POLYMER NANOCAPSULES SUITABLE FOR USE IN THERAPY, PARTICULARLY ANTICANCER THERAPY - Patent application

Abstract

Magnetic polymer nanocapsules, which are core-shell polymer nanocapsules containing magnetic iron oxide nanoparticles and carrying hydrophobic active agents, and their use in anti-cancer therapy are disclosed.

Description

[Technical Field]

[0001] The present invention relates to magnetic polymer nanocapsules and their use in anti-cancer therapy, the nanocapsules being core-shell polymer nanocapsules containing iron oxide nanoparticles carrying a hydrophobic active agent. [Background technology]

[0002] Core-shell magnetic nanocapsules containing iron oxide nanoparticles (wüstite-maghemite) are known from a research paper by E. Gumieniczek-Chopek, J. Odrobinska, T. Straczek, A. Radziszewska, S. Zapotoczny, and C. Kapusta, entitled "Hydrophobically Coated Superparamagnetic Iron Oxides Nanoparticles Incorporated into Polymer-Based Nanocapsules Dispersed in Water," Materials 2020, 13, 1219. Due to the use of biocompatible components, such systems can be tested in biological environments for magnetically controlled delivery of active hydrophobic substances. Polish Patent Specification PL 229276 B1 discloses a biocompatible polysaccharide based on a liquid oil core with a diameter of 1 μm or less, stabilized without the need for low-molecular surfactants, characterized by effective encapsulation of hydrophobic compounds and high stability in aqueous suspensions.

[0003] Polish Patent Application No. P.425141 disclosed nanocapsules containing a liquid oil core made of oleic acid and a stabilizing shell made of hydrophobically modified hyaluronic acid. The document further encompasses the use of the subject oil-core nanocapsules in anti-cancer therapy, particularly in breast cancer or melanoma.

[0004] U.S. Patent Application Publication No. 2016199308A1 discloses a magnetic capsule having an aqueous nanocore and a drug delivery shell containing multiple amphiphilic proteins (where the amphiphilic proteins have hydrophilic and hydrophobic chain ends), multiple iron oxide nanoparticles (Fe3O4), and hydrophilic and hydrophobic drugs. The shelled magnetic nanocore capsules of the present invention have high drug loading capacity and the ability to simultaneously encapsulate hydrophobic and hydrophilic drugs, and therefore can be used for targeted drug delivery, magnetic resonance imaging, and hyperthermia.

[0005] Many scientific groups are focusing their research on finding solutions that allow the body to deliver active substances in a controlled manner, while maintaining therapeutic concentrations and avoiding side effects associated with damage to healthy cells. Among the various systems dedicated to this type of application, a large group of systems made of polymeric materials should be distinguished. One example of such a carrier is a capsule based on a liquid oil core. The method for creating such systems involves the emulsification method, which consists in suspending oil droplets in an aqueous solution of appropriate pH. Due to their thermodynamic instability, the dispersed oil droplets that form the capsule core require the use of appropriate stabilizers. For biomedical applications, it is appropriate to abandon the use of low-molecular-weight surfactants for compound stabilization and instead use appropriately grafted biopolymers, for example, in the form of modified hyaluronic acid or chitosan. Due to their amphiphilic nature, compounds used in an aqueous environment are positioned with their hydrophilic backbone on the surface of the oil droplets, while hydrophobic side groups penetrate the interior of the carrier, forming a closed structure of the oil core / polymer shell type. Furthermore, the presence of charges on the backbone of the compounds used to stabilize the carriers allows for control of the surface charge of the entire system by using alternating oppositely charged layers of polyelectrolytes (LbL, layer-by-layer).

[0006] The systems obtained in this way exhibit high stability, suitable size, and the ability to encapsulate hydrophobic compounds [J. Szafraniec et al., Nanoscale, 2015, 7, 5525-5536]. To better target the delivery and release process of the encapsulated substances in the carriers shown above, it is possible to enhance the magnetic properties of these systems by incorporating magnetic nanoparticles into their structure. For biomedical applications, iron oxide nanoparticles are used for this purpose, and their hydrophobic coating allows the nanostructures to be placed in the oil core of the carrier. The extremely important strong magnetic properties, which allow for control of the entire system, are achieved by the superparamagnetic properties of the nanoparticles used [J. Odrobi-ska et al., ACS Appl. Mater. Interfaces 2019, 11, 10905-10913]. Among the products available on the medical market, there are several systems using magnetic nanoparticles, but at present, none of them are intended for magnetically navigated, targeted, and controlled delivery of active substances [DD Stueber et al., Pharmaceuticals 2021, 13, 943]. Highly stable nanocapsules based on a liquid oil core stabilized with appropriately modified biomolecules are well known in the literature, and magnetically controlled systems designed for targeted and controlled delivery of active substances have also been presented. However, the combination of these two solutions used in the present invention is not available.

[0007] Although many methods using magnetic nanoparticles and polymer nanocapsules can be found in the current state of the art, the development and implementation of effective and efficient anticancer therapies remains a challenge for modern science. The lack of polymer nanocapsules carrying active substances with high stability and magnetic properties for use in treatment, especially breast cancer treatment, is a major problem in anticancer therapy. Furthermore, the low efficiency of hydrophobic active substance encapsulation results in an insufficient concentration of active substance in the polymer capsules released into cancer cells corresponding to a therapeutic dose. Another problem is the inability to control (selective targeting / navigation) polymer nanocapsules carrying active substances to malignant cells, especially breast cancer cells, using static magnetic fields, resulting in the scarcity of targeted therapies. Another problem is the impossibility of releasing hydrophobic active substances in cancer cells using alternating magnetic fields. Furthermore, the side effects of currently used therapeutic drugs in anticancer therapy cannot be ignored, while widely used chemotherapy drugs have significant negative effects on the human body. In the previously known solutions using polymer capsules, the relatively large dimensions of the core-shell system hinder the penetration of the polymer containing the active substance into cancer cells, and the frequent occurrence of cytotoxicity of the polymer capsules towards normal cells in the body cannot be completely eliminated.Another problem is the inability to implement targeted and controlled anti-cancer therapy using magnetic polymer capsules carrying active substances. Summary of the Invention [Means for solving the problem]

