Magnetic polymer nanocapsules suitable for therapy, in particular anti-cancer therapy

Abstract

Disclosed are magnetic polymer nanocapsules, which are core-shell polymer nanocapsules carrying hydrophobic active substances, comprising magnetic iron oxide nanoparticles, and their use in anti-cancer therapy.

Description

Technical Field

[0001] The present invention relates to magnetic polymer nanocapsules and use thereof in anti-cancer therapy. The nanocapsules are core-shell polymer nanocapsules carrying hydrophobic active substances and contain nanoparticles made of iron oxide. Background Art

[0002] Core-shell magnetic nanocapsules comprising iron oxide nanoparticles (wüstite-maghemite phase) are known from research publications by the following authors: E. Gumieniczek- , J. Odrobinska, T. Straczek, A. Radziszewska, S. Zapotoczny, C. Kapusta, entitled “Hydrophobically Coated Superparamagnetic Iron Oxides Nanoparticles Incorporated into Polymer-Based Nanocapsules Dispersed in Water” Materials, 2020, 13, 1219, wherein such a system can test the magnetically controlled delivery of active hydrophobic substances in biological environments due to the use of biocompatible components.

[0003] Polish patent specification PL 229 276 B1 discloses biocompatible polysaccharide capsules based on a liquid oil core with a diameter not exceeding 1 μm, which are stabilized without the use of low-molecular surfactants and are characterized by efficient encapsulation of hydrophobic compounds and high stability in aqueous suspension.

[0004] Polish patent application P.425141 discloses nanocapsules comprising a liquid oil core made of oleic acid and a stabilizing shell made of hydrophobically modified hyaluronic acid. The document further covers the use of the subject oil-core nanocapsules in anticancer therapy, in particular in the treatment of breast cancer or melanoma.

[0005] US patent application US2016199308A1 discloses magnetic capsules with aqueous nanocores and drug delivery shells. These capsules contain multiple amphiphilic proteins (wherein 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 this invention have a high drug-loading capacity and can encapsulate both hydrophobic and hydrophilic drugs. Therefore, they can be used for targeted drug delivery, magnetic resonance imaging, and hyperthermia.

[0006] Many scientific groups are focusing their research on solutions that allow for the controlled delivery of active substances to the body, while maintaining concentrations consistent with therapeutic doses 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 from polymeric materials should be distinguished. An example of such a carrier is a capsule based on a liquid oil core. One method for creating such a system is to use an emulsification process, which involves suspending oil droplets in an aqueous solution with an appropriate pH. Due to thermodynamic instability, the dispersed oil droplets that form the capsule core require the use of appropriate stabilizers. In the case of biomedical applications, the correct approach is to abandon the use of low-molecular-weight surfactants to stabilize the compounds and instead use appropriately grafted biopolymers, such as modified hyaluronic acid or chitosan. Due to their amphiphilic nature, the compounds used in the aqueous environment align with their hydrophilic backbones on the surface of the oil droplets, while the pendant hydrophobic groups penetrate into the interior of the carrier, forming a closed structure of oil core / polymer shell. Furthermore, the presence of charges on the backbone of the compounds used to stabilize the carrier enables control of the surface charge of the entire system through the use of alternating layers of oppositely charged polyelectrolytes (LbL, layer-by-layer) techniques.

[0007] The system obtained in this way shows high stability, suitable size and ability to encapsulate hydrophobic compounds [J. Szafraniec et al., Nanoscale, 2015, 7, 5525-5536]. In order to better target the delivery and release process of the encapsulated substances in the above-mentioned carriers, the system can be enriched with magnetic properties by placing magnetic nanoparticles in the structure of these systems. Due to biomedical applications, iron oxide nanoparticles are used for this purpose, and their hydrophobic coating enables the nanostructure to be placed in the oil core of the carrier. It is very important that the strong magnetic properties of the entire system can be controlled by the superparamagnetism of the nanoparticles used [J. Odrobińska 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 none of them are currently used for magnetic navigation, targeting and controlled delivery of active substances [DD Stueber et al., Pharmaceutics 2021, 13, 943]. Very stable nanocapsules (based on liquid oil cores stabilized with appropriately modified biopolymers) are well known in the literature, and magnetic control systems designed for targeted and controlled delivery of active substances have also been proposed. However, the combination of these two solutions used in the present invention is new.

[0008] Numerous methods for using magnetic nanoparticles and polymer nanocapsules are available in the prior art, but 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 treating, in particular, breast cancer is a significant problem in the prior art. Furthermore, inefficient encapsulation of hydrophobic active substances results in insufficient concentrations of the active substances within the polymer capsules and released into cancer cells, corresponding to therapeutic doses. Another problem is that there are few existing targeted therapies, as it is impossible to control (selectively target / navigate) polymer nanocapsules carrying active substances to malignant cells, particularly breast cancer cells, using static magnetic fields. Another problem is that it is impossible to release hydrophobic active substances into cancer cells using alternating magnetic fields, and the side effects of currently used treatments in anticancer therapy cannot be ignored, while widely used chemotherapy has highly negative effects on the human body. In known solutions using polymer capsules, the relatively large size of the core-shell system hinders the penetration of the polymer capsules containing the active substance into cancer cells, and the frequent cytotoxicity of the polymer capsules to normal cells in the body cannot be completely eliminated. Another problem is that magnetic polymer capsules carrying active substances cannot be used for targeted and controlled anticancer therapy. Summary of the Invention

[0009] The object of the present invention is to provide polymer nanocapsules carrying active substances, characterized by very high stability and magnetic properties and high encapsulation efficiency for hydrophobic active substances, which allow to achieve therapeutic concentrations of active substances encapsulated in polymer capsules and released into cancer cells. Another object is to be able to navigate (selectively target) polymer capsules carrying active substances to cancer cells, in particular breast cancer cells, using a static magnetic field, and to provide the possibility of releasing hydrophobic active substances in cancer cells using an alternating magnetic field. Another object of the present invention is to completely eliminate the side effects of the therapy of magnetic polymer nanocapsules used so far in anticancer therapy. Another object of the present invention is to obtain very small size (nanosize) core-shell systems (capsules) for transporting active substances, which will easily penetrate into cancer cells and will not show cytotoxicity to normal cells in the body.

