Injectable suspension or emulsion of coated magnetite nanoparticles for use in the treatment of solid tumors

Abstract

The present invention relates to an injectable suspension or emulsion of magnetite nanoparticles coated with an organic layer, which can be used for the treatment of solid tumors.

Description

[Technical Field]

[0001] The present invention relates to an injection suspension or emulsion of magnetite nanoparticles coated with an organic layer, which can be used in a method for treating solid tumors. [Background technology]

[0002] Hyperthermia is one of the therapies proposed in oncology for the treatment of tumors. Simply put, the principle of this therapy is to induce apoptosis in tumor cells by heating them to a temperature range of approximately 41°C to 45°C; this therapy is particularly effective against cancer cells because cancer cells are more susceptible to temperature increases than healthy tissue, which has an effective cooling system due to vascularization. Furthermore, the vascular system of tumors is insufficient to ensure adequate oxygen and nutrient supply under thermal stress conditions. Hyperthermia can be induced by radio waves, microwaves, ultrasound, and other forms of energy.

[0003] Although this principle is conceptually simple, it has not been widely implemented in treatments or protocols to date due to limitations, side effects, and practical difficulties. The main obstacle to the application of this method is the need to heat the tissue with high selectivity in order to raise the temperature of tumor cells to an effective range without affecting the surrounding healthy tissue.

[0004] Due to these difficulties, this method has been applied only to a limited number of cases and only to local treatment. The first situation in which hyperthermia is used is when the tumor to be treated is close to the body surface; in this case, heating is directly generated in the tumor from an external device. The second application of hyperthermia is to insert a thin needle-like probe into the tumor for a short time, usually about 10 to 30 minutes; the tip of the probe emits high-frequency radiation, generating extremely high temperatures that destroy cells in the surrounding area.

[0005] Pure hyperthermia treatment cannot be applied to broader areas of the body, i.e., to a part of the body or the whole body. This method is used only as an adjunct to other major treatments such as chemotherapy or radiotherapy; in these cases, slight heating of the tissue is applied to kill tumor cells that have been weakened by the main treatment.

[0006] To overcome these difficulties, it has been proposed to use magnetic particles (generally nanoparticles) to reach the tumor site and generate heat through radio frequency irradiation.

[0007] Much research has focused on the fabrication and characterization of magnetic particles with controllable and reproducible properties; see, for example, the review “Iron oxide based MR contrast agents: from chemistry to cell labeling” (S. Laurent et al., Current Medicinal Chemistry, 2009, 16, 4712-4727); the article “Hydrothermal preparation and characterization of ultrafine magnetite powders” (Q. Yitai et al., Materials Research Bulletin, Vol.29(9), 1994, 953-957); the article “Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools” (R. Hergt et al., Journal of Magnetism and Magnetic Materials 293 (2005) 80-86); and patent application WO2011 / 073922 A1.

[0008] While these studies demonstrate the potential of using magnetic (nano) particles in diagnostic and therapeutic applications, the reported characterizations have been performed only on the particles themselves, or at best, in vivo; in particular, it has not been shown how actual therapeutic hyperthermia can be performed in vivo. Especially, practical details on how to selectively deliver particles to tumor sites have not been provided.

[0009] Patent application WO 2015 / 104664 A1 discloses a method for delivering magnetite nanoparticles to a tumor site. This method involves fabricating structures in which multiple magnetite nanoparticles are encapsulated within a biocompatible polymer shell; extracting immune system cells from a patient; allowing these cells to take up the structures; and reintroducing the cells thus taken up into the body to deliver them to the tumor site. Although this method is effective in principle, it is difficult to standardize and therefore unsuitable for widespread application because it requires extracting and reinjecting cells from a specific patient each time.

