The initial method of layer-by-layer application of MNPs onto the specified coating is based on the electrostatic attraction of oppositely charged layers, which are applied sequentially. In the first stage, the surface of polypropylene and polyvinyl difluoride was covered with a layer of positively charged polyelectrolyte gel (polyallylamine hydrochloride). Then the surface was washed with distilled water. Further, negatively charged MNPs were deposited on the surface, followed by washing in distilled water. Several cycles of the described process were carried out in accordance with the required number of layers. After the last application, the surface was dried.

EXAMPLE 4. EXPERIMENTAL DEVICE

An experimental device with air cooling for an in vitro experimental MG consists of: 1) a magnetic field generation system including an energy source, a generator, a magnetic coil (solenoid) and a control circuit, 2) a temperature measurement system, including a thermocouple and a control voltmeter, 3) a system cooling, including a water cooling system of the spiral, additional cooling fans of the generator and the energy source, and 4) an external control system including a personal computer and a switching device. Figure 3 shows a diagram of a device.

The inner diameter of the working hole of the magnetic field coil is 50 mm and the height is 80 mm. The device creates a magnetic field PT with an amplitude of 100 Oe and a frequency of 100 kHz.

The magnetic field generation system consists of a power source, a generator and a magnetic coil (solenoid). The voltage of the electric network decreases as it passes through the power source and reaches a generator that creates a signal with a frequency of 100 kHz in the solenoid. This leads to the appearance of a magnetic field with a strength of 100 Oe in the working hole of the coil.

The heating rate of MNPs was measured using a temperature measuring system that detects temperature changes caused by the action of a field. The temperature measurement system consisted of a differential thermocouple and an Agilent 3441 OA voltmeter connected to the host computer via a USB port. Then, using the model equation, the thermal parameters of the material were calculated.

EXAMPLE 5. MEASUREMENT OF MAGNETIC PROPERTIES

The magnetic properties of the MNPs used in the methods of the invention were investigated using a Lake Shore 7407 magnetometer with a vibrating probe in a magnetic field with a strength of up to 16 kOe at room temperature. Figure 4 shows the results of magnetization measurements for particles of Zn _x Mn _1-x Fe ₂ O ₄ with different Zn contents [18]. It was found that magnetization is maximum for a sample with 20% Zn substitution and then decreases with increasing Zn concentration. The results of measuring the coercive force are presented in Figure 5. The degree of Zn-substitution also affects the value of coercivity. In general, magnetization and coercive force decrease with increasing degree of Zn substitution. This is due to the fact that zinc ferrite has paramagnetic properties in a nanostructured state.

EXAMPLE 6. HEATING IN A MAGNETIC FIELD FR

The heating efficiency of the MNPs of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), which can be used in the described method, was evaluated using a graph of temperature versus time for various concentrations of Zn (x) (Figure 6) . The maximum temperature change in excess of 50 ° C was observed for MNPs with 20% Zn substitution, while for samples with a higher or lower Zn content, average heating was observed. Various factors were analyzed that presumably affect the heating efficiency (size, saturation magnetization, coercivity). It was shown that the first two factors are critical for heating efficiency [18].

The Figure 7 shows the values of the specific absorption coefficient (SAR) depending on the size of the MNPs, and Table 1 summarizes the SAR values for various concentrations of Zn. The following equation was used to calculate SAR:

where C is the specific heat of water, m _p is the mass of powder, m _w is the mass of water, dT / dt is the derivative of the temperature over time at the initial linear stage of heating. SAR values were calculated for anhydrous ferritic powders and for magnetic fluids based on 3 ferritic samples [18].

As follows from Table 1 and Figures 4-6, a sample with 20% Zn heats better than others due to its maximum coercivity and magnetic saturation. The Figure 8 shows the heating efficiency of particles of Zn _0.2 Mn _0.8 Fe ₂ O ₄ in the form of a graph of temperature versus time for various values of intensity H at a frequency f = 100 kHz. The results are also summarized in Table 2.

The heating efficiency of the MNPs of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9) used in the method according to the present invention was also compared with the heating efficiency of other compositions, such as compositions of polymer coated particles of Fe ₃ O ₄ [ 20], particles of Fe ₃ O ₄ without coating [21] and La _x Sr _1-x MnO ₃ [22]. The results are presented in a graph of the temperature versus time (Figure 9) and are summarized in Table 3.

SAR values with a concomitant approximation of the linear sections of the graph were calculated in accordance with the equation:

представляет собой отношение масса суспензии/масса МНЧ.where C is the specific heat of the suspension, dT / dt is the slope of the linear portion,

represents the ratio of the mass of the suspension / mass MNP.

