Hydration method of a water-insoluble polymer capable of containing intermediate water

Abstract

Provided is a means for causing a water-insoluble polymer, which is capable of containing intermediate water through hydration, to be hydrated using a method different from conventional methods.　This method for hydrating a water-insoluble polymer capable of containing intermediate water is characterized by comprising: a solution production step for dissolving a water-insoluble polymer, which is capable of containing intermediate water through hydration, in a polar organic solvent to obtain a solution; and a precipitation step for mixing the solution with an aqueous phase to hydrate and precipitate the water-insoluble polymer.

Description

Technical Field

[0001] The present invention relates to a method for hydrating a water-insoluble polymer capable of containing intermediate water, and a hydrate of the water-insoluble polymer obtained by the hydration method. This application claims priority based on Japanese Patent Application No. 2019-206564 filed in Japan on November 14, 2019, and incorporates its content by reference.

Background Art

[0002] Generally, when a biological component such as blood comes into contact with the surface of various materials, the surface of the material is recognized as a foreign substance, and non-specific adsorption, denaturation, multi-layer adsorption, etc. of proteins in biological tissues occur. As a result, it is known that activation of the coagulation system, complement system, platelet system, etc. occurs. On the other hand, it is known that on the surface generated when a polymer having a specific structure is hydrated, non-specific adsorption of the above proteins is suppressed, and as a result, biocompatibility such as difficulty in platelet adhesion is expressed.

[0003] When the polymer showing the above biocompatibility is hydrated, it has been clarified that water molecules can be contained in a form commonly called "intermediate water" (freezing-bound water, intermediate water). Intermediate water is characterized by the transfer of latent heat accompanying the regularization / irregularization of water molecules in the temperature range below the freezing point, and is understood as water molecules in a state showing intermediate properties with respect to unfrozen water strongly bound to the substance surface and free water hardly bound by the substance surface. It has been clarified that such intermediate water is also observed in various bio-derived substances in addition to the above polymers, and is considered to play an important role in the expression of biocompatibility (for example, see Non-Patent Document 1).

[0004] Regarding the mechanism by which water molecules in the form of intermediate water are generated when a polymer having a specific structure contains water, it has been clarified that the high molecular mobility exhibited by the polymer having the specific structure is related (for example, see Non-Patent Document 2). That is, it is considered that the high molecular mobility exhibited by the polymer when it contains water is the cause of the generation of water molecules in the form of intermediate water, and as a result, biocompatibility is exhibited.

[0005] On the other hand, the present inventors have found that even on a surface where it is difficult for blood components to adhere due to containing intermediate water, tumor cells such as metastatic cancer cells, stem cells, vascular endothelial cells, etc. contained in blood or the like can selectively adhere, and it is possible to selectively separate such cell types from blood or the like (Patent Document 1). Furthermore, it has been clarified that a surface containing intermediate water at a predetermined ratio is suitable for culturing various cells (Patent Document 2).

[0006] By utilizing various phenomena occurring between a polymer that retains intermediate water at a predetermined ratio as described above and various biological substances, the use of the polymer is expected in various applications other than imparting biocompatibility to the surface of medical devices.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] As described above, polymers capable of containing intermediate water by hydration are hydrophilic, and furthermore, many of them are known to exhibit water solubility. For example, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and methacryloyl phosphatidylcholine (MPC), which are known as polymers exhibiting biocompatibility, all exhibit water solubility. Therefore, when attempting to form a surface exhibiting biocompatibility using the above-mentioned PEG, MPC, etc., it is common to use a method such as forming a copolymer with other water-insoluble components (for example, see Patent Document 3, etc.) and fixing it in a film form on the surface of the substrate for use.

[0010] On the other hand, it is known that there are polymers such as poly(2-methoxyethyl acrylate) (PMEA) that exhibit biocompatibility by being hydrated and capable of containing intermediate water, while also exhibiting water insolubility. And for such water-insoluble biocompatible polymers, even in the state of a homopolymer, they can be dissolved in an organic solvent or the like and applied and fixed in a film form on the surfaces of various substrates for the purpose of forming a surface exhibiting biocompatibility.

[0011] However, for example, when a water-insoluble biocompatible polymer such as the above-mentioned PMEA is applied and fixed in a film form on the surface of a substrate, it takes a long time to bring water into contact with the PMEA film to make it saturated with water. That is, there is a trade-off relationship between being water-insoluble and containing intermediate water by hydration. And means for promoting the hydration of such water-insoluble biocompatible polymers are desired.

[0012] An object of the present invention is to provide a means for causing hydration of a water-insoluble polymer capable of containing intermediate water by hydration by a method different from the conventional method, and to provide a water-insoluble polymer hydrated by the method.

Means for Solving the Problems

[0013] In order to solve the above problems, the present invention has the following features. <1> A method for hydrating a water-insoluble polymer capable of containing intermediate water, comprising a solution generation step of dissolving a water-insoluble polymer capable of containing intermediate water by hydration in a polar organic solvent to obtain a solution, and a precipitation step of hydrating and precipitating the water-insoluble polymer by mixing the solution with an aqueous phase. <2> The method for hydrating a water-insoluble polymer capable of containing intermediate water, wherein the water-insoluble polymer capable of containing intermediate water by hydration is a polymer having at least a part of a side chain portion containing a chain ether structure or a cyclic ether structure with respect to the main chain skeleton. <3> The method for hydrating a water-insoluble polymer capable of containing intermediate water, wherein in the precipitation step, the water-insoluble polymer is precipitated in a colloidal state. <4> The method for hydrating a water-insoluble polymer capable of containing intermediate water, wherein the precipitation step is to mix the solution and the aqueous phase through a semipermeable membrane. <5> The method for hydrating a water-insoluble polymer capable of containing intermediate water, wherein a fat-soluble drug is further dissolved in the solution. <6> A composition containing hydrated water-insoluble polymer molecules, wherein the water-insoluble polymer capable of containing intermediate water by hydration is hydrated and precipitated by mixing a solution formed by dissolving the water-insoluble polymer in a polar organic solvent with an aqueous phase. <7> The composition containing the hydrated water-insoluble polymer molecules having a particle shape. <8> The composition containing the hydrated water-insoluble polymer molecules having an average particle diameter of 100 μm or less.