[0010] Therefore, a first object of the present invention is to provide a suspension or emulsion of magnetite nanoparticles coated with an organic layer that is ready for use in hyperthermia therapy, and a method for using the suspension or emulsion in the treatment of solid tumors. [Overview of the project]

[0011] According to the present invention, a suspension or emulsion of magnetite nanoparticles coated with an organic layer is provided, which is selected from the following: A) A water suspension containing a surfactant and one or more of the following: a buffer system and a stabilizer; and B) An aqueous lipid suspension or emulsion containing one or more components selected from egg phospholipids, medium-chain triglycerides, long-chain triglycerides, glycerol, linoleic acid, cholesterol, and sodium oleate. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the time course of average tumor volume in mice transplanted with IGROV1-luc cells (human ovarian cancer cell line), comparing mice treated according to the present invention with mice that were not treated according to the present invention. [Figure 2] This figure shows the time course of average tumor volume in mice transplanted with DETROIT-562 cells (subcutaneous pharyngeal cancer cell line), comparing mice treated according to the present invention with mice that were not treated according to the present invention. [Modes for carrying out the invention]

[0013] In the remainder of this specification, the following terms and abbreviations are used with the meanings set forth below: “room temperature” means a temperature in the range of 18 to 30°C; "NPs" stands for magnetite nanoparticles; For simplicity, unless otherwise specified, the suspensions or emulsions of the present invention shall be collectively referred to as "injectable compositions"; "Medium-chain triglycerides" refer to triglycerides obtained from C6-C12 fatty acids, while "long-chain triglycerides" refer to triglycerides obtained from C14-C60 fatty acids.

[0014] A first aspect of the present invention relates to a suspension or emulsion of magnetite (Fe3O4) NPs coated with an organic layer as described above.

[0015] The NPs before being coated with the organic layer have a size of 1 to 100 nm, preferably 10 to 60 nm, and more preferably 20 to 30 nm. These NPs can be produced by methods known in the art, such as those described in patent application WO2011 / 073922 A1. Briefly, this method is as follows: i) Attack an iron metal with an organic acid and treat it in a polyol-based solvent, such as glycerol, at a temperature of 130 to 200°C, and Fe 2+ Forming an ionic solution; ii) By bubbling an oxidizing gas (air) into the solution at a temperature below 100°C, Fe 2+ Ions Fe 3+ Oxidation into ions and filtration removal of residual metallic iron; iii) Water / Fe in the presence of mineral acid 3+ Treat with water so that the molar ratio of ions is 1.5 to 5. In this method, the size of NPs is water / Fe 3+ The process is controlled by the molar ratio of ions, and by operating within the aforementioned molar ratio range, it is guaranteed that particles within the desired size range can be obtained.

[0016] The NPs thus obtained are then coated with a layer of organic compound. In the coating process, the surface of the magnetite NPs is first functionalized with a catechol derivative having a lipophilic moiety on the phenyl ring; the two hydroxyl groups present in the catechol molecule form polar heads that electrostatically bond to the NP surface, while the lipophilic moiety allows the assembly to be fitted with a polymer that forms a shell around the NPs. Useful polymers for forming a shell around catechol-coated NPs include chitosan, sodium alginate, polymethyl methacrylate, and polysaccharides. A preferred polymer for producing the shell is a block copolymer of poly(lactic acid / glycolic acid)-polyethylene glycol carboxylate; for a detailed description of this copolymer and a method for coating NPs with the copolymer, see patent application WO2015 / 104664 A1.

[0017] The coated NPs thus obtained usually have an average particle size in the range of 35 - 60 nm and an average polydispersity index of 0.10 - 0.15, indicating a narrow particle size distribution.

[0018] The coated NPs obtained as described above are subsequently suspended by suspension means to prepare either the suspension or emulsion A) or B) of the present invention.

[0019] These injectable compositions must of course be characterized by a series of properties suitable for the intended use; in particular, - In order to avoid problems in case of leakage from the tumor mass to the surrounding healthy tissue, they must be sterile and free of pyrogenic substances; - For essentially the same reason, the injectable composition must have an osmotic concentration that is isotonic with body fluids. The preparation of isotonic compositions is within the knowledge of those skilled in the art and can be obtained, for example, by setting the concentration of NaCl to 0.9% by weight; - The pH of the injectable composition must be in the range of 8.0 ± 0.2, i.e., 7.8 - 8.2; the inventors have found that the polymer shell surrounding the NPs becomes unstable at different pH values; - Finally, in order to obtain an effective hyperthermia effect and to avoid affecting healthy tissues located at a medium distance from the injection site, the amount of NPs administered must be controlled; the inventors have found that these results are obtained with injectable compositions in which the concentration of NPs is in the range of 10 - 30 mM, preferably 15 - 30 mM. At concentrations higher than this range, excessive temperature increases occur, which can be harmful to healthy cells and tissues close to the treatment area; on the other hand, at concentrations lower than this range, an excessive injection volume is required to reach a useful amount of NPs, and if the amount of the injected suspension is too large, it may cause accidental rupture of tumor cells, and as a result, substances may leak out and be carried throughout the body.