Table 4 shows the results of an experimental measurement of heating, which was carried out for various types of MNP in physiological saline.

Thus, it can be concluded that Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _0.2 Mn _0.8 Fe ₂ O ₄ are the most promising compositions for use in the described method of magnetic hyperthermia.

EXAMPLE 7. HEATING CANCER CELLS

K562 human leukemia cells were cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum, 2 mM L-glutamine, penicillin at a concentration of 100 U / ml and streptomycin at a concentration of 100 μg / ml at 37 ° C in the presence of 5% CO ₂ . Cells (2 × 10 ⁵ in 1 ml of this solution) were incubated in 100 μl of saline at 43 ° C in the presence of Zn-substituted magnetic nanoparticles based on manganese ferrite Zn _0.2 Mn _0.8 Fe ₂ O ₄ (10 mg) under exposure to an alternating magnetic field (100 Oe) for 60 minutes Control cells (in the same density) were incubated at 37 ° C, 5% CO ₂ in a humidified atmosphere for 60 min.

Control cells included: intact cells (not exposed to nanoparticles and magnetic fields); cells that were incubated in the presence of Zn-substituted magnetic nanoparticles based on manganese ferrite (10 mg) (without exposure to a magnetic field); cells exposed to an alternating field with a strength of 100 Oe (without nanoparticles). Both heated and control samples were resuspended in 3 ml of fresh medium and further incubated at 37 ° C, 5% CO ₂ for an additional 24 hours. Control cells grew in a logarithmic progression and remained viable without morphological signs of cytotoxicity detected under the above conditions (Figure 10a). In contrast, severe damage to cells exposed to MNP-mediated hyperthermia was detected (Figure 10b). The number of cells was significantly less; all cells looked squeezed and their contours uneven. A large number of destroyed cells was observed.

The results indicate that Zn-substituted magnetic nanoparticles based on manganese ferrite can be successfully used to inhibit the growth of cancer cells or their destruction in magnetic hyperthermia methods, to ensure high heat production and, as expected, to reduce the frequency of severe adverse events due to the low toxicity of these nanoparticles.

CITED LITERATURE

[1] Hilger I. In vivo applications of magnetic nanoparticle hyperthermia. Int J Hyperthermia 2013; 29 (8): 828-834.

[2] Gray BN, Jones SK. Targeted hysteresis hyperthermia as a method for treating diseased tissue. US6565887 (2003).

[3] Mallory M, Gogineni E, Jones GC, Greer L, Simone CB. Therapeutic hyperthermia: the old, the new, and the upcoming. Crit Rev Oncol Hematol 2016; 97: 56-64.

[4] Arruebo M, Fernandez-Pacheco R, Ibarra MR, Santamaría J. Magnetic nanoparticles for drug delivery. Nanotoday 2007; 2: 22-32.

[5] Tomitaka A, Yamada T, Takemura Y. Magnetic nanoparticle hyperthermia using pluronic-coated Fe ₃ O ₄ nanoparticles: An in vitro study. Journal of Nanomaterials 2012; 2012: 1-5.

[6] Périgo EA, Hemery G, Sandre O, Ortega D, Garaio E, Plazaola F, Teran FJ. Fundamentals and advances in magnetic hyperthermia. Applied Physic Reviews 2015; 2 (4): 1-35.

[7] Hergt R, Dutz S, Muller R, Zeisberger M. Magnetic particle hyperthermia: nanoparticle magnetism and materials development for cancer therapy. J Phys: Condens Matter 2006; 18: S2919-S2934.

[8] Hergt R, Andra W, d'Ambly CG, Hilger I, Kaiser WA, Richter U, et al. Physical limits of hyperthermia using magnetite fine particles. IEEE Transactions on Magnetics 1998; 34 (5): 3745-54.

[9] Bae ST, Chung KW. Method for preparing engineered Mg doped ferrite superparamagnetic nanoparticles exhibiting AC magnetic induction heating at high temperature and Mg doped ferrite superparamagnetic nanoparticles engineered by the method. EP2377811 (2011).

[10] Ortega D, Pankhurst QA. Magnetic hyperthermia, in Nanoscience: Volume 1: Nanostructures through Chemistry. Nanoscience 2013; 1: 60-88.

[11] Rand RW. Inductive heating process for use in causing necrosis of neoplasms at selective frequencies. US 4,983,159 (1991).

[12] Kashevskij BE, Kashevskij SB, Prokhorov IV, Istomin YP, Ulaschik VS. Method of local hyperthermia of malignant tumor. EA019412 (2014).