Advantages of the Invention

[0014] By hydrating a water-insoluble polymer capable of containing intermediate water by a method different from the conventional method, the water-insoluble polymer can be rapidly hydrated and made into fine particles.

Brief Description of the Drawings

[0015]

Figure 1

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Embodiments for Carrying Out the Invention

[0016] (1) Regarding polymers suitable for hydration by the method according to the present invention For example, there are polymers that are water-insoluble but can contain intermediate water by hydration, such as PMEA and its analogs, and it is known that these polymers dissolve in specific polar solvents depending on the structure of each polymer. As described above, the present invention is characterized in that, for a polymer that is water-insoluble but can contain intermediate water by hydration, a solution in which the polymer is dissolved in a polar organic solvent capable of dissolving the polymer is used, and the polymer is hydrated in the process of mixing the solution with an aqueous phase.

[0017] In the hydration method according to the present invention, any polymer can be used without particular limitation as long as it exhibits water-insolubility when brought into contact with water in a synthesized bulk state, undergoes hydration with a predetermined amount of water molecules, and the presence of intermediate water is confirmed inside the polymer due to the hydration. As a polymer suitable for hydration by the method according to the present invention described above, in particular, those having a main chain such as an acrylic skeleton, a methacrylic skeleton, a polycarbonate skeleton, an alkylene skeleton, etc., and mainly having a chain ether structure or a cyclic ether structure, which are structural units constituting PEG, in their side chain portions, and containing a structure that contributes to the inclusion of intermediate water, are preferably used as polymers containing intermediate water when hydrated.

[0018] Examples of the above polymers include those having a (meth)acrylic skeleton and a chain ether structure in the side chain portion, such as those represented by the following formula (1). In the polymer represented by formula (1), a chain ether structure, which is a chain alkyl oxide (CH2-CH2-O), a constituent unit of PEG, is added to the main chain of the (meth)acrylic skeleton by an ester bond, and it is known that intermediate water can be contained in many structures. Examples of the polymer represented by formula (1) include poly(2-ethoxyethyl acrylate), poly(2-methoxyethyl acrylate), poly[2-(2-methoxyethoxy)ethyl methacrylate], poly[2-(2-ethoxyethoxy)ethyl acrylate], poly[2-(2-methoxyethoxy)ethoxy]ethyl methacrylate], poly[2-(2-(2-methoxyethoxy)ethoxy)ethyl acrylate], poly[2-(2-ethoxyethoxy)ethyl methacrylate], PMe2A (poly(2-(2-methoxyethoxy)ethyl acrylate-co-butyl acrylate)), PMe3A (poly(2-(2-methoxyethoxy)ethoxyethyl acrylate-co-butyl acrylate)), poly(2-(2-ethoxyethoxyethyl)acrylate), poly(2-ethoxyethyl vinyl ether), poly(tetrahydrofuran-3-ylmethyl acrylate), poly[2-(methoxyethoxy)ethyl methacrylate], etc. The polymer represented by the above formula (1) may be used as a polymer obtained by polymerizing only monomers having a single structure, or may be used as a copolymer containing a plurality of unit structures.

[0019] [Chemical formula] [In the formula, R 1 is a hydrogen atom or a methyl group, R 2 is a methyl group or an ethyl group, and n is from 1 to 3]

[0020] Among the polymers represented by the above formula (1), in particular, poly(2-methoxyethyl acrylate (PMEA)) represented by the following formula (2) and the like are particularly preferably used because they have excellent biocompatibility and have already been used in a plurality of bio-related applications. Further, PMe2A with the repeating number of ethylene glycol (n in formula (1)) in the side chain of PMEA being n = 2 and PMe3A with n = 3 are preferably used because they can contain intermediate water in a large proportion.

[0021]

Chemical formula

[0022] Further, examples of the above polymer include those having a vinyl skeleton and having a chain ether structure or a cyclic ether structure in the side chain portion, such as those represented by the following formulas (3) and (4). In the polymers represented by formulas (3) and (4), they have a repeating unit in which a side chain portion containing an ether structure is bonded to the main chain of the vinyl skeleton by an ether bond. The ether structure is a structure formed by a polyoxyalkylene group which may have a substituent, and examples thereof include a chain ether which is a chain alkyl oxide and a cyclic ether as one form thereof.

[0023]

Chemical formula

[0024] In general formula (3), R 3 is a linear or branched alkyl group having 1 to 4 carbon atoms, and preferably, R 3 means any one of CH2, C2H4, C3H6, or C4H8. Further, R 4 is H or a linear or branched alkyl group having 1 to 4 carbon atoms, and preferably, R 4means any one of H, CH3, C2H5, C3H7, or C4H9. m is a natural number from 1 to 10, preferably in the range of 1 to 4, and more preferably 1 or 2. Here, the part of (R 3 -O) is a part showing a chain ether structure which is a unit structure such as PEG.

[0025] In the polymer compound according to the present invention, R 3 , R 4 , and the m value can include repeating units represented by the general formula (3) that are different from each other. That is, in the polymer compound according to the present invention, for the main chain mainly composed of carbon, a monoether (m = 1) terminated with hydrogen or an alkyl group at the end, or a polyether (m ≧ 2) can be included as the organic component (R) of silsesquioxane in a structure bonded by an ether bond so as to form a side chain. For example, when R 3 is C2H4, the polymer compound of the present invention has a structure in which a chain ether (C2H4-O) which is a constituent unit of PEG terminated with an alkyl group or the like is bonded to the main chain by an ether bond. Examples of the polymer represented by the formula (3) include methoxyethyl vinyl ether and the like.

[0026] [Chemical formula]

[0027] In the general formula (4), R 5 has a structure selected from either CH2 or C2H4. Also, R 6 O k is a cyclic ether of any one of a 3-membered ring to a 6-membered ring, and the number of oxygen atoms (k) contained in the cyclic ether is k ≧ 1. Also, in the present invention, R 5 , R 6Those containing any hydrogen contained therein replaced by at least one of -OH, CH3, and C2H5 are included. That is, the repeating unit in this form has a structure in which a cyclic ether is bonded to the main chain by an ether bond.

[0028] In addition, by hydrating a polymer that exhibits biodegradability by having a polycarbonate skeleton in the main chain portion with the hydration method according to the present invention to form fine particle shapes, polymer fine particles that are particularly preferably used for drug delivery applications can be obtained.