[0020] In the first embodiment, the injectable composition of the present invention is an aqueous suspension A) containing NPs and a surfactant at concentrations within the range described above. At least one additional component selected from buffers and stabilizers is added to this basic composition.

[0021] Surfactants have the function of preventing the sedimentation of NPs and improving the stability of the suspension. Surfactants useful for the purposes of the present invention include sorbitan esters of fatty acids, commercially known under the trade name SPAN® (SPAN® 20: sorbitol monolaurate, SPAN® 40: sorbitan monopalmitate, SPAN® 60: sorbitan monostearate, SPAN® 65: sorbitan tristearate, and other grades sold under the trade names SPAN® 30, SPAN® 80, and SPAN® 85); polysorbate fatty acid esters, commercially available under the trade name TWEEN® (available in grades 20, 21, 40, 60, 65, 80, and 85); sorbitol and sorbitol esters, cyclodextrins, particularly β-cyclodextrin, sulfoalkyl ether cyclodextrin derivatives known as Captisol®; linoleic acid; and polyethylene glycol (PEG polyethylene Examples include glycol, particularly grades marketed under the name macrogol (e.g., macrogol 3350, macrogol 4000, and macrogol 6000). This component, if present, is added to suspension A) at concentrations ranging from 1% to 25% based on various selected excipients.

[0022] Buffer systems have the function of maintaining the pH of the suspension within the range of 8.0 ± 0.2. This range is suitable for injection into the body. Another function of buffers is to avoid degradation of magnetite NPs and the organic layer coating the surface of the NPs, achieved through pH stabilization. Useful buffer systems for maintaining the pH of the suspension within this range include, for example, phosphate buffer obtained from a Na2HPO4 / NaH2PO4 salt pair, citrate buffer obtained by mixing citrate and trisodium citrate (C6H8O7·H2O / Na3C6H5O7·H2O), histidine buffer, or so-called "universal buffers" that can adjust the pH over a wide range depending on the component ratio, such as SSC (sodium chloride-sodium citrate) buffer obtained from sodium chloride and trisodium citrate, with the pH adjusted with hydrochloric acid, or McIlvaine buffer obtained by mixing appropriate amounts of Na2HPO4 solution and citrate solution. The preparation of these buffer solutions is well known in the field and falls within the scope of the general technical knowledge of a chemist.

[0023] Finally, the stabilizer in injectable composition A) is a stabilizer having various functions or contributions, for example, Antioxidants, such as ascorbic acid at a concentration of 0.001 to 0.005% by weight (w / w); Chelating agents include, for example, EDTA at a concentration of 0.025-0.075% w / w, cyclodextrin (such as Captisol®) at a concentration of 1-15% w / w, or PEG (0.1-10% w / w); Preservatives include, for example, ethyl alcohol (10-15% w / w), benzyl alcohol (0.10-0.15% w / w), and sodium benzoate (0.10-0.15% w / w); That is the case.

[0024] Examples of aqueous suspensions useful for the purposes of the present invention are as follows. i) A suspension containing 3 mg of magnetite NPs; 2 g of disodium edetate (EDTA); 5 g of polysorbate 80 (polyoxyethylene (20) sorbitan monooleate, sold as TWEEN® 80); 9 mg of NaCl; a phosphate buffer in an amount such that the pH of the suspension is in the range of 8.0 ± 0.2; and water to bring the volume of the suspension to 1000 mL; ii) A suspension containing 3 mg of magnetite; 9 mg of NaCl; 2 mg of polyethylene glycol with an average molecular weight of 300 Da (commercially available as PEG Macrogol 300); an amount of phosphate buffer such that the pH of the suspension is in the range of 8.0 ± 0.2; and water to bring the volume of the suspension to 1000 mL; and iii) A suspension comprising 3 mg of magnetite; 9 mg of NaCl; 1 mg of polysorbate 80 (TWEEN® 80); a phosphate buffer in an amount such that the pH of the suspension is in the range of 8.0 ± 0.2; and water to make the volume of the suspension 1000 mL.