[13] Nat. Univ. Yokohama. Ni-Zn-based ferrite magnetic nanoparticle for thermotherapy, and drug-bound complex for thermotherapy using the same. JP 2013256405 (2013).

[14] Baldi D, Bonakki D, Innocenti F, Lorentsi D, Bitossi M, Ferruti P, Ranuchchi E, Richchi A, Komes FM. Magnetic nanoparticles to be used in hyperthermia, preparation and use it in magnetic systems for pharmacological application. RU 2481125 (2013).

[15] Tishin AM. Method for carrying out malignant neoplasm magnetic therapy. RU 2295933 (2007).

[16] Haik Y, Chen C. J. Magnetic particle composition for therapeutic hyperthermia. US 7,842,281 (2010).

[17] Lopez-Dominguez V, Hernandez JM, Tejada J, Ziolo RF. Colossal reduction in curie temperature due to finite-size effects in CoFeO nanoparticles. Chemistry of Materials 2013; 25 (1): 6-11.

[18] Elkhova TM, Yakushechkina AK, Semisalova AS, Gun'ko YK, Spichkin YI, Pyatakov AP, Kamilov KI, Perov NS, Tishin AM. Heating of Zn-substituted manganese ferrite magnetic nanoparticles in alternating magnetic field. Solid State Phenomena 2015; 233-234: 761-765.

[19] Sarwar A, Nemirovski A, Shapiro B. Optimal Halbach permanent magnet designs for maximally pulling and pushing nanoparticles. Journal of Magnetism and Magnetic Materials 2012; 324: 742-754.

[20] Dutz S, Andrä W, Hergt R, Millier R, Oestreich C, et al. Influence of dextran coating on the magnetic behavior of iron oxide nanoparticles. Journal of Magnetism and Magnetic Materials 2007; 311: 51-54.

[21] Müller R, Hergt R, Zeisberger M, Gawalek W. Preparation of magnetic nanoparticles with large specific loss power for heating applications. Journal of Magnetism and Magnetic Materials 2005; 289: 13-16.

[22] Andrade VM, Caraballo Vivas RJ, Costa-Soares T, Pedro SS, Rocco DL, Reis MS, Campos APC, Coelho AA. Magnetic and structural investigations on La _0.6 Sr _0.4 MnO ₃ nanostructured manganite: Evidence of a ferrimagnetic shell. J Sol St Chem 2014; 219: 87-92.

Claims (41)