[0010] Another object of the present invention is to develop a highly effective and efficient anticancer targeted therapy with the possibility of fully controlling the release of active substances by using magnetic polymer capsules carrying the active substances.

[0011] Unexpectedly, the present invention solves all the above technical problems. The subject of the present invention is: nanocapsules as defined in the appended claims and their medical use. The nanocapsules prepared according to the present invention are able to cross the cell membrane barrier under magnetic assistance without destroying the structure of the carrier (nanocapsule). During the research leading to the present invention, it was found that the negative surface charge of the capsule is necessary for the capsule to effectively cross the cell membrane into the cell and release the capsule content throughout the cell volume; see Figure 25 (positively charged capsules) and Figure 27 (Negatively charged capsules.) Positively charged capsules tend to concentrate more around or stay within the cell membrane rather than passing through it.

[0012] At the same time, it has been observed that application of an alternating magnetic field of appropriate strength leads to a rapid and complete release of the contents of the nanocapsules prepared according to the present invention into cells.

[0013] The present invention relates to the use of magnetically controlled polymer capsules based on liquid oil cores, which contain magnetic iron oxide nanoparticles and transported active substances in their structure. The carrier provided is a spherical core-shell system with a size of several hundred nanometers. These systems show high stability, can effectively encapsulate hydrophobic active substances, and can magnetically control (static magnetic field) delivery and release of the transported substances (effect of alternating magnetic field). The carrier obtains magnetic properties by placing spherical nanoparticles in the hydrophobic environment of the core. The spherical nanoparticles have a size of no more than 30 nanometers, are composed of iron oxide, and have an external lipophilic coating. Because their surface is made of biocompatible polysaccharides, the polymer capsules do not show cytotoxicity, but can be absorbed by cells, and their contents can be released into the cell interior under the effect of an alternating magnetic field. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The subject matter of the invention is shown in exemplary embodiments in the drawings, in which:

[0015] Figure 1 shows the temperature as a function of time during the thermal reaction of the decomposition of iron(III) oleate;

[0016] Figure 2 shows STEM images of nanoparticles obtained by thermal decomposition of organometallic precursors;

[0017] Figure 3 FT-IR spectra of magnetic nanoparticles and oleic acid are shown;

[0018] Figure 4 X-ray diffraction patterns of magnetic nanoparticles, microcrystalline magnetite, and wüstite are shown, with the Miller indices labeled.

[0019] Figure 5Spectra of magnetic nanoparticles obtained by Mössbauer spectroscopy are shown (black dots represent measurement results): a) measurement performed at room temperature, with the fitted sextet of wüstite (blue line), magnetite sextet (blue and green lines), and the total fitted line (red line); b) measurement of magnetic nanoparticles at room temperature 8 months after their synthesis; c) measurement at liquid nitrogen temperature, including the wüstite content (blue dots) and excluding the wüstite phase (red dots); d) spectrum of microcrystalline wüstite measured at liquid nitrogen temperature; e) spectrum of microcrystalline maghemite measured at liquid nitrogen temperature;

[0020] Figure 6 shows the magnetic susceptibility of the magnetic nanoparticles as a function of temperature measured in a 100 Oe field;

[0021] Figure 7 shows the magnetization curves of magnetic nanoparticles as a function of applied field strength measured in the temperature range of 4 K to 300 K;

[0022] Figure 8 Shown are the changes in hydrodynamic diameter values and zeta potential of magnetic cationic capsules measured over 48 weeks;

[0023] Figure 9 shows a cryoTEM image of an oil-core-based magnetic cation capsule;

[0024] Figure 10 is a schematic diagram of an oil-core-based anionic capsule with encapsulated magnetic nanoparticles;

[0025] Figure 11 Shown are the changes in hydrodynamic diameter values and zeta potential of magnetic anion capsules measured over 48 weeks;

[0026] Figure 12 Images of magnetic capsules taken with a confocal microscope are shown: A) cationic capsule (image taken on the day of capsule preparation); B) cationic capsule (image taken after 2 weeks of storage); C) anionic capsule (image taken on the day of capsule preparation); D) anionic capsule (image taken after 2 weeks of storage); scale: 1 μm;

[0027] Figure 13 shows the magnetization of cationic magnetic capsules as a function of applied field strength at 300 K;

[0028] Figure 14 The layout of a 24-well plate prepared for measuring the toxicity of capsules to 4T1 cell line cells is shown;

[0029] Figure 15 is a graph of cell survival of 4T1 cell line cells exposed to different concentrations of cationic magnetic capsules (determined by XTT assay);

[0030] Figure 16 is a graph of cell survival of 4T1 cell line cells exposed to different concentrations of anionic magnetic capsules (determined by XTT assay);

[0031] Figure 17 This is a schematic diagram of an experiment using a static magnetic field to pull cells;

[0032] Figure 18 Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRICT 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.

