Cross-linking agent, method of its manufacture and use, method of manufacturing polymer nanogels using this cross-linking agent and use of polymer nanogels as drug carriers

Abstract

The subject of the present invention is N,N'-bis(acryloyl)selenocystine represented by formula 1, a method of its manufacturing, and its use as a crosslinking agent in polymerisation reactions, preferably in nanogel polymerisation reactions. Another subject of the invention is a method of manufacturing a p(NIPA-BISeSe) nanogel, and the use of the p(NIPA-BISeSe) nanogel obtained by this method as a carrier of drugs, preferably anticancer drugs.

Description

[0001] Cross-linking agent, method of its manufacture and use, method of manufacturing polymer nanogels using this cross-linking agent and use of polymer nanogels as drug carriers

[0002] The invention concerns a seleno-cystine-based cross-linking agent, sensitive to reduction and oxidation, a method of its synthesis, and the use of this linker in polymerisation reactions to obtain environmentally sensitive nanogels that degrade under the influence of glutathione, which is present at elevated concentrations in most cancer cells. The invention also includes a method of synthesizing polymer nanogels using this cross-linking agent and the use of polymer nanogels as drug carriers, particularly anticancer drugs.

[0003] Polymer nanoparticles are known for the controlled delivery of drugs, genes, or imaging agents (International Journal of Molecular Sciences, 2021, 22(17), 9118). Thermosensitive polymers are known, i.e., those that exhibit a change in chemical and physical properties at a specific temperature (defined as the phase transition temperature, PTT) (Progress in Polymer Science, 2007, 32(10), 1205). One of the most intensively studied temperaturesensitive polymers is poly(N-isopropylacrylamide) - pNIPA, since it is a biocompatible polymer and its PTT is close to human body temperature. This means that pNIPA-based materials can find many medical applications, including as carriers for controlled drug release, base materials in tissue engineering, and new carriers in gene therapies (Polymers, 2011, 3, 1215).

[0004] Unfortunately, polymers typically dissolve in the body. Therefore, polymer cross-linking is one of the most commonly used strategies to eliminate the dissolution of polymer particles and prevent premature drug release at undesirable sites (Journal of Colloid and Interface Science, 2019, 535, 505). Cross-linked polymers, called nanogels, are typically stable under physiological conditions, respond to environmental stimuli, and can store large amounts of drugs and release them only under conditions found in cancer cells, such as pH ~4.5-6.5, elevated temperature, and elevated glutathione concentration (Applied Materials Today, 2017, 9, 516; Cells, 2021, 10(5), 1056).

[0005] Polymers are most often crosslinked using stable, non-degradable cross-linking agents. The most common linker is N,N'-methylenebisacrylamide. In turn, imine bonds, acylhydrazone bonds, S-S bonds, and Se-Se bonds are known to be unstable and breakable under certain conditions. Therefore, such bonds are attractive for creating degradable cross-linking agents or degradable polymers. Currently, much attention is being paid to nanogels based on degradable cross-linking agents. The degradation of such nanogels can be controlled by external stimuli such as pH, light, redox potential, or the presence of certain enzymes (Polymer, 2018, 137, 112; Journal of Controlled Release,

[0006] 2017, 259, 160; Colloids and Surfaces A, 2018, 558, 488; Reactive & Functional Polymers,

[0007] 2018, 133, 21).

[0008] Most cancer cells exhibit a reduced pH, a reducing environment, and elevated temperature (Journal of Cellular and Molecular Medicine 2019, 23, 59). Due to the above fact, nano-gels that degrade under the influence of oxidation / reduction, containing -S-S- bridges, and nano-gels that degrade at low pH, containing acetals, are very interesting. Thermosensitive nanogels based on thermosensitive polymers are also interesting as drug carriers and can be used in hyperthermia; when exposed to an external heat source, such a nanogel can shrink and release the drug previously stored within it.

[0009] It is known that the difference in redox potential in cells results from the gradient of glutathione (GSH) concentration between the inside of the cell and the concentration of GSH outside the cell. Furthermore, in some cancerous tissues, especially in drug-resistant tumours, the concentration of GSH is up to four times higher than in healthy tissues, hence many studies are devoted to obtaining redox-sensitive drug carriers (Biomarkers, 2012, 17(8): 671).