[0008] The objective of the present invention was to provide polymeric nanocapsules for carrying active substances, characterized by extremely high stability and magnetic properties, as well as high encapsulation efficiency for hydrophobic active substances, which allow for therapeutic concentrations of the active substance to be encapsulated and released into cancer cells. Another objective was to utilize a static magnetic field to deliver the active substance to cancer cells, particularly breast cancer cells, and an alternating magnetic field to enable navigation (selective targeting) of the polymeric capsules, providing the possibility of releasing the hydrophobic active substance in cancer cells. Another objective of the present invention was to completely eliminate the side effects of magnetic polymeric nanocapsules, which are conventionally used in anticancer therapy. Another objective of the present invention was to obtain a core-shell system (capsule) for transporting active substances with extremely small size (nanometer size) that would easily penetrate cancer cells and not be cytotoxic to normal cells in the body.

[0009] Another objective of the present invention was to develop efficient and effective targeted anti-cancer therapies with the possibility of fully controlling the release of active substances using magnetic polymer capsules to carry these substances.

[0010] Surprisingly, all of the above technical problems have been solved by the present invention. The subject of the present invention is nanocapsules and their medical uses, as defined in the appended claims. Nanocapsules produced according to the present invention can pass through magnetically assisted cell membrane barriers without damaging the structure of the carrier (nanocapsule). In the course of research leading to the present invention, it was found that a negative surface charge on the capsules is necessary for them to effectively pass through the cell membrane and enter the cell, and for the capsule contents to be released throughout the cell volume; see Figure 25 (positively charged capsules) and Figure 27 (negatively charged capsules). Positively charged capsules either become denser around the cell membrane or remain within the cell membrane, rather than passing through.

[0011] At the same time, application of an alternating magnetic field of appropriate strength has been confirmed to rapidly and completely release the contents of nanocapsules produced according to the present invention into cells. The present invention utilizes magnetically controlled polymer capsules based on a liquid oil core, whose structure contains magnetic iron oxide nanoparticles and the active substance to be transported. The carriers shown are spherical core-shell systems with dimensions on the order of several hundred nanometers. These systems exhibit high stability, enable effective encapsulation of hydrophobic active substances, and allow magnetically controlled (static magnetic field) delivery and release of the transport cargo (under the action of an alternating magnetic field). The carriers acquire magnetic properties by placing spherical nanoparticles in the hydrophobic environment of the core, have dimensions of less than 30 nanometers, and are composed of iron oxide and an outer lipophilic coating. Due to their surface made of biocompatible polysaccharides, the polymer capsules are not cytotoxic and can be absorbed by cells, and their contents can be released into cells under the action of an alternating magnetic field. The subject matter of the present invention in exemplary embodiments is shown in the following figures. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the temperature conditions as a function of time during the thermal reaction of iron(III) oleate decomposition. [Figure 2] 1 shows STEM images of nanoparticles obtained by pyrolysis of metalorganic precursors. [Figure 3] FT-IR spectra of magnetic nanoparticles and oleic acid are shown. [Figure 4] 1 shows the X-ray diffraction patterns of magnetic nanoparticles, microcrystalline magnetite and wustite, characterized by Miller indices. [Figure 5]Figure 1 shows the spectra of magnetic nanoparticles obtained by Mössbauer spectroscopy (black dots indicate measurements): a) Measurement performed at room temperature with fitted wüstite sextet (blue line), magnetite sextet (blue and green lines) and total fit (red line); b) Measurement of magnetic nanoparticles at room temperature 8 months after their synthesis; c) Measurement at liquid nitrogen temperature with wüstite content (blue dots) and exclusion of wüstite phase (red dots); d) Microcrystalline wüstite spectrum measured at liquid nitrogen temperature; e) Microcrystalline maghemite spectrum measured at liquid nitrogen temperature. [Figure 6] 1 shows the magnetic susceptibility of magnetic nanoparticles as a function of temperature measured in a magnetic field of 100 Oe. [Figure 7] Magnetization curves of magnetic nanoparticles as a function of applied magnetic field measured over the temperature range 4-300K are shown. [Figure 8] 1 shows the change in hydrodynamic diameter and zeta potential of magnetic cationic capsules measured over a 48 week period. [Figure 9] 1 shows cryo-TEM images of oil-core based magnetic cationic capsules. [Figure 10] FIG. 1 is a schematic diagram of an oil-core based anionic capsule with encapsulated magnetic nanoparticles. [Figure 11] 1 shows the change in hydrodynamic diameter and zeta potential of magnetic anionic capsules measured over a 48 week period. [Figure 12] Images of magnetic capsules taken with a confocal microscope: A) Cationic capsules (image taken on the day the capsules were manufactured); B) Cationic capsules (image taken two weeks after storage of the capsules); C) Anionic capsules (image taken on the day the capsules were manufactured); D) Anionic capsules (image taken two weeks after storage of the capsules). Scale: 1 μm. [Figure 13] 1 shows the magnetization of cationic magnetic capsules as a function of applied magnetic field at a temperature of 300K. [Figure 14]1 shows the layout of a 24-well plate created to determine the toxicity of capsules to 4T1 cell line cells. [Figure 15] 1 is a graph of cell viability (determined by XTT assay) of 4T1 cell line cells in contact with cationic magnetic capsules at various concentrations. [Figure 16] 1 is a graph of cell viability (determined by XTT assay) of 4T1 cell line cells in contact with anionic magnetic capsules at various concentrations. [Figure 17] Schematic diagram of an experiment in which cells are pulled by a static magnetic field. [Figure 18] Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRIC filter) fluorescent dyes after removal from the incubator and subjected to 5 and 15 minutes of static magnetic field exposure are shown. Scale: 20 μm. [Figure 19] Confocal microscopy images of cells subjected to an experiment in which fluorescently labeled magnetic cationic capsules were introduced using an external static magnetic field. Left column: transmitted light and TRITC filter; right column: TRITC filter. Scale: 20 μm. [Figure 20] Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRICT filter) fluorescent dyes after an experiment in which magnetic cationic capsules were introduced using an external static magnetic field are shown. Scale: 20 μm. [Figure 21] Confocal microscopy images of cells subjected to an experiment in which fluorescently labeled magnetic anionic capsules were introduced using an external static magnetic field. Left column: transmitted light and TRITC filter; right column: TRITC filter. Scale: 20 μm. [Figure 22] Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRIC filter) fluorescent dyes after an experiment in which magnetic anionic capsules were introduced using an external static magnetic field. Scale: 20 μm. [Figure 23]1 shows a schematic diagram of an experiment for the release of encapsulated substances within cells using an external AC magnetic field. [Figure 24] Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRIC filter) fluorescent dyes, subjected to 15 min of static magnetic field exposure followed by external AC magnetic field exposure, are shown. Scale: 20 μm. [Figure 25] Confocal microscopy images of cells subjected to a 15-minute external static magnetic field exposure followed by an associated alternating magnetic field exposure to test for the introduction and release of substances encapsulated in fluorescently labeled magnetic cationic capsules. Left column: transmitted light and TRITC filter; right column: TRITC filter. Scale: 20 μm. [Figure 26] Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRIC filter) fluorescent dyes were subjected to a 15-minute external static magnetic field exposure followed by an associated alternating magnetic field exposure to test for the introduction and release of substances encapsulated within magnetic cationic capsules. Scale: 20 μm. [Figure 27] Confocal microscopy images of cells subjected to an experiment to introduce and release substances encapsulated in fluorescently labeled magnetic anionic capsules using a 15-minute external static magnetic field exposure followed by an associated alternating magnetic field exposure. Left column: transmitted light and TRITC filter; right column: TRITC filter. Scale: 20 μm. [Figure 28] Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRIC filter) fluorescent dyes subjected to a 15-minute external static magnetic field exposure followed by an associated alternating magnetic field exposure to test for the introduction and release of substances encapsulated in magnetic anionic capsules. Scale: 20 μm. DETAILED DESCRIPTION OF THE INVENTION

