Gel-producing composition and its use

Abstract

A composition for producing a gel is provided, which can give a gel exhibiting excellent strength. [Solution] The gel-producing composition according to the present disclosure comprises a polyion complex and inorganic nanoparticles, and the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion.

Description

[Technical Field]

[0001] The present invention relates to a composition for producing a gel and its use. [Background technology]

[0002] Injectable gels are a promising biomaterial because they can be administered as a liquid and gel under specific conditions. These gels have potential applications in a variety of fields, including tissue regeneration, localized and sustained drug delivery, orthopedics, regenerative medicine, and cosmetic surgery.

[0003] Recently, polyion complexes (PICs) have become known as a technology that can be used to create such injectable gels. For example, a polyion complex containing a polycationic polymer and a polyanionic polymer, as described in Patent Document 1, forms stable micelles in an aqueous medium, and an aqueous solution of such micelles gels at around body temperature. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 167333 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the above-mentioned conventional techniques have room for further improvement in terms of the strength of the gel obtained. One aspect of the present invention aims to provide a composition for producing a gel, which can provide a gel exhibiting excellent strength. [Means for solving the problem]

[0006] In order to solve the above-mentioned problems, a gel-producing composition according to one embodiment of the present invention comprises a polyion complex and inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. [Effects of the Invention]

[0007] According to one aspect of the present invention, a composition for producing a gel can be provided, which can give a gel exhibiting excellent strength. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows GPC curves (solid lines) of P1 to P4 and a GPC curve (dashed line) of a PEG macroinitiator. [Figure 2] FIG. 1 shows 1H NMR spectra before and after deprotection of P4. [Figure 3] FIG. 1 shows the DLS measurement results (P1 to P4) of self-assembled nanoparticles (NanoLys / PSS). [Figure 4] FIG. 1 shows the DLS measurement results, transmission electron microscope (TEM) image, and 1H NMR spectrum (including the 1H NMR spectrum of NaPSS) of self-assembled nanoparticles (NanoLys / PSS(P2)). [Figure 5] FIG. 1 shows the effect of NaCl concentration on the temperature dependence of the elastic modulus and viscosity of self-assembled nanoparticles (NanoLys / PSS(P4)). [Figure 6] FIG. 1 shows the effect of the degree of polymerization of lysine on the temperature dependence of the elastic modulus of self-assembled nanoparticles (NanoLys / PSS). [Figure 7] FIG. 1 shows the results of DLS measurements of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-XS)). [Figure 8] FIG. 1 shows the results of DLS measurements of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-30)). [Figure 9] FIG. 1 shows the temperature dependence of the elastic modulus and viscosity of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-XS)) (the effect of NaCl concentration). [Figure 10] FIG. 1 shows the temperature dependence of the elastic modulus and viscosity of silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO(P4)) (the effect of silica nanoparticle size). [Figure 11] FIG. 1 shows the DLS measurement results of RIG and RIG / ST-XS (effect of addition of silica nanoparticles). [Figure 12] FIG. 1 shows the results of measuring the temperature dependence of the elastic modulus and viscosity of RIG and RIG / ST-XS (effect of the addition of silica nanoparticles). DETAILED DESCRIPTION OF THE INVENTION

[0009] An embodiment of the present invention will be described below, but the present invention is not limited thereto. In this specification, unless otherwise specified, "A to B" representing a numerical range means "A or more and B or less."

[0010] 1. Gel-producing composition A gel-producing composition according to one embodiment of the present invention comprises a polyion complex and inorganic nanoparticles. The polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. Hereinafter, polyethylene glycol will be abbreviated as PEG, and polyion complex will be abbreviated as PIC. The gel-producing composition may be a sol containing PIC and inorganic nanoparticles.

[0011] The PIC forms micelles through self-assembly in an aqueous medium. These micelles gel in response to temperature. The inventors have found that when inorganic nanoparticles are complexed with these micelles, they gel in response to temperature and ionic strength, and the resulting gels have superior strength compared to gels that do not contain inorganic nanoparticles. Gels that do not contain inorganic nanoparticles have insufficient mechanical strength and tend to flow after administration to the body, making them unsuitable for practical use. However, gels obtained using a gel-producing composition according to one embodiment of the present invention have sufficient strength to remain in the body. Furthermore, it has been confirmed that gels obtained by complexing inorganic nanoparticles in this way become irreversible gels that do not return to their original liquid state after gelation. Furthermore, inorganic nanoparticles are expected to improve drug-carrying capacity.