[0010] Many nanogels cross-linked with linkers containing disulphide bonds are known (Chemical Society Reviews, 2015, 44, 1948). In turn, diselenide bridges seem to be better than them. Selenium is an essential microelement that plays a key role in the human body. Most importantly, unlike the sulphur atom, the selenium atom in a diselenide bond has a larger atomic size and lower electronegativity. The bond energies of Se-Se (172 kJ mol”1) and C-Se (244 kJ mol”1) are lower than the bond energies of S-S (240 kJ mol-1) and C-S (272 kJ mol-1).

[0011] The lower binding energy of Se-Se and C-Se bonds makes them more susceptible to breaking (Macromolecules 2018, 51, 7435). Se-Se bonds can be more easily reduced by GSH or oxidised by reactive oxygen species (ROS) than sulphur bonds. It is known that ROS are present in cells affected by inflammation, including cancer cells. Therefore, nanogels based on selenium-containing linkers appear to be better drug carriers than carriers based on linkers with disulphide bonds.

[0012] Previous reports on diselenide-crosslinked nanogels concern cross-linking agents that are insoluble in water, which is why the synthesis of nanogels is carried out in toxic organic solvents (Polymer Chemistry, 2020, 11, 2360; Angewandte Chemie International Edition, 2019, 58, 9791). There is a need to develop a simple and rapid method for the synthesis of a cross-linking agent containing -Se-Se- bridges and soluble in an aqueous environment, which can be used to obtain oxidation- and reduction-sensitive nanogels.

[0013] The aim of this invention was to develop a new cross-linking agent based on selenocystine, sensitive to reduction and oxidation, for use in polymerisation reactions to obtain environmentally sensitive, degradable nanogels under the influence of glutathione, which is present in most cancer cells in elevated concentrations, as well as a method of synthesising this cross-linking agent.

[0014] This objective has been achieved by the subjects of the present invention.

[0015] Summary of the Invention

[0016] Therefore, the subject of the present invention is N,N'-bis(acryloyl)selenocystine represented by formula 1 :

[0017] Formula 1. Structural formula of N,N'-bis(acryloyl)selenocystine

[0018] Another subject of the invention is a method of manufacturing N,N'- bis(acryloyl)selenocystine with a structure represented by formula 1,

[0019] Formula 1 characterised in that it comprises the steps, in which:

[0020] - NaOH is added to seleno-L-cystine in an organic solvent and the mixture is cooled to 0°C; - acryloyl chloride is then added and left for 4 hours;

[0021] - the mixture is then brought to room temperature, filtered, and diethyl ether is added to the filtrate to precipitate a precipitate containing N,N'-bis(acryloyl)selenocystine. Preferably, the obtained precipitate containing N,N'-bis(acryloyl)selenocystine is subjected to further purification. The next subject of the invention is the use of N,N'-bis(acryloyl)selenocystine with the structure represented by formula 1:

[0022]

[0023] Formula 1 as a cross-linking agent in polymerisation reactions, preferably in polymerisation reactions of nanogels.

[0024] Another subject of the invention is a method of manufacturing a p(NIPA-BISeSe) nanogel, characterised in that it comprises the steps in which:

[0025] - in one reaction vessel, the main monomer, e.g., N-isopropylacrylamide, and N,N'- bis(acryloyl)selenocystine as a linker are dissolved in deionised water;

[0026] - in a second reaction vessel, a solution of potassium persulfate as an initiator in deionised water is prepared;

[0027] - both reaction vessels are heated to 70°C and subjected to deoxygenation;

[0028] - the monomer and linker solution are added to the second reaction vessel and the reaction is carried out for 2-4 hours, preferably 3 hours;

[0029] - the obtained nanogel is purified using dialysis.

[0030] Yet another subject of the invention is use of the p(NIPA-BISeSe) nanogel obtained by the method defined above as a carrier of drugs, preferably anticancer drugs.

[0031] Preferably, the anticancer drug is doxorubicin.