[0013] Example 1. High temperature synthesis of iron oxide nanoparticles with hydrophobic coating. The magnetic nanoparticles placed in the capsule structures are believed to impart magnetic properties to the entire carrier. These nanostructures were obtained by a two-stage synthesis involving the pyrolysis of pre-synthesized organometallic precursors in the presence of oleic acid. This method makes it possible to obtain nanoparticles of narrow size distribution composed of iron oxide and possessing strong magnetic properties. The presence of a hydrophobic substance during synthesis provides an adequate coating for the structures, allowing them to be placed in the oil core of the capsule.

[0014] The synthesis of iron(III) oleate was carried out based on the procedure previously described by Leszczyski et al. [Leszczyski, B. et al. The influence of oxidation process on exchange bias in egg-shaped FeO / Fe3O4 core / shell nanoparticles. J Magn Magn Mater 416, 269-274 (2016)]. To obtain the organometallic precursor, 30 mL of deionized water, 40 mL of ethanol, 70 mL of hexane, 3.25 g of anhydrous iron(III) chloride, and 18.25 g of sodium oleate were mixed together. The resulting solution was heated to 60 °C and maintained under constant stirring for 5 h. The dark hydrophobic fraction was then separated and removed, rinsed with deionized water, and heated to 40 °C to evaporate the remaining hexane. The synthesis leading directly to the formation of nanoparticles was carried out according to the procedure previously described by Park et al. [Park J. et al. Ultra-large-scale syntheses of monodisperse nanocrystals Nat Mater 3, 891-895 (2004)]. First, 42 mL of octadecane, 1.1 mL of oleic acid, and 7 g of pre-prepared iron(III) oleate were mixed. The entire reaction was carried out under inert gas conditions with constant stirring and under the temperature regime shown in the following diagram (Figure 1). The resulting product was cooled to room temperature and washed several times with ethanol. Unreacted substrate was removed by sonication in hexane (continuously, 5 min) and centrifugation (5,000 rpm, 5 min). The resulting nanoparticles were dried under vacuum.

[0015] Physicochemical properties of nanoparticles Imaging with a scanning transmission electron microscope (STEM) (Figure 2) confirmed the spherical shape of the individual nanoparticles, and analysis of the resulting images indicated an average size of the structures of 15 nm, with a narrow distribution of this parameter.

[0016] The choice of synthesis method for magnetic nanoparticles was strictly conditioned by the possibility of obtaining structures with an appropriate coating, allowing the particles to be placed in the hydrophobic interior of the capsule. To determine the proper adhesion of the oleic acid layer on the nanoparticle surface, Fourier transform infrared spectroscopy (FT-IR) measurements and TGA-DSC thermal analysis were carried out. The obtained spectrum of the nanoparticles was compared with the spectrum from the measurement of the oleic acid used during synthesis (Figure 3). For oleic acid, the peak at 3000 cm -1 The signal that appears at 2922 cm is the vibration of stretching the CH bond of the -C=CH group. -1 and 2853 cm -1 The band appearing at 1710 cm corresponds to the asymmetric and symmetric vibrations of the -CH group stretching. -1 The strong band at 1560 cm can be attributed to the vibration of the C=O bond. The spectrum measured on the magnetic nanoparticles shows the disappearance of the band arising from the C=O bond, whereas the band at 1560 cm corresponds to the asymmetric and symmetric stretching vibrations in the -COO- group. -1 and 1645cm -1Two new bands appear at wavelengths equal to 0.015 nm. The difference in the positions of these bands indicates the chelation of iron ions with the oleic acid -COO- groups [Premaratne, W., Priyadarshana, W., Gunawardena, S., De Alwis, A. Synthesis of Nanosilica from Paddy Husk Ash and Their Surface Functionalization. J. Sci. Univ. Kelaniya Sri Lanka 8, 33-48 (2013); Zhang, L., He, R., Gu, HClOleic Acid Coating on the Monodisperse Magnetite Nanoparticles. Appl. Surf. Sci. 253, 2611-2617 (2006); Bronstein, L. M. et al. Influence of Iron Oleate Complex Structure on Iron Oxide Nanoparticle Formation. Chem. Mater. 19, 3624-3632 (2007)].