1. A method of treating malignant neoplasms using magnetic hyperthermia, comprising the following stages: (i) delivery to the tumor region of magnetic nanoparticles in an effective concentration selected from the group of magnetic metal oxides, including Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9) whose average size is less than 40 nm, the Curie temperature is from 39 to 550 ° C, and the average value of the coercive force is from 5 to 250 Oe, (ii) exposing the tumor region to an AC magnetic field (AC) with an amplitude of from 14 to 300 Oe and a frequency of from 80 to 1000 kHz, where the exposure time of the specified magnetic field at a temperature of 38 to 51 ° C is from 15 to 60 minutes, to provide concomitant or subsequent selective damage or at least partial destruction of the tumor cells in the tumor area without damaging healthy surrounding tissue. 2. The method according to p. 1, characterized in that at stage (ii) the parameters of the electromagnetic field, including the strength and frequency, are selected depending on the type of tumor to provide damage or partial destruction of at least 80 to 90%, preferably 95 % tumor cells. 3. The method of claim 1, wherein in step (i) the magnetic nanoparticles are selected based on their characteristics, including the delivered concentration, average size, Curie temperature, average coercive force, and type of tumor to provide damage or partial destruction according to at least 80 to 90%, preferably 95% of the tumor cells. 4. The method according to p. 1, characterized in that stage (ii) includes one or more cycles. 5. The method according to p. 4, characterized in that in stage (ii) the time period and the number of exposure cycles are selected based on the type of tumor to provide damage or partial destruction of at least 80 to 90%, preferably 95% of the tumor cells. 6. The method according to p. 1, characterized in that said magnetic nanoparticles are Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _0.2 Mn _0.8 Fe ₂ O ₄ . 7. The method according to p. 1, characterized in that the average size of these magnetic nanoparticles is from 5 to 25 nm. 8. The method according to p. 1, characterized in that the said magnetic nanoparticles contain a protective shell or coating. 9. The method according to p. 8, characterized in that the protective shell or coating of magnetic nanoparticles consists of a biocompatible polymer material. 10. The method according to p. 1, characterized in that the Curie temperature of these magnetic nanoparticles is from 42 to 45 ° C. 11. The method according to p. 1, characterized in that the average value of the coercive force for these magnetic nanoparticles is from 10 to 30 E. 12. The method according to p. 1, characterized in that the said magnetic nanoparticles are delivered intravenously or into the tumor to specific parts of the body of the patient in need of said delivery. 13. The method according to p. 12, characterized in that the said magnetic nanoparticles are delivered by injection or infusion. 14. The method according to p. 1, characterized in that in stage (ii) the amplitude of the magnetic field of the PT is 100 Oe, and the frequency is 100 kHz. 15. The method according to p. 1, characterized in that the magnetic field of the PT is created using an external field source with a field gradient value from 3 to 100 T / m to accurately determine the movement and location of nanoparticles in the tumor area after the stage of delivery of magnetic nanoparticles to the tumor. 16. The method according to p. 15, characterized in that the external source of the field that creates the magnetic field of the PT is placed inside or outside the body of a patient in need of said treatment. 17. The method according to p. 1, characterized in that the method further includes the stage of localization and regulation of the concentration of nanoparticles in the tumor using an external field source based on a system of alkaline-earth permanent magnets using Halbach cylinders after the stage of delivery of magnetic nanoparticles to the tumor. 18. The method according to p. 17, characterized in that at the stage of localization and regulation of the concentration of nanoparticles, the local concentration of nanoparticles is selected depending on the characteristics of the nanoparticles, the type of tumor and magnetic field parameters. 19. The method according to p. 1, characterized in that at stage (ii) the exposure time of the tumor region to the magnetic field of the PT is from 30 to 45 minutes 20. The method according to p. 1, characterized in that the method further comprises the step of non-invasively measuring the temperature of the tumor during the stage of exposure of the tumor region to the magnetic field of the PT. 21. The method according to p. 1, characterized in that the method further comprises the step of invasively measuring the temperature of the tumor during the stage of exposure of the tumor region to the magnetic field of the PT. 22. The method according to p. 1, characterized in that the tumor area is heated to a temperature of 42 to 45 ° C. 23. The use of Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), the average size of which is less than 40 nm, the Curie temperature is from 39 to 550 ° C , and the coercive force is from 5 to 250 Oe, for the treatment of malignant neoplasms using magnetic hyperthermia. 24. The use of claim 23, wherein said magnetic nanoparticles are Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _0.2 Mn _0.8 Fe ₂ O ₄ . 25. The application of claim 23, wherein the average size of said magnetic nanoparticles is from 5 to 25 nm. 26. The application of claim 23, wherein said magnetic nanoparticles have a protective shell or coating. 27. The application of claim 23, wherein said protective shell or coating of magnetic nanoparticles consists of a biocompatible polymer material. 28. The application of claim 23, wherein the Curie temperature of said magnetic nanoparticles is from 42 to 45 ° C. 29. The application of claim 23, wherein the average value of the coercive force for these magnetic nanoparticles is from 10 to 30 E. 30. The application of claim 23, wherein said magnetic nanoparticles are administered intravenously or into the tumor into specific areas of the body of a patient in need of said administration. 31. A pharmaceutical composition for treating malignant neoplasms using magnetic hyperthermia, containing Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), the average size of which is less than 40 nm, the Curie temperature is from 39 to 550 ° C, and the coercive force is from 5 to 250 Oe, and a pharmaceutically acceptable carrier. 32. The pharmaceutical composition according to p. 31, characterized in that the said magnetic nanoparticles are Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _0.2 Mn _0.8 Fe ₂ O ₄ . 33. The pharmaceutical composition according to p. 31, characterized in that the composition further comprises a biologically active molecule. 34. The pharmaceutical composition according to p. 33, characterized in that the biologically active molecule is selected from an anticancer agent that recognizes tumor cells of an antibody or aptamer. 35. The pharmaceutical composition according to paragraphs. 31-34, characterized in that the composition may be presented in the form of a solution for infusion, a solution for injection, powder or lyophilisate. 36. A method for experimental damage to tumor cells using magnetic hyperthermia, comprising the following stages: (i) providing magnetic nanoparticles selected from the group of magnetic metal oxides, including Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), the average size of which is less than 40 nm, the Curie temperature is from 39 to 550 ° C, and the average value of the coercive force is from 5 to 250 Oe, (ii) adding said magnetic nanoparticles to the cell culture; (iii) exposing the indicated cell culture to a magnetic field of PT with an amplitude of from 14 O to 300 O and a frequency of from 80 to 1000 kHz, where the exposure time of the specified magnetic field at a temperature of 38 to 51 ° C is from 15 to 60 minutes, to ensure concomitant or subsequent damage or at least partial destruction of tumor cells.

Images