[0032] The method for synthesizing a polymeric nanogel is characterized in that a cross-linking agent as defined above is used during radical polymerisation of the monomers, wherein:

[0033] - a mixture of reactants including a cross-linking agent, a main monomer, e.g. N- isopropylacrylamide (NIPA) and a solvent is placed in the first reactor, air is removed, e.g. by using an inert gas flow, an inert gas (nitrogen or argon) is introduced and the temperature is set. The initiator and solvent are placed in the second reactor, an inert gas (nitrogen or argon) is introduced and the temperature is set, after which the radical polymerisation reaction is initiated by dropping the mixture from the first reactor into the second reactor containing the initiator; polymerisation is carried out, leading to obtaining nanogels, wherein to completely react the reactants, the reaction time must be longer than an hour, after which time the size of the gel particles usually does not change;

[0034] - the concentration of the initiator must be sufficiently low, too high a concentration of the initiator breaks / oxidises the selenide bridges and prevents obtaining nanogels;

[0035] - the drop-in rate cannot be too fast to ensure the correct degree of conversion of the monomers;

[0036] - the obtained nanogel is purified using dialysis membranes (10 kDa). Dialysis is usually performed for several days.

[0037] The radical polymerisation cross-linking agent, the method of its synthesis, and the method of synthesising exemplary environmentally sensitive and degradable nanogels using this linker are described in detail below, with reference to the accompanying figures.

[0038] Fig. 1. The spectrum of the BlSeSe linker obtained using high-resolution mass spectrometry.

[0039] Fig. 2. The spectrum of the BlSeSe linker obtained using1H NMR. nuclear magnetic resonance spectroscopy.

[0040] Fig. 3. (A) Micrographs of p(NIPA-BISeSe) nanogel and (B) its degradation products obtained using a transmission electron microscope after 24 hours of exposure of the nanogel to a 10 mM GSH solution. The nanogel was stained with uranyl acetate to visualize the distribution of negative charge. Fig. 4. Change in the particle size distribution of p(NIPA-BISeSe) nanogel after exposure to glutathione (10 mM GSH, 25°C), showing the progression of nanogel degradation over time.

[0041] Fig. 5. Hydrodynamic diameter of p(NIPA-BISeSe) nanogel measured as a function of temperature and pH at 25°C. The ionic strength was kept constant at 10 mM.

[0042] Fig. 6. DOX release profiles from p(NIPA-BISeSe) nanogel into buffer solutions at 37°C.

[0043] Fig. 7. MTT test results using MCF-7 and MCF-10A cell lines after 72 hours of cell interaction with free DOX drug, the drug-containing nanogel, and the drug-free nanogel. One-way (single-factor) ANOVA was used to test statistical significance. Differences from the control sample are indicated by the symbol while differences between groups are indicated by the symbol The difference was considered significant at p values < 0.05.

[0044] Fig. 8. Confocal microscopy images of MCF-10A and MCF-7 cell lines taken after 72 hours of incubation in the drug-containing nanogel. Cell nuclei were stained with Hoechest fluorescent dye (blue). For each cell line, separate fluorescent signals and a combined signal are shown. The red colour was emitted by DOX absorbed by the cells.

[0045] Fig. 9. Confocal microscopy images showing MCF-10A cells treated with DOX (left image) and treated with DOX nanogel (right image).

[0046] Detailed description of the invention.

[0047] The subject of the invention is an oxidation-sensitive, reduction and pH-sensitive crosslinking agent, N,N'-bis(acryloyl)selenocystine, containing two carboxyl groups and diselenide bridges, enabling the subsequent polymerisation of various types of monomers, as well as a synthetic route leading to obtaining of environmentally sensitive, degradable drug carriers.

[0048] The invention is a solution to a synthetic problem, consisting in the lack of a simple synthesis of selenium-based cross-linking agents for polymerisation in an aqueous environment, while simultaneously ensuring the stability of the particles obtained and pH sensitivity, without the need for additional comonomers during polymerisation. In the literature, only water-insoluble cross-linking agents with diselenide bridges can be found, the synthesis of which is additionally time-consuming.

[0049] An exemplary cross-linking agent containing diselenide bridges and polymerizable -C=C- double bonds is N,N'-bis(acryloyl)selenocystamine (Polymer Chemistry, 2020, 11, 2360). This linker is poorly soluble in water, therefore, for polymerisation in water, a mixture of water and an organic solvent in which the linker is highly soluble is used. Another example of a cross-linking agent containing -Se-Se- bridges is the diacryloyl derivative of bis-(11 -hydroxyundecyl) diselenide, also insoluble in water (Angewandte Chemie International Edition, 2019, 58, 9791). Furthermore, these cross-linking agents do not contain additional functional groups responsible for stability, pH sensitivity, or enabling further modification of the molecules obtained using them. The structures of these linkers are presented below.