[0029] Table 1 shows the solubility of PMEA and its analogs as examples of the above polymers in various solvents. Each n value in Table 1 means the n value in a polymer having the structure represented by formula (5) in the side chain portion.

[0030] [Chemical formula]

[0031] [Table 1]

[0032] As shown in Table 1, the polymer represented by formula (1) is known to dissolve in a specific polar solvent that is compatible with water depending on its structure. In addition to the solvents shown in Table 1, various polymers are soluble in polar solvents such as DMSO, THF, propanol, acetone, DMF, and acetonitrile, and can be mixed with water in an indefinite ratio.

[0033] (2) Regarding the hydration method of the water-insoluble polymer according to the present invention According to the hydration method of the present invention, it is possible to complete hydration in a shorter time compared to the method of hydrating a water-insoluble polymer formed in a film or the like. Further, by adjusting the conditions for hydrating the polymer dissolved in a polar organic solvent, it is possible to precipitate the hydrated polymer in the form of fine particles, and various functions can be imparted.

[0034] The mechanism by which the present invention can hydrate a water-insoluble polymer in a relatively short time is considered as follows. For example, when trying to hydrate the above PMEA or the like formed in a particulate or film form by bringing it into contact with water, since PMEA or the like is water-insoluble and non-permeable, the process of supplying water molecules to the PMEA molecules present inside the particles is that the water molecules contained in the PMEA molecules hydrated near the surface dissociate from the PMEA molecules and rehydrate with other PMEA molecules present deeper inside. Since it is rate-determined by the rate of such elementary steps, it is considered that a long time is required to hydrate the whole.

[0035] On the other hand, in the present invention, since the polymer is dissolved in a predetermined polar solvent and water molecules are supplied through the polar solvent to cause hydration, the supply of water molecules to the polymer molecules is rate-determined only by the diffusion rate of water molecules in the polar solvent. Since the diffusion of these water molecules proceeds rapidly, it is considered that the polymer can be hydrated in a relatively short time.

[0036] Furthermore, in the hydration method of the water-insoluble polymer according to the present invention, after supplying water molecules (aqueous phase) to the polar solvent in which the polymer is dissolved by an appropriate method to hydrate the polymer in the polar solvent, a sufficient amount of the aqueous phase is further supplied to substantially replace the solvent in which the polymer hydrate exists with the aqueous phase, thereby causing the solubility of the hydrate of the water-insoluble polymer to be lost and precipitating it.

[0037] In the present invention, the polar organic solvent used for dissolving the water-insoluble polymer can be used without particular limitation as long as it can dissolve the polymer and shows compatibility with water. In particular, methanol, DMSO, THF, propanol, acetone, DMF, acetonitrile, etc. are preferably used as those in which various polymers show solubility and compatibility with water.

[0038] With respect to the water-insoluble polymer handled in the present invention, the above polar solvent is a good solvent, and the aqueous phase corresponds to a poor solvent. Then, by mixing a solution in which a water-insoluble polymer or the like is dissolved in a good solvent with a poor solvent, the means of diffusing the good solvent into the poor solvent via an emulsion state and precipitating the polymer or the like as dried fine particles is known as the so-called "emulsion solvent diffusion method" or the like as a method for producing spherical solid fine particles. In the present invention, the elementary process by which the water-insoluble polymer dissolved in the polar organic solvent precipitates upon mixing with the aqueous phase is not necessarily clear, but depending on the surface tension between the two phases and the mutual diffusion coefficient when the polar organic solvent phase and the aqueous phase are mixed, etc., it is considered that the hydrated water-insoluble polymer precipitates in a spherical form via an emulsion state or the like.

[0039] The mixing of the polar organic solvent phase in which the water-insoluble polymer is dissolved and the aqueous phase can be carried out under appropriate conditions. As the polar organic solvent phase in which the water-insoluble polymer is dissolved, for example, by using a polar organic solvent phase in which various polymers are dissolved at a ratio of 0.1 to 1 wt%, rapid hydration can occur when the aqueous phase is mixed, and fine hydrates can be precipitated. In particular, by using a polar organic solvent phase in which the water-insoluble polymer is dissolved at a ratio of about 0.2 to 0.5 wt%, particles of about 0.1 to 100 μm can be stably produced as precipitates.

[0040] On the one hand, when the dissolved amount of the water-insoluble polymer in the polar organic solvent phase is about 0.1 wt% or less, it becomes difficult for aggregation to occur between the precipitated polymers, and thus there is a tendency that particulate precipitates are hardly obtained. On the other hand, when the dissolved amount of the water-insoluble polymer in the polar organic solvent phase is about 1 wt% or more, the aggregation between the precipitated polymers becomes remarkable, and there is a tendency to produce coarse precipitates. When producing particulate precipitates using a polar organic solvent phase in which the water-insoluble polymer is dissolved, it is preferable to use components for nucleation and surfactant components for stabilizing the fine particle shape in order to facilitate the formation of particulate precipitates.

[0041] For the polar organic solvent phase in which the above water-insoluble polymer is dissolved, for example, an aqueous phase of about the same volume is prepared, and by pouring the polar organic solvent phase into the aqueous phase, the polymer can precipitate in a colloidal state to obtain a turbid and uniform liquid phase. Alternatively, by pouring the aqueous phase into the polar organic solvent phase, a similar turbid and uniform liquid phase can also be obtained.

[0042] The polymer precipitated in particulate form by the above means typically has a particle diameter of about 0.1 to 100 μm as observed under a microscope and can stably exist in the aqueous phase for a long time. Preferably, after substantially replacing the liquid phase with an aqueous phase by supplying an additional aqueous phase to the liquid phase turbid due to the precipitation of the polymer, the precipitated polymer is concentrated by appropriate means such as centrifugation, and thus it can be used for various applications.

[0043] Also, in the mixing of the polar organic solvent phase in which the above polymer is dissolved and the aqueous phase, operations such as mixing a particularly small amount of the polar organic solvent phase into the aqueous phase are performed to cause a rapid replacement of the organic solvent and water, so that the precipitated polymer can be made to precipitate on the surface of the liquid phase instead of being dispersed in the liquid phase as described above, and subsequent recovery and use for various applications can be facilitated.