[0017] A crystal structure study of the obtained nanoparticles was performed based on powder X-ray diffraction (XRD) measurements. The obtained diffractograms were compared with those of microcrystalline magnetite and wüstite (Figure 4). The lines assigned to the magnetite phase (e.g., 2θ ≈ 30°) show no shift from the diffraction pattern of microcrystalline magnetite. However, in the case of wüstite, a maximum shift toward higher angles relative to the reference line can be observed. This shift suggests a reduction in the crystal lattice parameters due to compressive strain in the nanoparticle structure resulting from the core / shell structure. Given the fact that wüstite (FeO) under ambient conditions and at slightly elevated temperatures undergoes rapid oxidation to the magnetite (FeO) phase, its oxidized forms—maghemite (γ-FeO) and hematite (α-FeO), or a two-step disproportionation process leading to the formation of hematite and magnetite, we can conclude that the core of the nanoparticles is wüstite, while the shell is composed of the magnetite or maghemite phase. The strain generated in the nanoparticle structure can lead to an increase in the magnetocrystalline anisotropy. Based on Scherrer's equation, it was concluded that the average crystallite size of the magnetite / maghemite phase is 4.4 nm, and that of the wüstite phase is 6 nm. Considering the nanoparticle structure (core-shell) and using the determined crystallite size, the average size of the magnetic nanoparticles can be determined to be 14.8 nm.The performed fitting of the phases to the obtained diffractogram showed a 22% occupancy of the wüstite phase and a 77% occupancy of the magnetite / maghemite phase [Cornell, RM, Schwetmann, U. The Iron Oxides: Structure, Properties, Reactions, Occurrences and Uses. 2nd ed. WILEY-VCH GmbH & Co. KGaA, 139-183 (2003); Pichon, BP et al. Microstructural and Magnetic Investigations of Wuestite-Spinel Core-Shell Cubic-Shaped Nanoparticles. Chem. Mater. 23, 2886-2900 (2011); Sun, X., Frey Huls, N., Sigdel, A., Sun, S. Tuning Exchange Bias in Core / Shell FeO / Fe3O4 Nanoparticles. Nano Lett.12,246-251(2012);Scherrer,P.

[0018] Mössbauer spectroscopy studies of magnetic nanoparticles were performed under various temperature conditions (room temperature, liquid nitrogen temperature, and liquid helium temperature). The obtained spectra were compared with those of microcrystalline wüstite and microcrystalline maghemite (Figure 5). The spectrum measured at room temperature indicates the presence of a magnetically ordered phase through the presence of a broad sextet, while the singlet with an isomer shift of approximately 1 mm / sec suggests the presence of a second, but paramagnetic, phase. The spectra shown have a relaxational character, as indicated by the strong broadening of the inner lines and the lower amplitude of the outer lines than the theoretical 3:2:1 to 1:2:3 ratio, implying that the nanoparticles exhibit superparamagnetism at room temperature. The fluctuation frequency of the magnetization vector between the easy axes is 57 The relaxation spectrum occurs when the frequency is higher than the reciprocal of the Fe Mössbauer lifetime, i.e., 7 MHz. Fitting the obtained spectrum to the Blume and Tijon model yielded the set of parameters shown in Table 1. The determined isomer shift values indicate that the paramagnetic phase, represented as a singlet in the spectrum, is wüstite. On the other hand, in the spectrum measured at liquid nitrogen temperature, the singlet arising from the paramagnetic phase is absent, and the appearance of additional magnetically split constituent lines indicates the presence of a magnetically ordered phase. The observed lines are quite narrow, and the intensity ratio is close to the theoretical 3:2:1 to 1:2:3 ratio, which means that at this temperature, the nanoparticle magnetization vectors cannot overcome the energy barrier, and consequently, superparamagnetic fluctuations are not observed. After subtracting the 22% occupancy of the wüstite phase from the obtained spectrum, the iron(II) oxide phase present in the studied sample was determined to be maghemite.

[0019] The fluctuation frequency of 52 MHz determined based on the relaxation spectrum at room temperature indicates a relatively high superparamagnetic blocking temperature in the nanoparticles. Meanwhile, the high value of the asymmetry coefficient (ρ = 0.95), which corresponds to the relative remanence time of the nanoparticles' magnetic moment along the easy axis, implies a high magnetocrystalline anisotropy of the resulting system [Armstrong, RJ, Morrish, AH. Moessbauer Study Of Ferric Ions In The Tetrahedral And Octahedral Sites Of A Spinel. Phys. Lett. 23, 10, 414-416 (1966); Morup, S. Moessbauer Effect in Small Iron Particles. Hyperfine Interact. 60, 959-974 (1990); Redl, F. X. et al. Magnetic, Electronic, and Structural Characterization of Nonstoichiometric Iron Oxides at the Nanoscale. J. Am. Chem. Soc. 126, 14583-14599 (2004); Tucek, J., Zboril, R., Petridis, D. Maghemite Nanoparticles by View of Moessbauer Spectroscopy. J. Nanosci. Nanotechnol. 6, 926-947 (2006)].