[0033] Figure 19 Shown are confocal microscopy images of cells subjected to an experiment introducing fluorescently labeled magnetic cationic capsules, the cells being under a static external magnetic field, left column: transmitted light and TRITC filter, right column: TRITC filter, scale: 20 μm;

[0034] 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 are shown, the cells being under a static external magnetic field, scale: 20 μm;

[0035] Figure 21 Shown are confocal microscopy images of cells subjected to an experiment introducing fluorescently labeled magnetic anion capsules, the cells being under a static external magnetic field, left column: transmitted light and TRITC filter, right column: TRITC filter, scale: 20 μm;

[0036] Figure 22 Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRICT filter) fluorescent dyes after an experiment involving the introduction of magnetic anion capsules are shown, the cells being under a static external magnetic field, scale: 20 μm;

[0037] Figure 23 Schematic diagram showing the experiment of releasing encapsulated substances inside cells using an external alternating magnetic field;

[0038] Figure 24 Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRICT filter) fluorescent dyes, which were subjected to 15 minutes of static magnetic field exposure and then to external alternating magnetic field exposure, scale: 20 μm;

[0039] Figure 25 are confocal microscopy images of cells subjected to an experiment of introducing fluorescently labeled magnetic cationic capsules and releasing substances encapsulated in the fluorescently labeled magnetic cationic capsules, with exposure to an alternating magnetic field followed by exposure to a static external magnetic field for 15 minutes, left column: transmitted light and TRITC filter, right column: TRITC filter, scale: 20 μm;

[0040] Figure 26 Confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRICT filter) fluorescent dyes subjected to an experiment of introducing magnetic cationic capsules and releasing substances encapsulated therein are shown, said cells being exposed to a relevant alternating magnetic field after 15 minutes of static external magnetic field exposure, scale: 20 μm;

[0041] Figure 27 are confocal microscopy images of cells subjected to an experiment of introducing fluorescently labeled magnetic anion capsules and releasing substances encapsulated in the fluorescently labeled magnetic anion capsules, with associated alternating magnetic field exposure after 15 minutes of static external magnetic field exposure, left column: transmitted light and TRITC filter, right column: TRITC filter, scale: 20 μm; and

[0042] Figure 28 Shown are confocal microscopy images of cells stained with Hoechst (left column, DAPI filter) and propidium iodide (right column, TRICT filter) fluorescent dyes, subjected to an experiment of introducing magnetic anion capsules and releasing substances encapsulated in the magnetic anion capsules, the cells being exposed to a relevant alternating magnetic field after 15 minutes of static external magnetic field exposure, scale: 20 μm. DETAILED DESCRIPTION

[0043] Example 1: High temperature synthesis of iron oxide nanoparticles with a hydrophobic coating.

[0044] The magnetic nanoparticles, placed within the capsule structure, are believed to impart magnetic properties to the entire support. These nanostructures are obtained through a two-step synthesis process involving the thermal decomposition of previously synthesized organometallic precursors in the presence of oleic acid. This method allows for the production of nanoparticles with a narrow size distribution composed of iron oxides and exhibiting strong magnetic properties. The presence of a hydrophobic substance during the synthesis ensures sufficient coverage of the structures, allowing them to be placed within the oil core of the capsules.

[0045] The synthesis of iron (III) oleate was carried out based on the method previously proposed by Leszczyński et al. [Leszczyński, 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 kept under constant stirring for 5 hours. The dark hydrophobic portion was then separated, rinsed with deionized water, and heated to 40°C to evaporate the remaining hexane.

[0046] The synthesis leading directly to the production of nanoparticles was carried out according to a method previously described by Park et al. [Park J. et al., Ultra-large-scale syntheses of monodisperse nanocrystals, Nat Mater 3, 891-895 (2004)]. Initially, 42 mL of octadecane, 1.1 mL of oleic acid and 7 g of previously prepared iron (III) oleate were mixed. The entire reaction was carried out under inert gas conditions with constant stirring in a flask as shown in the accompanying drawings ( Figure 1 ) The resulting product was cooled to room temperature, washed several times with ethanol, and unreacted substrate was removed by sonication in hexane (continuous, 5 minutes) and centrifugation (5,000 rpm, 5 minutes). The obtained nanoparticles were vacuum dried.

[0047] Physicochemical properties of nanoparticles

[0048] Scanning electron transmission microscopy (STEM) imaging ( Figure 2 ) confirmed the spherical shape of the individual nanoparticles, while analysis of the images obtained showed that the average size of the structures was 15 nm with a narrow distribution of this parameter.

[0049] The choice of synthesis method for the magnetic nanoparticles strictly depends on the possibility of obtaining a structure with an appropriate coating that allows them to be placed in the hydrophobic interior of the capsule. In order to determine the correct deposition of the oleic acid layer on the surface of the nanoparticles, Fourier transform infrared spectroscopy (FT-IR) measurements and TGA-DSC thermal analysis were performed. The spectra of the nanoparticles obtained were compared with those measured for the oleic acid used during the synthesis ( Figure 3 ). For oleic acid, the signal appearing at 3000 cm-1 is the vibration of the C-H bond in the stretched -C=CH group, the bands appearing at 2922 cm-1 and 2853 cm-1 correspond to the asymmetric and symmetric vibrations of the stretched -CH2 group, and the strong band at 1710 cm-1 can be attributed to the vibration in the C=O bond. The spectrum measured on the magnetic nanoparticles shows that the band originating from the C=O bond disappears, while the band at 1560 cm-1 is equal to the band at 1710 cm-1. 1 and 1645cm- 1 Two new bands appear at a wavelength of , corresponding to the asymmetric and symmetric stretching vibrations in the -COO- group. The difference in the positions of these bands suggests a chelating linkage between the -COO- groups of oleic acid and the iron ions [Premaratne, W., Priyadarshana, W., Gunawardena, S., DeAlwis, 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, H.C., Oleic 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 (Effect of oleic acid iron complex structure on the formation of iron oxide nanoparticles), Chem. Mater. 19, 3624-3632 (2007)].