[0044] On the other hand, for a polymer solution in which a polymer is dissolved in a polar organic solvent, for example, by bringing an aqueous phase into contact with and mixing it through a semipermeable membrane or the like, a time lag for the reaggregation of hydrated and precipitated polymer molecules is ensured, which is effective in stably precipitating the hydrated polymer as fine particles. At that time, by dissolving a fat-soluble and hydrophobic low-molecular substance in the polar organic solvent, it is possible to carry the substance in the precipitated polymer particles. By utilizing this phenomenon, it is also possible to use the polymer precipitated according to the present invention as fine particles for drug delivery by using a polar organic solvent in which a substance that functions as a drug by exhibiting predetermined characteristics is dissolved.

[0045] The semipermeable membrane used above is not particularly limited as long as it is a membrane through which both the polar organic solvent and water can pass, and it can be a regenerated cellulose, acetyl cellulose, polyacrylonitrile, PTFE, polyester-based polymer alloy, or a porous membrane of polysulfone, etc., and a semipermeable membrane that can be used as a dialysis membrane can be used.

[0046] Any polymer that can contain intermediate water by hydration and has substantially no solubility in the aqueous phase can be hydrated and precipitated according to the present invention. For example, the polymers described in Table 1 above are water-insoluble and can be hydrated by the method according to the present invention. Also, for example, even a polymer that shows water solubility in the state of a homopolymer can be hydrated and precipitated according to the present invention by making it a copolymer or the like to be a polymer that has substantially no solubility in the aqueous phase.

[0047] (3) Use of the water-insoluble polymer hydrated according to the present invention As described in Patent Documents 1 and 2 above, on the surface of a polymer that contains intermediate water by hydration, the presence of the intermediate water exhibits an excellent function as a biointerface. The polymer hydrated according to the present invention can also be preferably used for forming a surface that requires biocompatibility as conventionally known.

[0048] For example, the surface obtained by applying the polymer hydrate hydrated by the present invention to the substrate surface can be preferably used as a support for cell culture that can adhere and maintain cells in a preferred form. That is, the support for cell culture composed of the surface obtained by applying the polymer hydrate hydrated by the present invention to the substrate surface can be used without particular limitation for cells that adhere and live on the substrate, such as epidermal cells, vascular endothelial cells, oral endothelial cells, esophageal epithelial cells, gastric epithelial cells, gastrointestinal epithelial cells such as intestinal epithelial cells, nasal mucosal epithelial cells, tracheal epithelial cells, respiratory epithelial cells such as alveolar epithelial cells, exocrine gland cells such as sweat gland cells, sebaceous gland cells, apocrine gland cells, mammary gland cells, salivary gland epithelial cells, lacrimal gland cells, pancreatic islet cells of Langerhans, adrenal medulla cells, adrenal cortex cells, pineal gland cells, pituitary gland cells, endocrine gland cells such as thyroid cells, hepatocytes, renal epithelial cells, pancreatic cells, adrenal cells and other visceral parenchymal cells, taste bud cells, olfactory epithelial cells, sensory organ cells such as hair cells, nerve cells and glial cells such as astrocytes, Schwann cells, muscle cells such as cardiomyocytes, skeletal muscle cells, smooth muscle cells, fibroblast cells, stromal cells, connective tissue cells, chondrocytes, osteoblasts and other mesenchymal cells, thymic epithelial cells, uterine epithelial cells, ovarian follicle cells, fallopian tube epithelial cells, seminiferous tubule epithelial cells, Leydig cells and other cells can be applied to cell culture.

[0049] In addition, the support for cell culture composed of the surface obtained by applying the polymer hydrate hydrated by the present invention to the substrate surface can be used for culturing various stem cells such as pluripotent stem cells having differentiation potential such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic carcinoma cells (EC cells), embryonic germ stem cells (EG cells), nuclear transfer ES cells, somatic cell-derived ES cells, hematopoietic stem cells, bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, other stromal-derived stem cells, Muse cells, neural stem cells and other tissue stem cells, pluripotent stem cells, and progenitor cells in various tissues such as the liver, pancreas, adipose tissue, bone tissue, cartilage tissue, etc.

[0050] In the culture of stem cells using a cell culture support composed of a surface obtained by applying a hydrated polymer hydrate to the surface of a substrate according to the present invention, due to the ability to adhere and maintain cells in a preferred form, etc., promotion or suppression of differentiation occurs according to the characteristics of the stem cells to be cultured, so that cell culture in accordance with the purpose of culture is possible.

[0051] In addition, fine particles composed of a water-insoluble polymer capable of containing intermediate water hydrated by the method according to the present invention exhibit selective adsorption properties such as selectively adsorbing to highly adhesive cells present in body fluids such as blood when compared with the surfaces of normal cells such as human dermal fibroblasts (NHDF). This property can be utilized for various purposes. For example, as described above, by loading a predetermined drug or the like into the precipitated polymer fine particles, it can be used as a carrier for drug delivery targeting a predetermined cell type.

[0052] In addition, when polymer fine particles precipitated by the method according to the present invention, particularly fine particles having a particle diameter of several μm or less, are used, they are also useful as a carrier for drug delivery targeting the above-mentioned predetermined cell type in that they exhibit the property of being efficiently incorporated into the adhered cells.

[0053] Examples of the highly adhesive cells present in body fluids such as the above-mentioned blood include metastatic cancer cells (CTC) contained in blood or the like, and tumor cells such as leukemia cells. It can also be used for the detection of cells such as stem cells, vascular endothelial cells, nerve cells, macrophages, dendritic cells, monocytes, neutrophils, smooth muscle cells, fibroblasts, cardiomyocytes, skeletal muscle cells, hepatocytes, non-parenchymal liver cells, pancreatic islet cells, and the detection of other biomarkers. Hereinafter, the method according to the present invention will be described in more detail using examples. Note that the examples shown below are one form of the present invention, and the present invention is not limited by the examples.

Examples

[0054] By the method shown below, a solution in which PMEA (poly(2-methoxyethyl acrylate)), a polymer that can contain intermediate water and exhibits water-insolubility, is dissolved in various polar organic solvents was mixed with an aqueous phase (purified water) to hydrate and precipitate PMEA dissolved in the polar organic solvent.