[0020] [Table 1]

[0021] Magnetic nanoparticles were analyzed using a vibrating sample magnetometer (VSM). The temperature dependence of the magnetic susceptibility was measured in the form of ZFC (zero-field cooling) and FC (field cooling) curves (Figure 6) at a static magnetic field of 100 Oe, as well as in the form of magnetization as a function of the applied magnetic field for temperatures ranging from 4 to 300 K (Figure 7). The temperature at which the magnetic susceptibility value significantly increases (230 K) corresponds to the transition of wüstite from the antiferromagnetic to the paramagnetic state, i.e., the Néel temperature. Its literature value for wüstite is approximately 192 K, but it can be increased by the presence of an additional phase in the form of ferrimagnetic maghemite around the wüstite nanoparticle core. From the maximum of the ZFC curve, it is possible to determine the average blocking temperature, i.e., the temperature above which the magnetic moment of the nanoparticles exhibits superparamagnetic fluctuations. The blocking temperature, determined based on the measured function, is 275 K. The shape of the magnetization curve above this temperature indicates the disappearance of hysteresis, which confirms the superparamagnetic state of the nanoparticles. During the measurements, the magnetization did not reach saturation, which is related to the presence of wüstite in the nanoparticle structure. The determined values of the coercivity field and remanence are shown in Table 2. Below the Néel temperature, unidirectional magnetic anisotropy appears, showing a shift in the hysteresis loop as a result of exchange bias coupling at the boundary between the antiferromagnetic wüstite core and the ferrimagnetic maghemite shell. This coupling occurs due to the presence of an antiferromagnetic phase, which is more difficult to remagnetize and depends on the core size, shell thickness, and shape of the nanoparticles [Pichon, B.P. et al. Microstructural and Magnetic Investigations of Wuestite-Spinel Core-Shell Cubic-Shaped Nanoparticles. Chem. Mater. 23, 2886-2900 (2011); Estrader, M. et al. Origin of the Large Dispersion of Magnetic Properties in Nanostructured Oxides: FexO / Fe3O4 nanoparticles as a Case Study. Nanoscale 7, 3002-3015 (2015)].

[0022] [Table 2]

[0023] Example 2. Preparation of liquid core-based cationic capsules with encapsulated magnetic nanoparticles. Capsules were obtained by an emulsification process, and hydrophobically and cationically modified chitosan derivatives, anionic chitosan derivatives, and magnetic nanoparticles obtained by thermal decomposition of organometallic precursors were used for their preparation.

[0024] The procedure for preparing liquid oil-core capsules based on the self-assembly of amphiphilic graft polymers was previously described by Szafraniec et al. [Szafraniec, J. et al. Chitosan-based nanocapsules of core-shell architecture. Polimery 62, 509-515 (2017); Szafraniec, J., Janik, M., Odrobi-ska, J., Zapotoczny, S. Nanocapsules templated on liquid cores stabilized by graft amphiphilic polyelectrolytes. Nanoscale 7, 5525-5536 (2015)]. For the encapsulation of magnetic nanoparticles, the procedure was modified. Thus, 10 μL of oleic acid containing 100 mg / mL of magnetic nanoparticles was added to 1 mL of 0.15 M sodium chloride (NaCl) solution containing 1 mg of cationically and hydrophobically modified chitosan (ChitC12). CChitC12 was obtained according to a previously described procedure (Karewicz A., Bielska D., Loboda A. et al.: Colloids and Surfaces B: Biointerfaces 2013, 109, 307. http: / / dx.doi.org / 10.1016 / j.colsurfb.2013.03.059). Diagram below, m:n:p=67.5:2:30.5. [ka] The resulting solution was subjected to vigorous mixing on a shaker, and then to impulse sonication (1 second on, 2 seconds off mode, room temperature) to precisely deposit a polymer layer on the surface of the oil capsule core. Unencapsulated magnetic nanoparticles were removed by magnetic separation. The resulting emulsion showed an off-white color.

[0025] Physicochemical properties of cationic capsules Dynamic light scattering (DLS) techniques were used to determine hydrodynamic diameter and zeta potential values. Measurements were repeated for 48 weeks, during which time the test samples were stored at 4°C. The hydrodynamic diameter and zeta potential values of capsules with positive surface charges remained at the same level throughout the study period (Figure 8). The average hydrodynamic diameter values of these systems were neither greater than 170 nm nor less than 140 nm, and the polydispersity index was less than 0.3, thereby demonstrating a relatively narrow size distribution. Meanwhile, the zeta potential values remained within the range of 30–40 mV throughout the entire measurement period.

[0026] The cationic magnetic capsules were imaged using cryo-transmission electron microscopy (cryoTEM) (Figure 9). The images obtained confirm the presence of magnetic nanoparticles within the carriers, and also reveal the spherical shape of the carriers, with sizes consistent with the DLS measurements.

[0027] Example 3. Preparation of liquid core-based anionic capsules with encapsulated magnetic nanoparticles. The second type of capsule studied was a system with a negative surface charge (Figure 10). Anionic capsules were produced based on the technique of applying oppositely charged layers. To prepare such carriers, 0.6 mL of a 1 g / L anionic chitosan solution (A Chit, prepared according to the procedure described above: Bulwan M., Zapotoczny S., Nowakowska M.: Soft Matter 2009, 5, 4726. http: / / dx.doi.org / 10.1039 / B909355A and diagram below, sulfonic acid group substitution degree 49 mol%) dissolved in 0.0015 M sodium chloride was added to 0.4 mL of the cationic capsule dispersion. The resulting solution was subjected to vigorous mixing on a shaker for 10 minutes. [ka]

[0028] Physicochemical properties of anionic capsules The hydrodynamic diameter and zeta potential measurements performed on the anionic capsules are shown in Figure 11. The measured mean hydrodynamic diameters ranged from 140 to 230 nm, with a polydispersity index of less than 0.4. The larger size distribution may be related to the formation of small amounts of aggregates or to a non-uniform coating of the capsules with anionic chitosan (AChit). The zeta potential values ranged from -35 to -45 mV. Both the higher mean hydrodynamic diameters and the more negative zeta potential values compared to the cationic capsules indicate a proper deposition of the anionic chitosan layer on the surface of the analyzed support.