[0050] The crystal structure of the obtained nanoparticles was studied based on X-ray powder diffraction (XRD) measurements. The obtained diffraction patterns were compared with those of microcrystalline magnetite and wüstite ( Figure 4 ). Lines assigned to the magnetite phase (e.g., for ) shows no shift in the diffraction pattern relative to that of microcrystalline magnetite. However, in the case of wüstite, a shift of the maximum toward higher angles relative to the reference line can be observed. This shift indicates a decrease in the lattice constant due to compressive strain in the nanoparticle structure caused by the core / shell structure. Given that wüstite (FeO) undergoes rapid oxidation under atmospheric conditions and even at slightly elevated temperatures to the magnetite (Fe3O4) phase, its oxidized forms—maghemite (γ-Fe2O3) and hematite (α-Fe2O3), or a two-stage disproportionation process resulting in the production of hematite and magnetite, it can be concluded that the core of the nanoparticle is wüstite, while the shell consists of a magnetite or maghemite phase. The strain occurring in the nanoparticle structure leads to an increase in magnetocrystalline anisotropy. Based on the Scherrer equation, the average crystallite size of the magnetite / maghemite phase was calculated to be 4.4 nm, and the average crystallite size of the wüstite phase was 6 nm. Considering the nanoparticle structure (core-shell) and using the determined crystallite sizes, the average size of the magnetic nanoparticles can be determined to be 14.8 nm. Phase fitting of the obtained diffraction pattern showed a 22% share of the wüstite phase and a 77% share of magnetite / hematite [Comell, R. M., Schwetmann, U., The Iron Oxides: Structure, Properties, Reactions, Occurrences and Uses, 2nd ed., WILEY-VCH GmbH & Co. KGaA, 139-183 (2003); Pichon, B. P. et al., Microstructural and Magnetic Investigations of Wüstite-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 / Fe3O4Nanoparticles (Tuning Exchange Bias in Core / Shell FeO / Fe3O4 Nanoparticles), Nano Lett. 12, 246-251 (2012); Scherrer, P. Bestimmung der und der inneren Struktur von Kolloidteilchen mittels ,Nachrichten von der Gesellschaft der Wissenschaften, .98-100(1918)].

[0051] The magnetic nanoparticles were studied by Mössbauer spectroscopy at different temperatures (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 by the presence of a broad sextet, while a single peak with an isotropic shift of about 1 mm / s indicates the presence of a second, but paramagnetic phase. The presented spectrum has a relaxation characteristic. This is indicated by a stronger broadening of the spectral lines towards the inside and lower amplitudes of the spectral lines at the outside (compared to the theoretical 3:2:1 to 1:2:3), which means that the nanoparticles exhibit superparamagnetic properties at room temperature. When the frequency of the fluctuation of the magnetization vector between the easy axes is greater than 57 The relaxation spectrum appears at the inverse of the Fe Mössbauer lifetime, or 7 MHz. The obtained spectrum was fitted to the Blume and Tijon model, resulting in the parameters presented in Table 1. The measured isotropic shift values indicate that the paramagnetic phase represented by a single peak in the spectrum is wüstite. On the other hand, in the spectrum measured at liquid nitrogen temperature, the single peak originating from the paramagnetic phase is absent, and the presence of additional magnetically split components indicates the presence of a magnetically ordered phase. The observed spectral lines are much narrower, with intensity ratios closer to the theoretical 3:2:1 to 1:2:3, indicating that the nanoparticle magnetization vector cannot overcome the energy barrier at this temperature, resulting in no observed superparamagnetic fluctuations. After subtracting the 22% contribution of the wüstite phase from the obtained spectrum, the second iron oxide phase present in the studied sample was identified as maghemite.

[0052] The 52 MHz fluctuation frequency determined from the room temperature relaxation spectrum indicates a relatively high superparamagnetic blocking temperature in the nanoparticles. On the other hand, the high value of the asymmetry coefficient (ρ = 0.95), which corresponds to the relative residence time of the nanoparticle magnetic moment along the easy magnetization direction, indicates a high magnetocrystalline anisotropy of the obtained system [Armstrong, RJ, Morrish, AH Study Of Ferric Ions In The Tetrahedral And Octahedral Sites Of ASpinel, Phys. Lett. 23, 414-416 (1966); S., Effect in Small Iron Particles, Hyperfine Interact, 60, 959-974 (1990); Redl, FX 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 Spectroscopy (Study on Magnetite Nanoparticles from Mössbauer Spectroscopy), J. Nanosci. Nanotechnol. 6, 926-947 (2006)].

[0053] Table 1: Parameters obtained after fitting the spectra obtained from Mössbauer spectroscopy measurements of magnetic nanoparticles, and summary of literature data for maghemite and wüstite.

[0054]

[0055] The magnetic nanoparticles were analyzed using a vibrating sample magnetometer (VSM). The temperature dependence of the magnetic susceptibility measured in the form of ZFC (zero field cooling) and FC (field cooling) in a static field of 100 Oe was measured ( Figure 6 ), and the magnetization curves as a function of applied magnetic field strength in the temperature range of 4K to 300K ( Figure 7). The temperature at which the magnetization value increases significantly (230K) corresponds to the transition of pyrite from the antiferromagnetic state to the paramagnetic state, i.e. the Neel temperature. The literature value for pyrite is about 192K, however, the presence of an additional phase in the form of ferrimagnetic hematite surrounding the pyrite nanoparticle core can lead to an increase in this temperature. The maximum of the ZFC curve allows the determination of the average blocking temperature, i.e. the temperature above which the magnetic moment of the nanoparticle exhibits superparamagnetic fluctuations. The blocking temperature value determined based on the measured function is 275K. Above this temperature, the shape of the magnetization curve indicates that the hysteresis disappears, which proves the superparamagnetic state of the nanoparticles. During the measurement, the magnetization did not reach saturation, which is related to the presence of pyrite in the nanoparticle structure. The measured values of the coercive field and remanence are shown in Table 2. Below the Neel temperature, due to the exchange bias coupling at the boundary of the antiferromagnetic pyrite core and the ferrimagnetic hematite shell, the nanoparticles show a shift in the hysteresis loop, resulting in the emergence of unidirectional magnetic anisotropy. This coupling is caused by the presence of an antiferromagnetic phase, which is more difficult to remagnetize, and it depends on the size of the core, the thickness of the shell, and the shape of the nanoparticle [Pichon, BP et al., Microstructural and Magnetic Investigations of Wüstite-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: Fe x O / Fe3O4 nanoparticles as a Case Study x O / Fe3O4 nanoparticles as a case study), Nanoscale 7, 3002-3015 (2015)].