[0055] The PMEA used was synthesized by a known method (for example, refer to JP-A-2012-105579). The PMEA was put into methanol, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF), which are polar organic solvents, so as to have a concentration of 0.2 wt% and dissolved. PMEA dissolved in all of the solvents, and a transparent PMEA solution was produced.

[0056] Each of the above PMEA solutions (1 mL) was dropped into the same volume of purified water at a rate of 0.1 mL / second using a mechanical pipette (hereinafter, the mixing by this method may be referred to as "Oil in Water"). The mixed solutions produced by dropping each PMEA solution into purified water all became cloudy in a colloidal state.

[0057] Conversely, the same volume of purified water as each PMEA solution (1 mL) used was dropped into each PMEA solution at a rate of 0.1 mL / second using a mechanical pipette (hereinafter, the mixing by this method may be referred to as "Water in Oil"). The mixed solutions produced by dropping purified water into each PMEA solution all became cloudy in a colloidal state.

[0058] Fig. 1 shows the state of the mixed solutions obtained by mixing purified water and the PMEA solution above. (a) in Fig. 1 shows the mixed solution when the PMEA solution was mixed in purified water (Oil in Water), and (b) shows the mixed solution when purified water was mixed in the PMEA solution (Water in Oil). It can be seen that all of the mixed solutions obtained above became cloudy in a colloidal state. A tendency was observed for the state of the colloids produced to change depending on the difference in the polar organic solvents used and the difference in the mixing method. Figure 2 shows the phase-contrast microscopic image of the white turbid aggregates obtained by mixing purified water and PMEA solution. Each scale bar in Figure 2 indicates a length of 10 μm. It was confirmed that spherical aggregates with a particle diameter of about 0.1 to several tens of μm were formed regardless of the preparation method. Also, due to the differences in the polar organic solvents used and the mixing methods, there was a tendency for the average particle diameter and the state of particle diameter dispersion to change.

[0059] The PMEA solution (DMSO) was dropped into purified water (Oil in Water) to form a colloidal white turbid state, and it was allowed to stand at room temperature for a predetermined time (10 minutes, 2, 6, 24 hours), and then centrifuged at 1,500 rpm for 3 minutes to precipitate the white turbid aggregates and remove the supernatant. Further, after sufficiently removing the excess water adhering to the precipitate with filter paper, 1H NMR measurement was performed on the sample dissolved in deuterated chloroform.

[0060] Figure 7 shows, as an example of the results of 1H NMR measurement, the results (B) for the white turbid aggregates allowed to stand for 2 hours after colloidal purification, compared with the results (A) of 1H NMR measurement of a PMEA solution in deuterated chloroform. In the results (A) of 1H NMR measurement of the PMEA solution in deuterated chloroform, peaks (a - d) derived from the structures of each part of PMEA and peaks derived from H2O mixed in due to the moisture absorption of PMEA are observed. On the other hand, in the results (B) of 1H NMR measurement for the above-mentioned white turbid aggregates, in addition to the peaks (a - d) derived from the above PMEA, peaks derived from DMSO remaining in the white turbid aggregates are observed, and it is observed that the peak intensity of H2O-derived peaks becomes stronger.

[0061] From the results shown in Figure 7, it was shown that the white turbid aggregates obtained in Example 1 above were mainly composed of PMEA and water molecules, and it was considered that they were hydrates in which PMEA was hydrated. Figure 8 shows a graph plotting the number of H2O and DMSO molecules present per side chain of PMEA, calculated from the integral intensity ratios of the peaks at 3.36 ppm (derived from the methoxy group of PMEA), 1.60 ppm (derived from H2O), and 2.61 ppm (derived from DMSO) in the 1H NMR measurement of the above precipitate, against the time of retention as a colloid. As shown in Figure 8, regardless of the elapsed time after colloid formation, it was shown that about 5 molecules of water were contained per side chain of PMEA, and it was considered that the hydration of PMEA was completed immediately after mixing the DMSO solution of PMEA and the aqueous phase. When the film-like PMEA was saturated with water, the water content was about 9 wt%, whereas the water content in the above-mentioned white turbid aggregates corresponded to about 40 wt%. This indicates that according to the hydration method of the present invention, in addition to being able to hydrate the water-insoluble polymer in a short time, it is possible to hydrate a larger proportion of water molecules.

Example

[0062] In the methods shown below, a polymer solution in which PMEA, a polymer that can contain intermediate water by hydration and is water-insoluble, and various analogs thereof are dissolved in methanol, and an aqueous phase (purified water) were mixed through a dialysis membrane to hydrate and precipitate each polymer. By mixing the polymer solution and purified water through a dialysis membrane, the mixing rate can be adjusted, and by using an excessive amount of purified water, it is possible to substantially recover the precipitate in the aqueous phase.

[0063] PMEA, PMC3A (poly(3-methoxypropyl acrylate)), PEEA (poly(2-ethoxyethyl acrylate)), PEt2A (poly(2-(2-ethoxyethoxy)ethyl acrylate)), PEt2MA (poly(2-(2-ethoxyethoxy)ethyl acrylate)), and PTHFA (poly(tetrahydrofurfuryl acrylate)) used for evaluation were each dissolved in methanol to a concentration of 0.2 wt% and filtered through a 0.2 μm filter, and these were used as each polymer solution. 5 mL of each polymer solution was placed in a dialysis membrane made of regenerated cellulose (molecular weight fractionation: 3,500) and dialyzed by immersing it in a large excess (about 20 times) of purified water. The purified water as the external solvent was exchanged 4 times, and dialysis was carried out for a total of 2 days. Colloidal turbidity was observed inside the dialysis membrane after dialysis, regardless of which polymer was used.

[0064] Figure 3 shows the phase contrast microscope images of the white turbid aggregates formed inside the dialysis membrane described above. Each scale bar in Figure 3 indicates a length of 20 μm. Regarding the white turbid aggregates precipitated when using the 0.2 wt% PMEA solution, the Feret diameter by microscopic observation was about 1.2 μm, the particle diameter by dynamic light scattering measurement (DLS) was about 0.3 - 1.5 μm, and the particle diameter by volume measurement using a flow cytometer was about 5 μm.