[0029] Example 4. Preparation of liquid fluorescent core-based capsules with encapsulated magnetic nanoparticles. For the purpose of confocal microscopy imaging, fluorescent modification of the capsules to be studied was necessary. The manufacturing procedure for both cationic and anionic capsules was extended by a step involving the addition of a fluorescent dye to the oil suspension of magnetic nanoparticles that would later form the carrier core. In the case of capsules enriched with perylene dye, 1 mg of dye was added to 1 mL of oil mixture containing dispersed magnetic nanoparticles.

[0030] Imaging of capsules further containing a hydrophobic fluorescent dye in the form of perylene in the oil core was performed by confocal microscopy (Figure 12). The image shown shows the blue fluorescent emission of the labeled oil capsule core. The images obtained confirm the possibility of placing hydrophobic substances, especially hydrophobic active substances, within the magnetic capsule core, and the images obtained two weeks after capsule production confirm the feasibility of longer-term encapsulation of substances that are soluble in a hydrophobic environment.

[0031] Example 5. Preparation of concentrated dispersions of liquid core-based cationic capsules with encapsulated magnetic nanoparticles. To verify the magnetic properties of the capsules, vibrating sample magnetometer (VSM) measurements were performed. To obtain an adequate signal-to-noise ratio, it was necessary to prepare capsule suspensions containing increased concentrations of magnetic nanoparticles, and therefore increased amounts of carrier suspended in solution. Sample preparation was performed following a procedure similar to that for cationic capsule formation, using 30 μL of oleic acid with dispersed magnetic nanoparticles at a concentration of 100 g / L and 1 mL of chitosan derivative (CChitC12) at a concentration of 10 g / L in 0.15 M NaCl solution.

[0032] The curves obtained from magnetometer measurements confirm the magnetic nature of the carriers, and the absence of a hysteresis loop is associated with superparamagnetic nanoparticles in the capsule core (Figure 13).

[0033] Example 6. Experimental studies involving cell lines. In vitro studies were performed using the 4T1 cell line, a cancer cell line derived from mouse mammary gland tissue. Cultures were maintained at 37°C under sterile conditions in the presence of 5% CO2. Cell culture was performed using medium supplemented with antibiotics in the form of penicillin and streptomycin solution (1%) and fetal bovine serum (5%). All reagents used in cell studies were warmed in a 37°C water bath. In the first step, cells were thawed for culture, and the cell suspension was transferred to a centrifuge tube. 5 mL of medium was added and diluted with dimethyl sulfoxide (DMSO). The cells were then centrifuged (1000 rpm, 5 min), the solution covering the cell pellet was decanted, and the cells were resuspended in 1 mL of medium. The cells thus prepared were seeded into culture dishes pre-filled with 10 mL of medium. Cell growth and status during culture were monitored under an optical microscope, and the medium was changed every two days. Trypsinization was performed for subculture. The dish on which the cells were spread was rinsed twice with PBS solution. Next, 0.8 mL of trypsin solution was added, removed, and 0.8 mL of trypsin was added again. The treated dish was placed in an incubator, and after 3 minutes, it was completely removed and placed back in the incubator. After another 3 minutes, the dish was removed again, and the degree of cell detachment from the substrate was monitored under a microscope. After adding 3 mL of medium to inactivate the trypsin, the detached cells were transferred from the dish to a Falcon tube. The resulting cell suspension was centrifuged (1000 rpm, 5 minutes), the liquid was poured off, and the pelleted cells were suspended in 1 mL of medium. This resulted in a cell suspension, which was used in subsequent experiments.

[0034] Example 7. Cytotoxicity study of capsules against 4T1 line cancer cells (XTT). To determine the cytotoxicity of cationic and anionic capsules against the 4T1 cell line, an XTT assay was performed. This assay allows for the determination of cellular mitochondrial metabolic activity based on the spectrophotometric measurement of the reduction of XTT tetrazole salt to formazan due to mitochondrial dehydrogenase activity. The day before the direct determination of capsule toxicity against the selected cell line, two 24-well plates containing the 4T1 cell line and appropriate dispersion concentrations of cationic capsules (Plate 1) and anionic capsules (Plate 2) were prepared. The plates were prepared by adding 0.9 mL of medium to each well, followed by 100 μL of cell suspension. Each well was thoroughly mixed by pipetting and shaking the plate several times. The inoculated plate was placed in an incubator for 4 hours. The next step was the addition of the agent in the form of capsules. For this, the liquid was aspirated from each well of the plate, and 1 mL of medium was added. 0.1 mL of agent was then added to each well, capsules diluted in 0.15 M NaCl (for cationic capsules) or 0.0015 M NaCl (for anionic capsules), and 0.15 M NaCl (cationic capsules) or 0.0015 M NaCl (anionic capsules) solution to reach the concentrations shown in Figure 14, and the resulting solution was mixed in the well by pipetting. The prepared plates were placed in an incubator for 24 hours.

[0035] Prior to the experiment, the reagents required for the XTT assay were prepared. According to the manufacturer's instructions, the reagents were preheated to 37°C, and 5 mL of XTT labeling reagent and 0.1 mL of coupling reagent were mixed. Before adding the solution, the plate prepared the previous day was removed from the incubator, the liquid was removed with a pipette, and 0.2 mL of fresh medium followed by 0.1 mL of XTT solution was added to each well. The plate was gently mixed and placed back in the incubator. After 1 hour, spectrophotometric measurements of absorbance were performed. The results, shown graphically in Figures 15 and 16, clearly demonstrate the relationship between the decreasing concentration of the agent and the subsequent increase in cell viability. Based on the obtained values, it can be concluded that capsules with a negative surface charge are less toxic to cells. However, in both the anionic and cationic capsules, a 2% agent concentration did not induce a negative response in the assayed cells.