[0056] Table 2: Coercive field and remanence values determined from the magnetization curves of magnetic nanoparticles.

[0057]

[0058] Example 2: Preparation of liquid-core based cationic capsules with encapsulated magnetic nanoparticles.

[0059] The capsules were obtained by emulsification process and prepared using hydrophobic and cationic modified chitosan derivatives, anionic chitosan derivatives and magnetic nanoparticles obtained by thermal decomposition of organometallic precursors.

[0060] The method for preparing liquid oil core capsules based on the self-assembly phenomenon of amphiphilic grafted 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., Odrobińiska, J., Zapotoczny, S., Nanocapsules templated on liquid cores stabilized by graft amphiphilic polyelectrolytes, Nanoscale 7, 5525-5536 (2015)]. The above method was modified due to the additional encapsulation of magnetic nanoparticles. Therefore, 10 μL of oleic acid with dispersed 100 mg / mL magnetic nanoparticles was added to 1 mL of a 0.15 M sodium chloride (NaCl) solution with 1 mg of dissolved cationically and hydrophobically modified chitosan (CChitC12). CChitC12 was obtained according to a previously described method [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]. Figure below, m:n:p = 67.5:2:30.5.

[0061]

[0062] The resulting solution was vigorously mixed in an oscillator and then subjected to pulsed sonication (1 second on, 2 second pause mode at room temperature) to accurately deposit the polymer layer on the surface of the oil capsule core. Unencapsulated magnetic nanoparticles were removed by magnetic separation. The resulting emulsion appeared off-white.

[0063] Physical and chemical properties of cationic capsules

[0064] The measurements were performed using dynamic light scattering (DLS) technology to determine the hydrodynamic diameter and zeta potential values. The measurements were repeated for 48 weeks, during which the test samples were stored at 4°C. The hydrodynamic diameter and zeta potential values of the capsules with positive surface charge remained at the same level throughout the study period ( Figure 8 The average hydrodynamic diameter of these systems does not exceed 170 nm and is not less than 140 nm, and the polydispersity index is less than 0.3, which demonstrates a relatively narrow size distribution. On the other hand, the zeta potential values are within the range of 30 mV-40 mV throughout the measurement period.

[0065] Cryogenic transmission electron microscopy (cryoTEM) was used to image the cationic magnetic capsules. Figure 9 The images obtained confirmed the presence of magnetic nanoparticles inside the carriers, and the spherical shape of the carriers could also be observed, whose size was consistent with the DLS measurements.

[0066] Example 3: Preparation of liquid-core based anionic capsules with encapsulated magnetic nanoparticles.

[0067] The second type of capsule studied was a system with a negative surface charge ( Figure 10 Anionic capsules are prepared based on a technique that applies layers of opposite charge. To create such a carrier, 0.6 mL of a 1 g / L anionic chitosan solution (AChit, prepared according to a previously described method: Bulwan M., Zapotoczny S., Nowakowska M.: Soft Matter 2009, 5, 4726, http: / / dx.doi.org / 10.1039 / B909355A, below, with a 49 mol% degree of substitution of sulfonic acid groups) dissolved in 0.0015 M sodium chloride was added to 0.4 mL of a cationic capsule dispersion. The resulting solution was vigorously mixed on an oscillator for 10 minutes.

[0068]

[0069] Physical and chemical properties of anionic capsules

[0070] The hydrodynamic diameter and zeta potential values of the anionic capsules were measured and shown in Figure 11 The measured mean hydrodynamic diameters ranged from 140 nm to 230 nm, with a polydispersity index of less than 0.4. The larger size distribution could be related to the formation of a small amount of aggregates or to the non-uniform coating of the capsules with anionic chitosan (AChit). The zeta potential values ranged from -35 mV to -45 mV. The higher mean hydrodynamic diameters and negative zeta potential values compared to cationic capsules indicate proper deposition of the anionic chitosan layer on the analyzed support surfaces.

[0071] Example 4: Preparation of liquid fluorescent core-based capsules with encapsulated magnetic nanoparticles.

[0072] For confocal microscopy imaging, it was necessary to fluorescently modify the capsules under investigation. The preparation method for both cationic and anionic capsules was expanded by adding a fluorescent dye to an oil suspension of magnetic nanoparticles, which subsequently formed the core of the carrier. In the case of capsules enriched with perylene dyes, 1 mg of dye was added per 1 mL of the oil mixture containing the dispersed magnetic nanoparticles.

[0073] Capsules containing an additional hydrophobic fluorescent dye in the form of perylene in an oil core were imaged using confocal microscopy ( Figure 12 The images presented show the blue fluorescent glow of the labeled oil capsule core. The images obtained demonstrate the feasibility of placing hydrophobic substances, particularly hydrophobic active substances, within the magnetic capsule core. Furthermore, images obtained two weeks after capsule preparation demonstrate the feasibility of longer-term encapsulation of substances that are soluble in a hydrophobic environment.

[0074] Example 5: Preparation of concentrated dispersions of liquid-core based cationic capsules with encapsulated magnetic nanoparticles.