[0025] In a second embodiment, the injectable composition of the present invention is a lipid suspension or emulsion B), the main component of the liquid phase being oleic acid, linoleic acid, stearic acid, polyethylene glycol, or a mixture thereof, to which one or more components selected from medium-chain triglycerides, long-chain triglycerides, glycerol, and sodium oleate can be added in an amount ranging from 0.1 to 10% by weight (w / w).

[0026] An example of the lipid composition of the present invention is as follows. iv) A suspension containing 3 mg of magnetite and 1 g of linoleic acid.

[0027] In a preferred embodiment, the injectable composition of the present invention may be provided in kit form (kit-of-parts), as follows: - A first container containing the liquid phases of each composition A) and B), i.e., water in composition A) and oleic acid in composition B); - One or more containers containing a predetermined amount of freeze-dried powder of surface-coated magnetite NPs, and at least one component selected from surfactants, excipients, salts or compounds that form a buffer system, and stabilizers; Includes.

[0028] The surfactant, buffering system components, and stabilizers used in preparing the kit of the present invention are the same as those described above with respect to composition A). The excipient may be selected from mannitol, lactose, sucrose, and glycine.

[0029] Providing the injectable composition of the present invention in kit form offers the advantage of completely avoiding the effects of deterioration over time of liquid compositions, such as sedimentation of solid components and chemical interactions between components. Subsequently, the liquid composition can be reconstituted by simply mixing the contents of the various containers in the kit, usually with gentle stirring, at the time of use.

[0030] The composition of the present invention is preferably stored at a temperature below room temperature, and more preferably in the range of 2 to 8°C.

[0031] The compositions of the present invention are useful for the treatment of solid tumors, particularly head and neck cancers, which is carried out by directly injecting one of the aforementioned compositions into the tumor mass and then irradiating the injection site with radiofrequency radiation.

[0032] Head and neck cancer is the most suitable form of cancer to be treated by the method of the present invention because the tumor masses located in these regions are relatively close to the body surface and can be easily reached with the tip of an injection needle.

[0033] The injectable composition of the present invention is administered percutaneously by direct injection into the tumor mass through a needle; the injection volume of the above composition is in the range of 20 μL to 1 mL, and the tumor volume is 100 to 500 mm². 3In this case, the volume is preferably about 50 to 200 μL. For tumors exceeding this volume, it is recommended to administer multiple injections to different sites on the tumor mass, ensuring that each injection volume remains within the above range.

[0034] Subsequent irradiation is typically performed at radio frequencies (RF) in the range of 10 to 400, with a magnetic field strength H0 of 20 to 25 kA / m, for 20 to 60 minutes, preferably 30 ± 5 minutes.

[0035] Regarding the treatment schedule, the inventors found that the best results in terms of suppressing tumor growth are obtained with a 7-cycle injection / irradiation program, in which one irradiation event is performed after the injection in each cycle, with intervals of 1 to 7 days between cycles. [Examples]

[0036] The features and advantages of the present invention will become clearer from the following embodiments.

[0037] Experimental apparatus, method, and experimental conditions The following studies were conducted in accordance with good laboratory practice principles using 5-7 week old female athymoid nude mice provided by Envigo RMS Srl, San Pietro al Natisone, Udine, Italy. The animals were labeled with indelible ink, randomly assigned based on tumor volume and clinical observations, and euthanized at the end of the study. All animal experimental procedures (feeding, health monitoring, restraint, administration, etc.) and ethical reviews were carried out in accordance with Italian Legislative Decree No. 26 of March 4, 2014, which implements Directive 2010 / 63 / EU (Directive on the protection of animals used in biomedical research).

[0038] RF irradiation was performed using a NovaStar 5W device manufactured by Ameritherm Inc. in Scottsville, New York, USA.

[0039] MS analysis was performed using an Applied Biosystems / Sciex triple quadrupole mass spectrometer equipped with an Agilent Technologies 1100 HPLC pump and a CTC PAL autosampler.

[0040] Example 1 This embodiment relates to the preparation of a suspension of coated magnetite NPs.