[0036] Example 8. Directing capsules to cancer cells using a static magnetic field. The feasibility of magnetic control of capsules containing magnetic nanoparticles was verified by conducting experiments using a static non-uniform magnetic field applied to a cuvette filled with capsules suspended in culture medium and a microscope slide containing live cells on its surface. To eliminate the effect of gravity on the penetration of capsules into the cells, the slide with the cell culture was placed in a vertical position.

[0037] Before starting the main experiment, slide cultures had to be prepared; for this purpose, appropriately cut sterile microscope cover glasses were used and placed in 6-well cell culture plates. The cell culture slides were then placed on the side walls of sterile cuvettes, into which 1 mL of medium and 0.1 mL of a 2% capsule dispersion in 0.15 M NaCl (for cationic capsules) and 0.0015 M NaCl (for anionic capsules) were poured.

[0038] The cuvettes thus prepared were subjected to an external static magnetic field (5 and 15 minutes) according to the scheme shown in Figure 17. The distance between the sample and the magnet was 1 cm, which corresponds to a magnetic induction of 143 mT. Experiments were also carried out to verify the spontaneous penetration of the capsules into the cells and the effect of the external static magnetic field on the condition of the cells.

[0039] After the experiment was completed, the cells on the slides were fixed for subsequent imaging using a confocal microscope. For fixation, the cell slides were transferred to a 6-well plate and washed twice with 2 mL of PBS solution. Then, 2 mL of a 10% formalin solution in PBS was added to each well. After 10 minutes, the solution was removed, the slides were rinsed with PBS solution, and placed on glass microscope slides with the edges protected with varnish.

[0040] Capsules further enriched with a fluorescent probe in the form of Nile Red dye were used in the experiments. The procedure for preparing such capsules consisted in adding the dye at a concentration of 5 mg / mL to an oil suspension of magnetic nanoparticles before starting to add it to a solution of chitosan (CChitC12) dissolved in water.

[0041] Cell viability was also verified after the external magnetic field experiments. To this end, two solutions were prepared: 10 mg of Hoechst dissolved in 1 mL of deionized water and diluted with PBS at a volume ratio of 1:2000; and 1 mg of propidium iodide dissolved in 1 mL of deionized water and diluted with PBS at a volume ratio of 1:3000. The cells on the slides fixed after the external magnetic field experiments were then incubated with the respective dyes for 5 minutes, rinsed three times with PBS, placed on glass microscope slides, and protected with varnish. The specimens thus obtained were imaged using a confocal microscope.

[0042] Example 9. Verification of the effect of a static magnetic field on the state of cells for cationic and anionic capsules. In the first phase of the study, the effect of an external static magnetic field on cancer cells of the 4T1 cell line was evaluated in the absence of a carrier. The images collected (Figure 18) clearly ruled out any negative effects of the static magnetic field, as evidenced by the lack of fluorescent signal in cells stained with propidium iodide, which penetrates only damaged cell membranes.

[0043] Next, a study was conducted to verify the possibility of using an external static magnetic field to navigate cationic capsules to cancer cells. The images shown (Figure 19) indicate a slight tendency for the cationic capsules to spontaneously penetrate, and the use of an external static magnetic field increases the effectiveness of the studied phenomenon, primarily contributing to the accumulation of capsules in the vicinity of cancer cells.

[0044] The effectiveness of the cationic capsule permeation process assisted by an external static magnetic field was verified by fluorescent staining of the cells after the experiment. The obtained images (Figure 20) confirm that 15 minutes of exposure to a static magnetic field in the presence of magnetic cationic capsules significantly contributes to the disruption of cell membrane integrity.

[0045] Similar experiments of carrier navigation using an external magnetic field were performed for anionic capsules. The images obtained (Figure 21) show the possibility of spontaneous penetration of capsules with negative surface charges into cells by endocytosis, but the use of an external static magnetic field increases the effectiveness of the penetration process.

[0046] Fluorescent labeling of cells exposed to an external static magnetic field in the presence of anionic capsules and without an external magnetic field was performed and is shown in Figure 22. The obtained images confirm the increased tendency of spontaneous penetration of the anionic capsules into the cells. The visible bright red light emission after application of the external static magnetic field indicates significant disruption of the integrity of the cell membrane, confirming the successful introduction of the carrier into the cancer cells.

[0047] Example 10. Alternating magnetic field assisted release of encapsulated substances from capsules placed within cancer cells. The possibility of releasing the substance encapsulated in the carrier was evaluated by applying an alternating magnetic field. The use of an external alternating magnetic field was carried out as a continuation of the static magnetic field. After 15 minutes of exposure to the static magnetic field, the above-mentioned system was transferred for 5 minutes to an external alternating magnetic field with a frequency of 50 Hz and various induction values as shown in Table 3.

[0048] [Table 3]

[0049] A so-called blank experiment was also performed on cells not in contact with the capsule to rule out any harmful effects of the AC magnetic field. A diagram of the experimental procedure is shown in Figure 23. After the experiment was completed, the slides with the cells were fixed according to the previously described protocol. Hoechst dye and propidium iodide were also used to verify cell viability after the experiment, similar to the verification described above.

[0050] Example 11. Verification of the effect of an AC magnetic field on the state of cells. Confocal microscopy images are shown in Figure 24, demonstrating the deleterious effects of alternating magnetic fields on cancer cells. The images confirm the maintenance of cell membrane integrity after exposure to an external alternating magnetic field, as indicated by the absence of fluorescence in cells stained with propidium iodide. Figure 25 shows the experimental results of 15-minute exposure of fluorescently labeled cationic capsules and cancer cells to an external static magnetic field and 5-minute exposure to an alternating magnetic field with various parameters. The images show that, in the case of cationic capsules, application of a strong alternating magnetic field contributes to significant permeability of the cell membrane barrier but does not lead to effective release of the encapsulated material within the cells. The disruption of cell membrane integrity is confirmed by images of fluorescently stained cells after an experiment involving exposure of cells to external static and alternating magnetic fields in the presence of cationic capsules (Figure 26). The images demonstrate the possibility of introducing capsules into cells with damaged cell membranes, which may contribute to their death.