[0065] Also, the white turbid aggregates precipitated using the 0.2 wt% PMEA methanol solution were freeze-dried and dried under reduced pressure, and then the mass of the remaining PMEA polymer was measured. As a result, the content of PMEA in the white turbid aggregates was about 1.56 mg / mL, indicating that PMEA was hydrated. In addition, the colloidal solution inside the dialysis membrane after dialysis was diluted 10 - 100 times in water, phosphate buffered saline (PBS), and cell culture medium (containing 10% fetal bovine serum) and held for 1 week. As a result, the spherical shape was not broken and the particle size was maintained in all cases. Note that Figure 3 above shows the state when the colloidal solution inside the dialysis membrane after dialysis was diluted in PBS.

Example

[0066] The degree of accumulation of the white turbid aggregates containing each polymer obtained in Example 2 was evaluated against human cervical cancer HeLa cells and human normal fibroblasts (NHDF).

[0067] HeLa cells and NHDF were adjusted to 1.0×10 5Seeded in a 24-well plate dish to form (cells / well) and left standing in an incubator for 24 hours (37 °C, 5% CO2). The culture medium used was DMEM / F12 (containing 10% fetal bovine serum, penicillin, and streptomycin). To the above HeLa cells and NHDF cells, a turbid aggregate containing each of the above polymers was added so that the polymer concentration became 150 μg / mL, and the cells were left standing in an incubator for 24 hours to attempt adsorption of the turbid aggregate to each cell.

[0068] Figure 4 shows the phase-contrast microscope images of each cell after adding the turbid aggregate and leaving it standing. Figure 4(a) shows HeLa cells, and Figure 4(b) shows NHDF cells. Also, the scale bars in Figure 4 each indicate a length of 10 μm. As shown in Figure 4, among the polymers evaluated, PMEA, PEt2A, and PEt2MA showed excessive accumulation on HeLa cells, while no similar accumulation was observed on NHDF cells. Instead, a distribution avoiding the cells was observed. Also, accumulation was observed in human lung cancer cells A549 and human fibrosarcoma HT1080 with the turbid aggregates containing the above polymers. On the other hand, no accumulation was observed for PMC3A, PTHFA, or PEEA on either HeLa cells or NHDF cells.

[0069] From the above results, it is considered that the turbid aggregates containing PMEA, PEt2A, and PEt2MA have cancer cell-selective accumulation. As described in Patent Document 1, it is known that the cell types that can be adsorbed change according to the amount of intermediate water contained in polymers and the like. The above results are considered to show that the accumulation on various cells changed according to the amount of intermediate water contained in the polymer hydrated by the hydration method according to the present invention.

Example

[0070] Each water-insoluble polymer used in Example 2 is a polymer that exhibits biocompatibility by containing a predetermined amount of intermediate water when hydrated. On the other hand, the white turbid aggregates containing each polymer precipitated by hydration in Example 2 are hydrated in a state dissolved in a polar solvent, so there is a possibility that the polar solvent remains in the white turbid aggregates and exhibits toxicity to cells. Therefore, below, the hemolytic test and the evaluation of cell viability were performed on the white turbid aggregates containing each polymer obtained in Example 2, and the safety of the white turbid aggregates was evaluated.

[0071] (a) Hemolytic test Red blood cells (4×10 8 cells) derived from human whole blood (purchased blood) were diluted in Hank's buffer and seeded in a 96-well plate dish. A dilution of the white turbid aggregates containing each polymer obtained in Example 2 in Hank's buffer was mixed with the red blood cells derived from human whole blood diluted in the above Hank's buffer so that each polymer concentration was ~1000 μg / mL, and left standing in an environment at 37°C for 2 hours. Thereafter, the 96-well plate dish was centrifuged at 1,500 rpm, and the absorbance of the supernatant at a wavelength of 540 nm was measured. Taking the absorbance when treating red blood cells with 2% sodium dodecyl sulfate showing high hemolysis as 100%, the hemolysis rate (%) shown by the white turbid aggregates containing each polymer was calculated. At that time, Triton X-100 used as a positive control for hemolysis was used as a comparison target.

[0072] Figure 5A shows the results of the hemolytic test shown by the white turbid aggregates containing each polymer. As shown in Figure 5A, no hemolysis was observed even when the white turbid aggregates containing each polymer used were present at a concentration of 1000 μg / mL.

[0073] (b) Cytotoxicity test HeLa cells were seeded at 1.0×10 4 (cells / cm 2) were seeded into 96-well plate dishes and left standing in an incubator for 24 hours (37 °C, 5% CO2). Next, the cloudy aggregates containing PMEA obtained in Example 2 were added to the above HeLa cells to a polymer concentration of ~3 mg / mM and left standing in an incubator for 24 hours. The intracellular metabolic activity of HeLa cells coexisting with the cloudy aggregates containing PMEA at each polymer concentration was measured using a commercially available cell counting kit-8 (DOJINDO), and the cell viability was evaluated.

[0074] Figure 5B shows the viability of HeLa cells coexisting with the cloudy aggregates containing PMEA. As shown in Figure 5B, in the range of the polymer concentration of PMEA from 0.01 to 3 mg / mM, the viability of HeLa cells coexisting with the cloudy aggregates containing PMEA did not show a decrease. Therefore, it was considered that the cytotoxicity of the cloudy aggregates containing PMEA was not recognized.

[0075] As described above, regarding the cloudy aggregates generated when hydrating a water-insoluble polymer in a polar solvent by the hydration method according to the present invention, since hemolytic activity and cytotoxicity were not observed, the concentration of the polar solvent remaining in the cloudy aggregates was considered to be below the concentration showing hemolytic activity and cytotoxicity.

[0076] (c) Evaluation of surfactant action on cells When the turbid aggregates containing the above polymers were mixed with cells and cultured, for the purpose of evaluating the surfactant action exerted by the turbid aggregates on the cells, in Example 3, for PMEA, PEt2A in which cancer cell-selective accumulation was observed, and PEEA which did not show accumulation, after the above 24-hour incubation, the HeLa cells were washed twice with PBS, and after lysing the cells with RIPA buffer, the total cholesterol amount in the HeLa cells was quantified by Amplex Red assay. Also, for comparison, untreated HeLa cells (Untreated), HeLa cells incubated for 1 hour in a medium dissolved with the hemolytic agent Me-β-CD (10 mM), HeLa cells incubated for 6 hours in a medium dissolved with cholesterol (0.77 mM), and HeLa cells incubated for 6 hours after mixing with polystyrene beads (PSt) in the medium were each subjected to the same evaluation.