[0041] Magnetite nanoparticles (NPs) were prepared according to the polyol synthesis route described in patent application WO 2011 / 073922 A1; after preparation, the NPs were first surface-functionalized with the organic ligand N-(3,4-dihydroxyphenethyl)-dodecanamide (DDA dodecanamide) and then dispersed in tetrahydrofuran (THF tetrahydrofuran). The organically coated magnetite nanoparticles were then encapsulated in a polylactic acid-coglycolic acid-copolyethylene glycol (PLGA-b-PEG-COOH) matrix of the polymer described in patent application WO 2011 / 073922 A1. This process resulted in the formation of a stable phosphate-buffered suspension of the coated nanoparticles according to a nanoprecipitation method using a THF:water ratio of 1:10. The resulting suspension was dialyzed in a tangential flow membrane to remove organic solvents, impurities, and unreacted reagents, and then concentrated to an Fe3O4 concentration of 0.3% (w / w) (150 mL); finally, 2 mg / mL of EDTA and 5 mg / mL of TWEEN 80® were added to the suspension.

[0042] The coated NPs in the suspension were then fully characterized.

[0043] The average particle size and average polydispersity index of the coated NPs were measured by dynamic light scattering (DLS) and found to be 50.8 ± 0.6 nm and 0.11 ± 0.01, respectively, indicating a narrow particle size distribution. Transmission electron micrographs (TEM) of the coated NPs showed clusters of a small number of inorganic particles uniformly dispersed within the polymer matrix, with an average diameter of 50 nm. The zeta potential showed a negative surface charge of -43.0 ± 0.2 mV, which is thought to be due to the presence of multiple carboxylic acid groups on the outer surface. The iron content was measured by inductively coupled plasma atomic emission spectroscopy and confirmed to be 2.7 mg / mL.

[0044] The resulting suspension was dispensed into syringes for use in subsequent biological tests.

[0045] Example 2 This embodiment relates to the suppression of ovarian cancer proliferation achieved by the method of the present invention.

[0046] 32 female athymoid nude mice were given 5 × 10 in 200 μL of culture medium. 6 IGROV1-luc cells (a human ovarian cancer cell line) were injected subcutaneously into the left abdomen on day 0. IGROV1-luc cells are commercially available from companies such as Creative Biolabs, BPS Biosciences, or Affimetrix.

[0047] Animals were monitored daily for tumor development, mortality, and clinical symptoms. Toxicity was assessed based on signs of physiological or behavioral distress (e.g., weight loss of 20% or more), paralysis, hypotonia, tremors, bleeding from any orifice, respiratory distress, decreased activity, neurological symptoms, and kyphosis. No unexpected deaths occurred during the study period.

[0048] Tumor progression was evaluated by caliper measurement. Tumor volume and body weight were evaluated twice a week. Tumor volume was calculated according to the following formula 1. Tumor volume (mm 3 ) = major diameter (Length) (mm) × minor diameter 2 (Width 2 )(mm 2 ) × 0.5 (1)

[0049] The tumor growth inhibition rate (%T.G.I.: Tumor Growth Inhibition) was calculated according to the following formula 2. %T.G.I. = 100 - (Mtt / Mtc × 100) (2) Here, Mtt = average tumor volume of the treatment group; Mtc = average tumor volume of the control group.

[0050] When the tumor reached an average volume of 482 mm 3 at the time point (day 8), the mice were randomized and assigned to the experimental groups with a target of 8 mice per group.

[0051] On day 9, 50 μL of the suspension of Example 1 was injected once into the tumors of the mice in the second to fourth groups. The mice in the first group (control) were not injected with the NPs suspension.

[0052] The radiotherapy of the mice in the third and fourth groups was started 1 hour after the intratumoral injection of the NPs suspension. The mice were anesthetized by inhaling isoflurane in a mouse chamber equipped with active exhaust. The parameters of the radiation device were frequency = 360 kHz and magnetic field strength (H0) = 21 kA / m.

[0053] The mice in the second group were not irradiated with radiation. The mice in the third group received a single 40-minute radiation treatment, and the mice in the fourth group received 40-minute radiation treatments continuously for 5 days at 24-hour intervals.

[0054] The treatment conditions of the high-frequency irradiation are summarized in Table 1 below. [Table 1] All mice were euthanized when they reached their respective humane endpoint criteria, and no macroscopic internal lesions were observed during macroscopic autopsy.

[0055] Volume (cm³) per day for all mice in the 4 groups 3 These are shown in Tables 2 to 5 below, corresponding to Group 1 to Group 4 mice, respectively. [Table 2] [Table 3] [Table 4] [Table 5]

[0056] Table 6 shows the average tumor volume (cm³) for each group. 3 The standard error (SE) and standard deviation (SD) are shown as functions of the number of test days. [Table 6]

[0057] Average tumor volume (mm) as a function of the number of days of the trial 3 The increase ± SE is also shown in graph form in Figure 1. The numbers in the figure represent the group (1-4) that the data refers to; the data is shown along with the significance level.