[0051] The possibility of targeted and magnetically controlled release of encapsulated substances within cells was also verified using capsules with negative surface charges. The images shown (Figure 27) were obtained after 15 minutes of exposure of cells surrounded by anionic capsules containing fluorescent dyes to a static magnetic field and after 5 minutes of exposure to each of the alternating magnetic fields. The results obtained with samples exposed to a strong alternating magnetic field confirm the possibility of effective release of encapsulated substances within cancer cells.

[0052] The results of cell membrane evaluation after experiments aimed at magnetically controlled introduction and release of substances carried within anionic capsules are shown in Figure 28. Bright red light emission confirms significant damage to the cell membrane, especially when a strong alternating magnetic field is applied. These damages affect all cells in the image that were subjected to the alternating magnetic field, ultimately inducing cancer cell death.

[0053] List of reagents used: 1) Chitosan (molar mass: 50-190 kDa, Sigma-Aldrich), 2) glycidyltrimethylammonium chloride (GTMAC, ≥90%, Sigma-Aldrich); 3) N-dodecyl aldehyde (92%, Sigma-Aldrich); 4) Sodium cyanoborohydride (NaCNBH3, 95%, Igma-Aldrich), 5) Carboxymethyl chitosan (CMC, acetylation degree 90%, AK Scientific), 6) sulfur trioxide-trimethylamine complex (TMST, 99%, Sigma-Aldrich); 7) Sodium bicarbonate (NaHCO3, pa, Sigma-Aldrich), 8) Sodium hydroxide (NaOH, pa, Avantor Performance Materials Poland SA), 9) Iron(III) chloride (FeCl3, anhydrous, Merck), 10) Sodium oleate (>97%, TCI), 11) Octadecane (90%, Alfa Aesar), 12) Oleic acid (OA, 99.5%, Alfa Aesar), 13) Perylene (Pe, Gold Label, 99.9%, Sigma-Aldrich), 14) Nile Red (NR, for microscopy, Sigma-Aldrich); 15) n-octadecane (pa, Polyscience Corp.), 16) Dulbecco's modified Eagle's medium (DMEM, high glucose, Sigma-Aldrich); 17) fetal bovine serum (FBS, HyClone); 18) Penicillin-streptomycin solution (Symbios), 19) Trypsin (HyClone), 20) Mixture of salts for preparing phosphate-buffered saline (PBS, tablets, Sigma-Aldrich), 21) Dimethyl sulfoxide (DMSO, ≥99.7%, Sigma-Aldrich); 22) XTT (Cell Proliferation Kit II, Sigma-Aldrich), 23) Formalin (36.5-38% aqueous solution, Sigma-Aldrich), 24) Hoechst (Sigma-Aldrich), 25) Propidium iodide (PI, Sigma-Aldrich), 26) Acetic acid (99.5%, Chempur); 27) Sodium chloride (NaCl, pa, Lachner), 28) Ethanol (96%, Chempur), 29) Acetone (pa, Chempur), 30) Methanol (pa, Chempur), 31) Hexane (pa, Chempur), 32) Cellulose dialysis tubing (cutoff 14,000 g / mol, Sigma Aldrich); 33) Inert gas - argon, 34) All cell assays were performed with a breast cancer cell line (4T1 cell line, ATCCCRL-2539) derived from the mammary gland tissue of the BALB / c mouse strain. 35) All aqueous solutions were prepared in deionized water.

Claims

1. 1. Magnetic core / shell polymer nanocapsules containing iron oxide nanoparticles and chitosan derivatives, characterized in that the nanocapsules consist of a liquid oil core in which magnetic iron oxide nanoparticles, preferably wustite crystallites and crystallites of magnetite-maghemite phases, preferably in a percentage ratio of 22:77, respectively, are suspended, with an inner shell containing a cationic chitosan derivative and a negatively charged outer shell containing an anionic chitosan derivative, the oil core containing a known hydrophobic active substance or dye.

2. 2. The nanocapsule of claim 1, wherein the concentration of the iron oxide nanoparticles in the oil core is at least 100 g / L.

3. 2. The nanocapsule of claim 1, wherein the dye is perylene or Nile Red and the active substance is a known hydrophobic anticancer drug, preferably selected from paclitaxel, lapatinib, fulvestrant or a mixture thereof.

4. 2. The nanocapsule of claim 1, wherein the oil core comprises oleic acid.

5. 2. The nanocapsules according to claim 1, wherein the cationic chitosan derivative comprises N-[(2-hydroxo-3-trimethylamino)propyl]chitosan chloride or n-dodecyl hydrophobic group.

6. The nanocapsule of claim 1 , wherein the anionic chitosan derivative comprises carboxymethylchitosan.

7. 10. The nanocapsules of claim 1, which disintegrate under the influence of an alternating magnetic field having a frequency of 50 Hz, a magnetic induction of 222 mT or more, and a magnetic field duration of at least 5 minutes.

8. The nanocapsules according to claim 1, characterized in that the size of the iron oxide nanoparticles is 15 to 30 nm.

9. 2. The nanocapsule according to claim 1, wherein the wustite crystallite size is 6 nm or less.

10. 2. The nanocapsule according to claim 1, wherein the crystallite size of the magnetite-maghemite is 4.4 nm or less.

11. 2. The nanocapsules of claim 1, wherein the hydrodynamic size of the polymer nanocapsules is between 140 and 230 nm.

12. 2. The nanocapsules of claim 1, wherein the nanocapsules have a dispersion coefficient of 0.4 or less in the anionic system.

13. 2. The nanocapsules according to claim 1, characterized in that the zeta potential value of the polymer nanocapsules in the anionic system is −35 to −45 mV.

14. A pharmaceutical composition comprising the magnetic polymer nanocapsules according to any one of claims 1 to 14.

15. Magnetic polymer nanocapsules according to any one of claims 1 to 14 for use in pharmacy or in diagnostics, especially for use in the treatment or prevention of cancer, especially breast cancer.

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