[0077] Figure 9 shows the results of the above Amplex Red assay. As shown in Figure 9, in the HeLa cells treated with Me-β-CD, cholesterol was eluted by the treatment, and the amount of cholesterol remaining decreased compared to untreated HeLa cells. On the other hand, in the HeLa cells incubated in the presence of PMEA, PEt2A, and PEEA hydrated by the method according to the present invention, since the amount of cholesterol observed in each case was comparable to that of untreated HeLa cells, it was considered that they did not show a surfactant action on HeLa cells and the like.

[0078] (d) Evaluation of internalization into cells As shown in Example 3, for the turbid aggregates (PMEA hydrate) in which cancer cell-selective accumulation was observed when mixed with cells and cultured, the state of being taken up and internalized by the cells during the culture was evaluated by the following method. Fluorescently labeled PMEA was synthesized by modifying the thiol end of PMEA (number average molecular weight: 22,000) polymerized by reversible addition-fragmentation chain transfer polymerization (RAFT) using 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid with a fluorescent group (Bodipy). Fluorescent PMEA was synthesized by mixing the fluorescently labeled PMEA with the unmodified PMEA at 5 wt% to obtain fluorescent PMEA (EF-PMEA). The fluorescent PMEA was then hydrated by the method described in Example 2 to obtain cloudy aggregates. The cloudy aggregates were added to HeLa cells in the same manner as in Example 3, and the cells were cultured by leaving them stationary in an incubator.

[0079] After 24 hours of incubation, the cells were washed twice with PBS, and the intracellular organelles, lysosomes, endoplasmic reticulum, and mitochondria, were stained with LysoTracker, ER-Tracker, and MitoTracker, respectively, and the localization of fluorescent PMEA to intracellular organelles was evaluated using a confocal laser scanning microscope. As an index of colocalization rate, the Pearson's correlation coefficient between fluorescent PMEA and each organelle was calculated by image analysis using ImageJ.

[0080] FIG. 10 shows the correlation coefficients with fluorescent PMEA in lysosomes, endoplasmic reticulum, and mitochondria calculated above. As shown in FIG. 10, it was observed that fluorescent PMEA exhibited a high correlation coefficient with lysosomes and endoplasmic reticulum. From this, it was inferred that fluorescent PMEA entered cells via a pathway mediated by endocytosis. Furthermore, the fact that fluorescent PMEA exhibited a high correlation coefficient with endoplasmic reticulum is considered to indicate that fluorescent PMEA that entered lysosomes was further transferred to the endoplasmic reticulum via a pathway mediated by endosomal escape or the like.

[0081] When attempting to directly deliver a drug to organelles or the like present within the cytoplasm by means of a so-called DDS (Drug Delivery System), a route that penetrates the cell membrane via endocytosis and enters the cell is promising, and thereafter, it is necessary to further migrate to cell organelles such as the endoplasmic reticulum. In the turbid aggregates generated by hydrating fluorescent PMEA by the hydration method according to the present invention, as described above, the occurrence of entry into the cell via endocytosis and subsequent migration to organelles such as the endoplasmic reticulum suggests that the turbid aggregates function effectively as a carrier when performing DDS, and it is expected that the same action will occur in non-fluorescent PMEA and the like.

[0082] When performing DDS starting from intravenous administration or the like, it is necessary to suppress the interaction between the DDS carrier and blood, and in particular, to prevent platelets and the like from adsorbing and aggregating on the DDS carrier, and it is necessary to impart biocompatibility (blood compatibility) to the surface of the DDS carrier. As a specific means of imparting biocompatibility to the surface of the DDS carrier, a technique of coating the surface of the DDS carrier with PEG showing biocompatibility has been studied.

[0083] The intracellular uptake rate and the like of fluorescent PMEA hydrated by the hydration method according to the present invention were evaluated in comparison with PEG by the method described below. As a comparison, fluorescent PEG (EF-PEG) in which one end of PEG with an Mn of 20,000 was fluorescently labeled with a fluorescent group (Bodipy) was used so that the number average molecular weight (Mn) was approximately the same as that of the fluorescent PMEA. The cells used for the evaluation were untreated NHDF and HeLa cells, and in addition, for the purpose of evaluating the influence of cell membrane glycans, HeLa cells pretreated for one hour with a culture medium (DMEM / F12 (containing 10% fetal bovine serum, penicillin and streptomycin)) supplemented with Heparinase (2U) that degrades heparin, Hyaluronidase (10U) that cleaves hyaluronic acid, and Neuraminidase (0.1U) that cleaves polysialic acid were used.

[0084] A 12-well plate dish was used, and each of the above cells was seeded with a culture medium (DMEM / F12 (containing 10% fetal bovine serum, penicillin-streptomycin)). For each cell, a turbid aggregate (150 μg / mL) prepared using fluorescent PMEA in the same manner as above, or the above fluorescent PEG was added, and the cells were left standing in an incubator for 3 hours. At this time, the number of fluorescent molecules contained in the fluorescent PEG was made equal to the number of fluorescent molecules contained in the fluorescent PMEA so that the absorbance would be equal. After washing each of the standing cells twice with PBS, the cells were detached from the dish by trypsin treatment, and the integrated value of the fluorescence intensity per cell was measured with a flow cytometer.

[0085] Table 2 shows the relative values of the average fluorescence intensity measured above. As shown in Table 2, when EF-PEG was mixed, in both HeLa cells and NHDF cells, compared with the case where no fluorescent component was mixed (untreated), there was no substantial change in the fluorescence intensity after culturing. On the other hand, when cultured with EF-PMEA mixed, an increase in fluorescence intensity was observed. From this result, it was inferred that EF-PMEA is taken up into cells more rapidly compared to EF-PEG. In addition, when cultured with EF-PMEA mixed, in HeLa cells, the degree of increase in fluorescence intensity was greater compared to NHDF cells, and this was inferred to be the result of EF-PMEA selectively adsorbing to HeLa cells. Also, since changing the cell membrane sugar chains of HeLa cells caused a difference in fluorescence intensity, it was inferred that the structure of the cell surface is related to the uptake of EF-PMEA into cells.