[0050] N,N'-bis(acryloyl)selenocystamine

[0051] Diacryloyl derivative of bis-(11-hydroxyundecyl)diselenide

[0052] The use of linkers containing diselenide bridges allows for the polymerisation of various types of monomers and obtaining polymer materials that degrade under appropriate conditions. Such materials obtained using the method according to the invention, with the appropriate selection of polymers, can have various properties and be thermosensitive, thanks to which they can find a number of applications, including medical applications, as carriers for controlled drug release, base materials in tissue engineering, carriers in gene therapies, or as materials for constructing "smart membranes." The solution according to the invention is presented below in the embodiments that do not limit its scope or application.

[0053] Example 1. SYNTHESIS of the BlSeSe linker - N,N'- bis(acryloyl)selenocystine

[0054] To a solution of 516 mg of seleno-L-cystine (1.54 mmol) in 10 ml of methanol, 276 mg of NaOH was added and cooled to 0°C. 303 pl of acryloyl chloride was added dropwise to the reaction mixture and allowed to stand for 4 hours. After this time, the reaction mixture was brought to room temperature, filtered, and the filtrate was precipitated in diethyl ether. The resulting precipitate was drained and dried under reduced pressure. 820 mg of a light orange precipitate was obtained. The amount of precipitate obtained indicates the presence of an inorganic salt (NaCI) formed during the reaction. The BlSeSe content in the precipitate, ranging from 68% to 75%, was calculated based on the selenium content in the sample.

[0055] The molecular structure of BlSeSe, (CnH NiOsSei), was confirmed experimentally by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy (Fig. 1 and 2):

[0056] HRMS [M-H]“: expected M / Z = 440.92783, measured M / Z = 440.92642

[0057] 1H NMR (300 MHz, D2O): 8 6.35-6.05 (m, 2H), 5.80-5.65 (m, 1 H), 4.60-4.47 (m, 1 H), 3.40-3.35 (m, 1 H), 3.20-3.05 (m, 1 H)

[0058] 1H NMR (75 MHz, D2O): 8 176.3, 168.1, 129.8, 128.1, 55.2, 30.7.

[0059] Example 2. POLYMERISATION LEADING TO THE FORMATION OF DEGRADING p(N I PA- BlSeSe) NANOGELS

[0060] Polymerisation of p(NIPA-BISeSe) nanogels was carried out using two three-necked flasks equipped with a reflux condenser, inert gas inlet and outlet, and a magnetic stirrer. The main monomer N-isopropylacrylamide and the linker N,N'-bis(acryloyl)selenocystine (3 mol%) were dissolved in 35 ml of deionised water and placed in one of the flasks. The second flask contained 5 mg of the initiator - potassium persulfate dissolved in 15 ml of deionised water. Both solutions were heated to 70°C and deoxygenated for 30 min using argon. Then, the monomer and linker solution were added to the initiator solution using a pump (at a rate of 0.7 ml min-1). The final concentration of monomers (after they were into the reactor) and initiator (after mixing with the monomer solution) were 70 and 0.3 mM, respectively. After the addition was complete, the reaction was continued for another 3 hours. The obtained nanoparticles were purified by dialysis. For this purpose, the suspension was placed in a dialysis membrane and transferred to 5 litres of deionised water. The water was replaced daily for 4 days.

[0061] The obtained cross-linking agent was characterised by1H NMR. spectroscopy and high- resolution mass spectrometry. In turn, nanogels obtained with the participation of the cross-linking agent as a carrier for the anticancer drug doxorubicin were characterised by dynamic light scattering and electron microscopy, and drug release studies were performed using UV-Vis spectroscopy and cytotoxicity tests.