[0075] Vibrating sample magnetometer (VSM) measurements were performed to verify the magnetic properties of the capsules. To achieve an appropriate signal-to-noise ratio, capsule suspensions containing increasing concentrations of magnetic nanoparticles were prepared, thereby increasing the amount of carrier suspended in the solution. Sample preparation was performed according to a method similar to the cationic capsule formation method, using 30 μL of oleic acid containing dispersed magnetic nanoparticles at a concentration of 100 g / L and 1 mL of a chitosan derivative (CChitC12) at a concentration of 10 g / L in a 0.15 M NaCl solution.

[0076] The curves obtained from magnetometry confirm the magnetic nature of the carrier, while the absence of the hysteresis loop is associated with the presence of superparamagnetic nanoparticles in the capsule core ( Figure 13 ).

[0077] Example 6: Experimental studies involving cell lines.

[0078] In vitro studies were performed using the 4T1 line, a cancer cell line derived from mouse mammary tissue. The cultures were maintained under sterile conditions at 37°C and 5% CO2. Cell culture was performed using a culture medium supplemented with antibiotics (1%) and fetal bovine serum (5%) in the form of penicillin and streptomycin solutions. All reagents used in the cell studies were heated in a 37°C water bath. In the first step, the cells were thawed for culture, the cell suspension was transferred to a centrifuge tube, and 5 mL of culture medium was added to dilute dimethyl sulfoxide (DMSO). The cells were then centrifuged (1000 rpm, 5 minutes), the solution covering the cell pellet was decanted, and the cells were resuspended in 1 mL of culture medium. The cells prepared in this way were inoculated onto a culture dish pre-filled with 10 mL of culture medium. The proliferation of the cells and their condition during culture were monitored using an optical microscope, and the culture medium was replaced every 2 days. Trypsinization was performed to perform passage. The culture dish with the expanded cells was rinsed twice with PBS solution. Subsequently, 0.8 mL of trypsin solution was added, withdrawn, and 0.8 mL of trypsin was added again. The culture dish of processing is placed in incubator, after 3 minutes, it is thoroughly tapped and put back in incubator.After another 3 minutes, tap the culture dish again, monitor the degree of separation of cell and substrate under the microscope.After adding 3mL culture medium for making trypsin inactivation, the cell of separation is transferred in falcon pipe from culture dish.The cell suspension obtained is centrifuged (1000rpm, 5 minutes), fluid is poured out, and the cell suspension of precipitation is in the culture medium of 1mL.Thus, obtain cell suspension, it is used in experiment subsequently.

[0079] Example 7: Cytotoxicity study of the capsule on 4T1 cancer cells (XTT).

[0080] The XTT assay was performed to determine the cytotoxicity of cationic and anionic capsules against the 4T1 line. This assay allows for the determination of the metabolic activity of cellular mitochondria based on the spectrophotometric measurement of the reduction of XTT tetrazolium salt to formazan by the activity of mitochondrial dehydrogenases. The day before the direct determination of the toxicity of the capsules against the selected cell lines, two 24-well plates were prepared containing 4T1 cell line cells and appropriately dispersed concentrations of cationic capsules (plate 1) and anionic capsules (plate 2). The plates were prepared by adding 0.9 mL of culture medium to each well and then adding 100 μL of cell suspension. Each well was thoroughly mixed by pipetting and shaking the plate several times. The inoculated plates were placed in an incubator for 4 hours. The next step was to add the agent in the form of capsules. To this end, the fluid was aspirated from each well of the plate and 1 mL of culture medium was added. 0.1 mL of the agent was then added to each well, which was a capsule diluted with 0.15 M NaCl (for cationic capsules) or 0.0015 M NaCl (for anionic capsules) to achieve a 4% toxicity. Figure 14The indicated concentrations were added, as well as 0.15 M NaCl (for cationic capsules) or 0.0015 M NaCl (for anionic capsules) solutions alone, and the resulting solutions were mixed in the wells by pipetting. The prepared plates were placed in an incubator for 24 hours.

[0081] Before the experiment, prepare the reagents required for the XTT assay. According to the manufacturer's instructions, preheat the reagents to 37°C and mix 5 mL of XTT labeling reagent and 0.1 mL of coupling reagent. Before adding the solution, remove the well plate prepared the day before from the incubator and remove the liquid in it, add 0.2 mL of fresh culture medium to each well, and then add 0.1 mL of XTT solution to each well. Mix the well plate gently and place it in the incubator. After 1 hour, perform absorption spectrophotometric measurement. In Figure ( Figure 15 and Figure 16 The obtained results, presented in Figure 3, clearly show a relationship between a decrease in drug concentration and a subsequent increase in cell survival. Based on the values obtained, it can be concluded that capsules with a negative surface charge are less toxic to the cells. However, it can be seen that in the case of both anionic and cationic capsules, a drug concentration of 2% did not induce a negative response in the cells tested.

[0082] Example 8: Using a static magnetic field to introduce capsules into cancer cells.

[0083] The possibility of magnetically controlling capsules containing magnetic nanoparticles was verified by performing experiments using a static inhomogeneous magnetic field applied to a cuvette (filled with capsules suspended in culture medium) and a microscope slide (containing living cells on its surface). To exclude the influence of gravity on the penetration of capsules into cells, the slide with cell culture was placed in a vertical position.

[0084] Before starting the main experiment, it is necessary to generate a slide culture. To do this, appropriately cut sterile microscope coverslips are used and placed in a 6-well cell culture plate. The cell culture slide is then placed on the side of a sterile cuvette and 1 mL of culture 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) are poured into it.