[0086] [Table 2]

[0087] (e) Evaluation of the interaction between the turbid aggregate according to the present invention and sugar chains As described above, since it was observed that the degree of uptake of the white turbid aggregates obtained by hydrating the fluorescent PMEA by the hydration method according to the present invention changes according to the state of the cell membrane sugar chain, the interaction between the white turbid aggregates and the sugar chain was evaluated by the method described below. Hyaluromic acid (weight average molecular weight: 30,000) and dextran-70 (weight average molecular weight: 70,000) were used as sugar chains, and each was fluorescently labeled (EF-Hyaluromic acid, EF-dextran) by condensation reaction with a fluorescent molecule having an amino group using DMT-MM and Carbonyldiimidazole (CDI), respectively. 1 mg of each was dissolved in 900 μL of PBS. The solution was mixed with the white turbid aggregates (polymer weight: 100 mg) obtained by hydrating PMEA by the method described in Example 2, and after standing for 6 hours, the localization of the fluorescent molecule was evaluated by a fluorescence microscope. FIG. 11 shows a phase contrast image and a fluorescence image of the white turbid aggregates mixed with each of the above fluorescently labeled sugar chains, compared with the white turbid aggregates not mixed with the sugar chain. As shown in FIG. 11, it was observed that each sugar chain adsorbed and existed in the white turbid aggregates, indicating that there was an affinity between the white turbid aggregates and the sugar chain.

Example

[0088] In this example, an attempt was made to carry a substance that exhibits a predetermined function as a drug in the white turbid aggregates generated when hydrated by the hydration method according to the present invention.

[0089] 2 mg of PMEA and 0.5 mg of doxorubicin (DOX), a lipophilic anticancer agent, were dissolved in 1 mL of methanol to prepare a polymer solution. This was placed in a dialysis membrane made of regenerated cellulose (molecular weight fractionation: 3,500) in the same manner as in Example 2 and dialyzed against a large excess (about 20 times) of purified water. Dialysis was carried out for 3 days while exchanging the purified water until the coloring of the external solvent due to the elution of DOX disappeared sufficiently. Colloidal turbidity was observed inside the dialysis membrane after dialysis, and white turbid aggregates were formed.

[0090] Figure 6 shows the results of observing the obtained turbid aggregates under a fluorescence microscope. The scale bars in Figure 6 indicate lengths of 10 μm each. As shown in Figure 6, in the fluorescence microscope observation (after 12 hours) of the obtained turbid aggregates, red fluorescence of doxorubicin (excitation wavelength 480 nm, fluorescence wavelength 590 nm) was observed only at the same position as the spherical aggregates, so it was considered that doxorubicin was incorporated into the aggregates. Furthermore, the turbid aggregates were diluted with PBS and allowed to stand in an incubator at 37°C, and then the encapsulation of doxorubicin was confirmed by fluorescence microscope observation (after 3 days).

[0091] The turbid aggregates after 3 days had elapsed since the above generation were dissolved by adding an excessive amount of methanol, and the amount of doxorubicin present was determined by measuring the absorbance (480 nm). The encapsulation efficiency of doxorubicin incorporated into the turbid aggregates was calculated from the ratio to the amount of doxorubicin used during the above hydration. As a result, the encapsulation efficiency of doxorubicin into the turbid aggregates containing the above PMEA was 26.4%. The above encapsulation of doxorubicin is considered to be the result of incorporating lipophilic doxorubicin when PMEA dissolved in a good solvent was replaced from the good solvent to purified water by dialysis and hydrated to precipitate. Similar encapsulation was also confirmed for mitoxantrone, an anticancer agent, and fluorescent molecules such as fluorescein, rhodamine, 6-p-toluidinylnaphthalene-2-sulfonate, and the fluorescent lipid molecule laurdan, in addition to doxorubicin.

[0092] From the above, it is considered that in the aggregates precipitated in the hydration method according to the present invention, it is possible to encapsulate a wide range of lipophilic (hydrophobic) drugs, physiologically active substances, lipids, and synthetic compounds. Together with the selective accumulation in cancer cells and the like due to the presence of intermediate water, it can be used for applications such as carriers for drugs having anticancer properties and separation of physiologically active substances near cancer tissues.

Industrial Applicability

[0093] The water-insoluble polymer hydrated by the method according to the present invention can be preferably used for forming a surface that requires biocompatibility. Also, by utilizing the selective adhesiveness to cells, it can be used for detecting various cells and the like.

Claims

1. A solution generation step of dissolving a water-insoluble polymer capable of containing intermediate water by hydration in a polar organic solvent to obtain a solution, and A precipitation step of hydrating and precipitating the water-insoluble polymer by mixing the solution with an aqueous phase, characterized in that it includes a method for hydrating a water-insoluble polymer capable of containing intermediate water.

2. The water-insoluble polymer capable of containing intermediate water by hydration is a polymer having at least a part of a side chain portion containing a chain ether structure or a cyclic ether structure with respect to the main chain skeleton, characterized in that it is the method for hydrating a water-insoluble polymer capable of containing intermediate water according to Claim 1.

3. In the precipitation step, at least a part of the water-insoluble polymer is precipitated in a colloidal state, characterized in that it is the method for hydrating a water-insoluble polymer capable of containing intermediate water according to Claim 1 or 2.

4. The precipitation step is characterized in that the solution and the aqueous phase are mixed through a semipermeable membrane, and it is the method for hydrating a water-insoluble polymer capable of containing intermediate water according to any one of Claims 1 to 3.

5. In the solution, a fat-soluble drug is further dissolved, characterized in that it is the method for hydrating a water-insoluble polymer capable of containing intermediate water according to any one of Claims 1 to 4.

6. A composition containing hydrated water-insoluble polymer molecules, characterized in that a solution obtained by dissolving a water-insoluble polymer capable of containing intermediate water by hydration in a polar organic solvent and an aqueous phase are mixed, and the water-insoluble polymer is hydrated and precipitated.

7. Characterized by having a particle shape, it is the composition containing hydrated water-insoluble polymer molecules according to Claim 6.

8. Characterized in that the average particle diameter is 100 μm or less, it is the composition containing hydrated water-insoluble polymer molecules according to Claim 6 or 7.

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