[0058] Table 7 shows the tumor growth inhibition rate (%TGI) values ​​calculated from the data in Table 6. [Table 7]

[0059] From day 15 onward, all mice in this study exhibited mild weight loss and mild erythema around the tumor. The weight loss curve showed a decreasing trend from around day 15, but the localized erythema showed no signs of recovery until the end of the study.

[0060] The control mice (Group 1) showed an exponential increase in tumor burden.

[0061] The mice in group 2 did not show any significant difference compared to the mice in group 1.

[0062] The mice in group 3, which received a single dose of radiation, did not show any significant difference compared to the mice in group 1.

[0063] The mice in group 4 showed a significant increase in antitumor effect, reaching 43.0% on day 18.

[0064] No toxic effects were observed in any of the mice studied, confirming that injection of the coated NPs according to the present invention does not cause harmful side effects.

[0065] Example 3 This embodiment relates to the suppression of subcutaneous pharyngeal carcinoma growth achieved by the method of the present invention.

[0066] 49 female athymoid nude mice were given 2 × 10 in 200 μL of culture medium. 6 One DETROIT-562 cell line (subcutaneous pharyngeal cancer cell line) was injected subcutaneously into the left abdomen on day 0.

[0067] Monitoring of the animals during the first few days of cancer growth followed the same general procedure detailed in Example 2. No unexpected deaths occurred during the study period.

[0068] The tumor had an average volume of 113 mm². 3At the point when they reached this stage (day 7), the mice were randomly assigned to seven experimental groups, with a target of seven mice per group.

[0069] From day 8, mice from groups 2 through 7 were injected multiple times into the tumor with different solutions or suspensions according to two different schedules: - Once a week x 4 times (q7dx4); and, - Every 4 days x 7 times (q4dx7).

[0070] Specifically, groups 2 and 3 were injected multiple times with 50 μL of buffer solution (phosphate-buffered saline, PBS); groups 4 through 7 were injected multiple times with 50 μL of the suspension from Example 1. Subsequently, only the mice in groups 6 and 7 were anesthetized and subjected to RF irradiation under the same conditions as shown in Example 2.

[0071] Group 6 mice received intratumoral therapy with q7dx4 NPs; after each treatment, they received three consecutive 40-minute radiation doses at 24-hour intervals.

[0072] Group 7 mice received intratumoral therapy with q4dx7 NPs; a single dose of radiation therapy was administered after each treatment.

[0073] The mice in Group 1 (control group) did not receive any injections.

[0074] The injection / irradiation conditions are summarized in Table 8 below. [Table 8]

[0075] Table 9 shows the average tumor volume (cm³) for each group. 3 The standard error (SE) and standard deviation (SD) are summarized as functions of the number of test days.

[0076] Average tumor volume (mm) as a function of the number of days of the trial 3The increase ± SE is also shown in graph form in Figure 2. In the figure, the graphs for groups 1 through 5 are almost overlapping, but the graphs for groups 6 and 7 are identified by their corresponding numbers in the figure; the data are shown along with the significance level. [Table 9]

[0077] Table 10 shows the tumor growth inhibition rate (%TGI) values ​​calculated from the data in Table 9. [Table 10]

[0078] Compared to the control group (Group 1), neither treatment with PBS (Groups 2 and 3) nor treatment with coated NPs without radiation (Groups 4 and 5) showed a significant antitumor effect.

[0079] On the other hand, intratumoral injection of coated NPs followed by multiple radiation treatments actually enhanced the antitumor effect. Progressive delay in tumor growth was observed up to day 24, at which point significant suppression of tumor growth of up to 37.2% (p<0.01) and 42.8% (p<0.01), respectively, was observed in groups 6 and 7. The Mann-Whitney Test revealed that the treatment effects of groups 6 and 7 were significantly greater than those of group 1 (control group). No significant antitumor effect was observed in the other groups.

[0080] No weight loss or side effects were observed.

[0081] Analysis of the results The data obtained from the series of tests in Examples 2 and 3 demonstrate that the method of the present invention, consisting of intratumoral injection of a composition containing magnetite NPs followed by RF irradiation, is capable of significantly suppressing the growth and progression of cancer.