[0085] according to Figure 17 Following the protocol presented in [ 1 ], the prepared cuvettes were subjected to an external static magnetic field for 5 and 15 minutes. The distance between the sample and the magnet was 1 cm, corresponding to a magnetic induction intensity of 143 mT. Furthermore, experiments were performed to verify the spontaneous penetration of the capsule into cells and the effect of the external static magnetic field on the cell condition.

[0086] Once the experiment is complete, the cells on the slides are fixed so they can be subsequently imaged using a confocal microscope. To fix the cells, the slides are transferred to a 6-well plate and washed twice with 2 mL of PBS. Then, 2 mL of 10% formalin in PBS is added to each well. After 10 minutes, the solution is removed, and the slides are rinsed with PBS and placed on a glass microscope slide, with the edges protected with varnish.

[0087] Additionally, capsules enriched with a fluorescent probe in the form of Nile Red dye were used in the experiments. The method used to generate such capsules involved adding the dye to an oil suspension of magnetic nanoparticles at a concentration of 5 mg / mL and then initially adding chitosan (CChitC12) dissolved in water to the solution.

[0088] Cell viability was also verified after experiments with an external magnetic field. To this end, two solutions were prepared: 10 mg of Hoechst was dissolved in 1 mL of deionized water and diluted with PBS at a volume ratio of 1:2000, and 1 mg of propidium iodide was dissolved in 1 mL of deionized water and diluted with PBS at a volume ratio of 1:3000. Cells on glass slides fixed after the external magnetic field experiment were then incubated with the corresponding dye for 5 minutes, rinsed three times with PBS, placed on microscope glass slides, and protected with varnish. The resulting samples were imaged using a confocal microscope.

[0089] Example 9: Verification of the effect of a static magnetic field on cell conditions in the case of cationic and anionic capsules.

[0090] In the first phase of the study, the effects of an external static magnetic field on 4T1 cancer cell line were evaluated in the absence of a carrier. Images collected ( Figure 18 ) clearly ruled out a negative influence of a static magnetic field, as evidenced by the lack of fluorescent signal in cells stained with propidium iodide, which permeates only across damaged cell membranes.

[0091] Then, studies were conducted to verify the feasibility of navigating cationic capsules into cancer cells using an external static magnetic field. The presented image ( Figure 19 ) showed a slight tendency for spontaneous penetration of cationic capsules, while the use of an external static magnetic field increased the validity of the studied phenomenon and mainly facilitated the accumulation of capsules in the vicinity of cancer cells.

[0092] After the experiment, the effectiveness of the cationic capsule penetration process assisted by the static external magnetic field was verified by fluorescence staining of the cells. Figure 20 ) demonstrated that exposure to a static magnetic field for 15 min in the presence of magnetic cationic capsules significantly contributed to the disruption of cell membrane integrity.

[0093] Similar experiments were performed on anion capsules using carrier navigation using an external magnetic field. The images obtained ( Figure 21 ) suggest the possibility that capsules with negative surface charges spontaneously penetrate into cells via endocytosis, but the use of an external static magnetic field increases the effectiveness of the penetration process.

[0094] Cells exposed to a static external magnetic field in the presence of anionic capsules and in the absence of an external field were fluorescently labeled and are shown in Figure 22 The images obtained demonstrate the increased tendency of the anionic capsules to spontaneously penetrate the cells. The bright red glow visible after the application of an external static magnetic field indicates significant disruption of the cell membrane integrity, confirming the successful introduction of the vector into the cancer cells.

[0095] Example 10: Alternating magnetic field assisted release of encapsulated substances from capsules placed inside cancer cells.

[0096] The potential for releasing substances encapsulated in the carrier was evaluated by applying an alternating magnetic field. This external alternating magnetic field served as a continuation of the static magnetic field experiments. As shown in Table 3, after the system was exposed to a static magnetic field for 15 minutes, it was transferred to an external alternating magnetic field with a frequency of 50 Hz and varying induction values for 5 minutes.

[0097] Table 3: Magnetic induction values in experiments with an alternating magnetic field.

[0098] field Induction value [mT] weak 22 medium 67 powerful 222

[0099] Experiments were also performed to exclude the harmful effects of the alternating field on the cells by performing experiments called blank experiments on cells that were not in contact with the capsules. A schematic diagram of the experimental method is shown in Figure 23 Once the experiment was complete, the slides with cells were fixed according to the previously described protocol. Similar to what was described previously, the viability of the cells was also verified after the experiment using Hoechst dye and propidium iodide.

[0100] Example 11: Verify the effect of alternating magnetic field on cell conditions.

[0101] The experimental results that can verify the harmful effects of alternating magnetic fields on cancer cells are presented in confocal microscope images ( Figure 24 ). The images obtained confirmed the integrity of the cell membrane after exposure of the cells to an external alternating magnetic field, as indicated by the lack of fluorescence in cells stained with propidium iodide.

[0102] The experimental results of exposing fluorescently labeled cationic capsules and cancer cells to an external static magnetic field for 15 minutes and to an alternating magnetic field with different parameters for 5 minutes are shown in Figure 25The images obtained show that, in the case of cationic capsules, the applied strong alternating magnetic field facilitates significant penetration of the cell membrane barrier but does not lead to an efficient release of the encapsulated material inside the cell.

[0103] After the cells in the presence of cationic capsules were exposed to static external magnetic fields and alternating magnetic fields, the destruction of cell membrane integrity was confirmed by fluorescence-stained cell images ( Figure 26 ). The images obtained indicate the possibility of introducing the capsule inside cells and disrupting their membranes, which would promote their death.

[0104] For capsules with negative surface charge, the possibility of targeted and magnetically controlled release of encapsulated substances inside cells was also verified. Figure 27 ) was obtained after the cells were exposed to a static magnetic field for 15 minutes and to a corresponding alternating magnetic field for 5 minutes, the cells being surrounded by anionic capsules containing fluorescent dyes. The results obtained for the samples exposed to a strong alternating magnetic field clearly demonstrate the possibility of effective release of the encapsulated material inside the cancer cells.