[0062] The morphology of the p(NIPA-BISeSe) nanogel examined by transmission electron microscope confirmed the formation of spherical nanogel particles ranging in size from 56 to 100 nm. The average particle size was 81 nm. In order to visualise the distribution of carboxyl groups, the nanogel was stained with uranyl acetate. It was observed that more uranyl acetate particles and thus more carboxyl groups were present on the surface of the nanogels (darker colour). Treatment of the nanogels with 10 mM glutathione (reducing agent) confirmed the reduction of diselenide bridges and thus the degradation of the nanogels. TEM micrographs of the nanogel before and after one day of interaction with glutathione are shown in Fig. 3A and 3B. The micrographs confirmed the degradation of the nanogels.

[0063] Degradation of the nanogels was also confirmed by measuring the particle size distribution after various exposure times to the reducing agent glutathione (10 mM). The measurements were performed using dynamic light scattering at room temperature. The results are presented in Fig. 4. The degradation process of the nanogels depended on the time of their interaction with glutathione. In the first stage, a slight increase in particle size was observed; the reduction of some diselenide bonds led to the loosening of the polymer network of the nanogels. As the degradation process progressed and subsequent diselenide bonds were reduced, a decrease in the size of the nanogel was observed, caused by the breakdown of the nanogels into smaller particles.

[0064] The sensitivity of the nanogel to pH, due to the presence of carboxyl groups derived from the cross-linking agent, was confirmed using dynamic light scattering. Typical changes in the hydrodynamic diameter of the nanogel as a function of temperature, measured at three selected pH values, are shown in Fig. 5. As can be seen in this figure, the hydrodynamic diameter and the volumetric phase transition temperature (VPTT) of the nanogel (from the swollen to the shrunken state) depended on pH. At low pH, the diameter of the nanogel particles and the VPTT were smaller than at higher pH. At alkaline pH, most of the carboxyl groups in the nanogel were ionised, and repulsive forces between like charges caused the nanogel to swell and an increase in the temperature required for the nanogel to shrink. In turn, at low pH, deprotonation of the carboxyl groups caused a decrease in the VPTT of the nanogels and their hydrodynamic diameter. Furthermore, as the temperature increased at a pH of approximately 2, the nanogels aggregated (a large increase in size). This was caused by a significant decrease in the negative charge of the nanogels, the presence of which is responsible for the stability of a nanogel.

[0065] The usefulness of a nanogel as a carrier for anticancer drugs was investigated by placing the model anticancer drug doxorubicin into the nanogel. Drug loading into the nanogel was performed at pH 7.4. At this pH, doxorubicin interacts with the nanogel via electrostatic forces, as ionised carboxyl groups in the nanogel derived from the crosslinking agent attract the protonated amino groups of doxorubicin. The sorption capacity of the nanogel, i.e., the ratio of the drug mass bound to the nanogel mass (DLC), was 25%. In turn, the sorption efficiency of the nanogel, i.e., the ratio of the drug bound to the mass of the drug used during sorption (EE), was approximately 81%. The effect of pH and GSH on the efficiency of DOX release from the nanogel was studied at 37°C. The results are presented in Fig. 6. Drug release depended on the pH of the solution and the presence of glutathione. At physiological pH (7.4), release was lowest - below 20%. At this pH, most DOX particles were positively charged and electrostatically bound to the ionised carboxyl groups of the nanogel. However, at pH 5.0, close to the pH of cancer cells, some carboxyl groups underwent protonation, and the electrostatic interactions between the nanogel particles and DOX were weakened, which resulted in an increase in drug release to approximately 40%. The addition of GSH caused degradation of the nanogel particles and a further increase in release. At pH 7.4, in the presence of 40 mM of GSH, the cumulative release reached approximately 70%. Most importantly, under conditions typical of most cancer cells (pH 5, 40 mM GSH), the highest drug release was observed, reaching approximately 80%.