[0082] One advantage of this method is that the surrounding tissue of the treated tumor mass remains essentially unaffected by the treatment, allowing it to be repeated multiple times as needed; this differs from known hyperthermia treatments achieved with single RF irradiation, which employ much higher frequency ranges, causing changes in areas throughout the body and therefore cannot be repeated over time.

Claims

1. An injectable composition for percutaneous administration to solid tumors by direct injection into the tumor mass, comprising a suspension or emulsion of magnetite nanoparticles coated with an organic layer, selected from the following: A) A water suspension containing a surfactant and one or more of the following: a buffer system and a stabilizer; and B) Aqueous lipid suspension or emulsion containing one or more components selected from egg phospholipids, medium-chain triglycerides, long-chain triglycerides, glycerol, linoleic acid, cholesterol, and sodium oleate. The injectable composition contains the following properties: - It must be sterile and free of pyrogenic substances; - It is isotonic with body fluids; - The pH of the injectable composition shall be 7.8 to 8.2; - The concentration of magnetite nanoparticles in the injectable composition shall be in the range of 10 to 30 mM.

2. The injectable composition according to claim 1, wherein the magnetite nanoparticles have a size of 1 to 100 nm, preferably 10 to 60 nm, and more preferably 20 to 30 nm.

3. The injectable composition according to claim 1 or 2, wherein the surface of the magnetite nanoparticles is first functionalized with a catechol derivative to form an aggregate surrounded by a polymer shell, the polymer being selected from chitosan, sodium alginate, polymethyl methacrylate, polysaccharides, block copolymers of poly(lactic acid / glycolic acid) and carboxylated polyethylene glycol, and combinations thereof.

4. The injectable composition according to any one of claims 1 to 3, wherein the surfactant in suspension A) is selected from sorbitan esters of fatty acids, polysorbate fatty acid esters, sorbitol, sorbitol esters, cyclodextrin, sulfoalkyl ether cyclodextrin derivatives, linoleic acid, polyethylene glycol, and mixtures thereof, and when the surfactant is present in suspension A), it is added to suspension A) at a concentration of 1 to 25% by weight.

5. The injectable composition according to any one of claims 1 to 4, wherein the buffer system in suspension A) is selected from the following: Sodium salt Na ２ HPO ４ / NaH ２ PO ４ Phosphate buffer formed by the salt pair; C ６ H ８ O ７ ·H ２ O / Na ３ C ６ H ５ O ７ ·H ２ O Citrate buffer formed by citric acid and trisodium citrate; Histidine buffer; Physiological saline-citrate buffer formed by sodium chloride, trisodium citrate and hydrochloric acid; and Na ２ HPO ４ McIlvaine buffer formed by the solution and citric acid solution.

6. The injectable composition according to any one of claims 1 to 5, wherein the stabilizer in suspension A) is at least one of the following: - Antioxidant at a concentration of 0.001–0.005% by weight; - A chelating agent selected from ethylenediaminetetraacetic acid (EDTA) at a concentration of 0.025–0.075% by weight, cyclodextrin at a concentration of 1–15% by weight, polyethylene glycol (PEG) at a concentration of 0.1–10% by weight, and mixtures thereof; - A preservative selected from ethyl alcohol at a concentration of 10–15% by weight, benzyl alcohol at a concentration of 0.10–0.15% by weight, sodium benzoate at a concentration of 0.10–0.15% by weight, and mixtures thereof.

7. The injectable composition according to any one of claims 1 to 3, wherein in the suspension or emulsion B), the component is added to the composition at a concentration of 0.1 to 10% by weight.

8. An injectable composition in kit form comprising the following, the injectable composition according to any one of claims 1 to 7: - A first container containing either composition A) or composition B), i.e., composition A) contains water, and composition B) contains one or more of oleic acid, linoleic acid, stearic acid, and polyethylene glycol; - One or more containers comprising a predetermined amount of freeze-dried powder of surface-coated magnetite nanoparticles, and at least one component selected from surfactants, excipients, salts or compounds that form a buffer system, and stabilizers.

9. The kit-type injectable composition according to claim 8, wherein the excipient is selected from mannitol, lactose, sucrose, glycine, and mixtures thereof.

10. 100-500mm ３ An injectable composition according to any one of claims 1 to 9, administered in a range of 20 μL to 1 mL relative to the tumor volume.

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