[0105] Figure 28 Figure 2 shows the results of cell membrane assessments following experiments targeting the magnetically controlled introduction and release of substances carried within anion capsules. A bright red fluorescent glow confirms significant damage to the cell membrane, particularly when a strong alternating magnetic field is applied. This damage affects all cells in the image subjected to experiments using a strong alternating magnetic field and ultimately leads to cancer cell death.

[0106] List of reagents used:

[0107] 1) Chitosan (molar mass: 50 kDa-190 kDa, Sigma-Aldrich);

[0108] 2) glycidyltrimethylammonium chloride (GTMAC, ≥90%, Sigma-Aldrich);

[0109] 3) n-dodecanal (92%, Sigma-Aldrich);

[0110] 4) sodium cyanoborohydride (NaCNBH3, 95%, Sigma-Aldrich);

[0111] 5) carboxymethyl chitosan (CMC, degree of deacetylation 90%, AK Scientific);

[0112] 6) Sulfur trioxide-trimethylamine complex (TMST, 99%, Sigma-Aldrich);

[0113] 7) sodium bicarbonate (NaHCO3, pa, Sigma-Aldrich);

[0114] 8) sodium hydroxide (NaOH, pa, Avantor Performance Materials Poland SA);

[0115] 9) Iron (III) chloride (FeCl3, anhydrous, Merck);

[0116] 10) Sodium oleate (>97%, TCI);

[0117] 11) Octadecane (90%, Alfa Aesar);

[0118] 12) oleic acid (OA, 99.5%, Alfa Aesar);

[0119] 13) Perylene (Pe, gold-labeled, 99.9%, Sigma-Aldrich);

[0120] 14) Nile red (NR, for microscopy, Sigma-Aldrich);

[0121] 15) n-octadecane (pa, Polyscience Corp.);

[0122] 16) Dulbecco's modified Eagle's medium (DMEM, high glucose, Sigma-Aldrich);

[0123] 17) Fetal bovine serum (FBS, HyClone);

[0124] 18) Penicillin-streptomycin solution (Symbios);

[0125] 19) Trypsin (HyClone);

[0126] 20) a mixture of salts for preparing phosphate buffered saline (PBS, tablets, Sigma-Aldrich);

[0127] 21) dimethyl sulfoxide (DMSO, ≥99.7%, Sigma-Aldrich);

[0128] 22) XTT (Cell Proliferation Kit II, Sigma-Aldrich);

[0129] 23) Formalin (36.5%-38% aqueous solution, Sigma-Aldrich);

[0130] 24) Hoechst (Sigma-Aldrich);

[0131] 25) Propidium iodide (PI, Sigma-Aldrich);

[0132] 26) acetic acid (99.5%, Chempur);

[0133] 27) sodium chloride (NaCl, pa, Lachner);

[0134] 28) ethanol (96%, Chempur);

[0135] 29) acetone (pa, Chempur);

[0136] 30) methanol (pa, Chempur);

[0137] 31) Hexane (pa, Chempur);

[0138] 32) Cellulose dialysis tubing (molecular weight cut-off 14,000 g / mol, Sigma Aldrich);

[0139] 33) Inert gas - argon;

[0140] 34) All cell assays were performed on a breast cancer cell line derived from mammary tissue of the murine BALB / c strain (strain 4T1, ATCC CRL-2539);

[0141] 35) All aqueous solutions were prepared with deionized water.

Claims

1. A magnetic core / shell polymer nanocapsule comprising iron oxide nanoparticles and chitosan derivatives, characterized in that: The nanocapsule is composed of a liquid oil core provided with an inner shell comprising a cationic chitosan derivative and a negatively charged outer shell comprising an anionic chitosan derivative, the oil core containing a known hydrophobic active substance or dye, and magnetic iron oxide nanoparticles suspended in the oil core, preferably, wurtzite crystallites and magnetite-maghemite phase crystallites are suspended in the oil core, preferably, the ratio of the percentage of the wurtzite crystallites to the percentage of the magnetite-maghemite phase crystallites is 22:77 respectively.

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

3. The nanocapsule according to claim 1, characterized in that 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. The nanocapsule according to claim 1, characterized in that The oil core includes oleic acid.

5. The nanocapsule according to claim 1, characterized in that The cationic chitosan derivative includes an N-[(2-hydroxy-3-trimethylamino)propyl]chitosan chloride group or an n-dodecyl hydrophobic group.

6. The nanocapsule according to claim 1, characterized in that The anionic chitosan derivatives include carboxymethyl chitosan.

7. The nanocapsule according to claim 1, characterized in that The nanocapsules disintegrate under the influence of an alternating magnetic field having a frequency of 50 Hz, a magnetic induction intensity of not less than 222 mT, and a magnetic field duration of at least 5 minutes.

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

9. The nanocapsule according to claim 1, characterized in that The size of the wüstite crystallites is no greater than 6 nm.

10. The nanocapsule according to claim 1, characterized in that The crystallite size of the magnetite-maghemite phase is no greater than 4.4 nm.

11. The nanocapsule according to claim 1, characterized in that The hydrodynamic size of the polymer nanocapsules is 140 nm to 230 nm.

12. The nanocapsule according to claim 1, characterized in that In the anionic system, the dispersion coefficient of the polymer nanocapsules is no more than 0.

4.

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

14. A pharmaceutical composition comprising the magnetic polymer nanocapsules as defined in claims 1 to 14.

15. Magnetic polymer nanocapsules as defined in claims 1 to 14, for use in medicine or diagnostics, in particular for use in the treatment or prevention of cancer, in particular breast cancer.

Images