[0066] To test the cytotoxicity of selected materials against cancer and healthy cells, an MTT assay was performed. The cells were incubated in various concentrations of the tested materials for 72 hours. MCF-7 and MCF-10A, breast cancer cells and non-cancerous epithelial cells, respectively, were selected as the cells. The assay was performed against a control sample (the cells treated with medium). The obtained results showed that the free nanogel was not toxic to the cells across the entire concentration range tested. For both MCF-10A and MCF-7 cells, the percentage of viability was approximately 80% or higher for all nanogel concentrations tested (see Fig. 7). The concentration of the tested compound is considered safe for the body if cell viability is equal to or higher than 70%. Such high cell viability values can be regarded as evidence of good biocompatibility of the unloaded / drug-free nanogel. Completely different behaviour was found for the DOX- loaded nanogels and DOX drug in free state. As seen in Fig. 7, the value of cell viability decreased with increasing drug concentration. For MCF-7 cell lines, the IC50 value was 0.13 and 0.08 pM for DOX-loaded nanogels and free DOX, respectively. The most interesting results were obtained for healthy cells - MCF-10A. When DOX alone was used, the IC50 value reached 0.16 pM. When healthy cells were treated with drug-loaded carriers, cytotoxicity significantly decreased: an IC50 value of 9.65 pM was obtained. The results show that the carrier of the present invention increases the efficacy of cancer treatment with DOX, since a lower drug concentration is required. At the same time, high protection of healthy cells bas been achieved.

[0067] To visualise the effect of the drug-loaded nanogel on selected cells, confocal microscopy measurements were also performed. The images in Fig. 8 show MCF-7 and MCF-10A cells after 72 hours of incubation in the drug-loaded nanogel. The nuclei of cells that had previously been treated with Hoechest dye emitted blue-cyan light, while DOX emitted red light. As can be seen, in healthy cells (MCF-10A), the drug accumulated mainly in the cytoplasm, while in cancer cells (MCF-7), DOX was observed in the nucleus.

[0068] To test the differences between the effects of free DOX and the nanogel doped with DOX on healthy MCF-10A cells, the experiment was also performed for DOX alone (Fig.

[0069] 9). Differences were observed between the morphology of healthy cells treated with DOX alone and a DOX-loaded nanogel. In the first case, the cells were small and round, whereas in the latter case, the cells retained their proper elongated shape. Taking into account the above research, we can conclude good effectiveness of nanogels as carriers targeting cancer cells.

[0070] Glossary of abbreviations used:

[0071] BlSeSe - N,N'-bis(acryloyl)selenocystine

[0072] NIPA - (N-isopropylacrylamide)

[0073] DLS - Dynamic light scattering

[0074] TEM - Transmission electron microscope

[0075] SEM - Scanning electron microscope pNIPA - poly(N-isopropylacrylamide)

[0076] MCF-7 - breast cancer cells

[0077] MCF-10A - normal breast cells

[0078] PTT - phase transition temperature

[0079] GSH - glutathione

[0080] DOX - doxorubicin

[0081] NMR. - nuclear magnetic resonance

[0082] DLC - drug sorption capacity

[0083] EE - encapsulation efficiency

Claims

Claims1. N,N'-bis(acryloyl)selenocystine represented by formula 1:Formula 12. A method of manufacturing N,N'-bis(acryloyl)selenocystine with a structure represented by formula 1,Formula 1 characterised in that it comprises the steps, in which:- NaOH is added to seleno-L-cystine in an organic solvent and the mixture is cooled to 0°C;- acryloyl chloride is then added and left for 4 hours;- the mixture is then brought to room temperature, filtered, and diethyl ether is added to the filtrate to precipitate a precipitate containing N,N'-bis(acryloyl)selenocystine.

3. A method according to claim 2, characterised in that the obtained precipitate containing N,N'-bis(acryloyl)selenocystine is subjected to further purification.

4. Use of N,N'-bis(acryloyl)selenocystine with the structure represented by formula 1 :Formula 1 as a cross-linking agent in polymerisation reactions, preferably in polymerisation reactions of nanogels.

5. A method of manufacturing a p(NIPA-BISeSe) nanogel, characterised in that it comprises the steps in which:- in one reaction vessel, the main monomer, e.g., N-isopropylacrylamide, and N,N'- bis(acryloyl)selenocystine as a linker are dissolved in deionised water;- in a second reaction vessel, a solution of potassium persulfate as an initiator in deionised water is prepared;- both reaction vessels are heated to 70°C and subjected to deoxygenation;- the monomer and linker solution are added to the second reaction vessel and the reaction is carried out for 2-4 hours, preferably 3 hours;- the obtained nanogel is purified using dialysis.

6. Use of the p(NIPA-BISeSe) nanogel obtained by the method defined in claim 5 as a carrier of drugs, preferably anticancer drugs.

7. The use according to claim 6, wherein the anticancer drug is doxorubicin.

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