[0012] That is, the gel-producing composition and gel can be administered into the body as a liquid and used as an injectable gel that gels in response to body temperature and ionic strength within the body. Such injectable gels can be applied in fields such as tissue regeneration, pharmaceuticals (local and sustained drug delivery), antibacterial agents, orthopedics, regenerative medicine, cosmetic surgery, supplements, and cosmetics. The injectable gel can be used in the form of, for example, subcutaneous injections, spray formulations, coating materials, scaffolds for cell regeneration, drug delivery matrices, matrices for cosmetic surgery, surgical adhesion barriers, and the like. The injectable gel may be a redox injectable gel (RIG).

[0013] <1-1. Triblock copolymer> The first triblock copolymer has a structure represented by polycation-block-polyethylene glycol-block-polycation. The second triblock copolymer has a structure represented by polyanion-block-polyethylene glycol-block-polyanion. In this specification, the connector "-block-" means that the segments connected by this "-block-" each constitute a block, and is hereinafter also abbreviated as "-b-". In other words, in the first triblock copolymer, it can be said that the polycation segment, PEG segment, and polycation segment are connected in this order. Furthermore, the second triblock copolymer has a structure represented by a polyanion segment, PEG segment, and polyanion segment.

[0014] The PEG segment is -(OCH2CH2) m Here, m is an integer of preferably 20 to 800, more preferably 30 to 500, and even more preferably 40 to 400.

[0015] Examples of polymers constituting polycation segments include polymers having a substituent in the side chain that can be protonated in water. The main backbone of the polymer is not limited, but examples include polyamino acids, polymethacrylic acid derivatives, polyacrylic acid derivatives, polystyrene derivatives, etc. Examples of the substituent include primary amino groups, secondary amino groups, etc., specifically amino groups, imidazole groups, etc. Examples of the polyamino acids include polylysine, polyarginine, polyornithine, polyhistidine, polytryptophan, etc.

[0016] The polycation segment may have a cyclic nitroxide radical as a part of the pendant group. Examples of the cyclic nitroxide radical include 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-yl, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxazolidin-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl, and 2,4,4-trimethyl-imidazolindin-3-oxyl-2-yl. The cyclic nitroxide radical may be o- or p-phenylene-C 1~6 Alkylene-NH-(C 1~6 alkylene)q- (where q is 0 or 1) to the polycation segment.

[0017] Examples of polymers constituting the polyanion segment include polyacrylic acid, polymethacrylic acid, polysulfonic acid, polystyrene sulfonic acid, poly(vinyl benzoate), polyanionic polysaccharides, and anionic proteins. Examples of polyanionic polysaccharides include chondroitin sulfate, carrageenan, heparin, carboxymethyl dextran, xanthan gum, and hyaluronic acid. Examples of anionic proteins include polyaspartic acid and polyglutamic acid.

[0018] The degree of polymerization (the number of repeating monomer units) of the polycation segment or polyanion segment is not limited from the viewpoint of the stability of the PIC micelle in an aqueous medium, but is preferably an integer of 10 to 200, more preferably 15 to 150, even more preferably 15 to 100, and particularly preferably 20 to 100.

[0019] Each segment can be connected by a linking group. The linking group can be any divalent organic group as long as it does not adversely affect the formation of the PIC. Examples of the linking group include -O-(CH2) a -NH-, -O-(CH2) a -O-, -(CH2) a -NH-, -(CH2) a -O-,

[0020] [ka]

[0021] Here, a is an integer of 1 to 6, preferably 1 to 3.

[0022] The terminal of the triblock copolymer, i.e., the terminal opposite to the terminal connected to the PEG segment in the polycationic or polyanionic segment, may be, for example, H, an amino group, a linear or branched C 1~6 Alkyl group, phenylthiocarbonylthio group, C 1~6 Alkyloxyalkylthiocarbonylthio group, C 1~6 It may be an alkyloxythiocarbonylthio group, a sulfanyl group, or the like. 1~6 Alkyl group, phenylthiocarbonylthio group, C 1~6 Alkyloxyalkylthiocarbonylthio group, C 1~6 The alkyloxythiocarbonylthio group may be unsubstituted or substituted, and when substituted, the substituents are C 1~4 Alkyl group, C 1~4 Alkyloxy group, hydroxyl group, carboxyl group, cyano group, nitro group, halogen atom group, or mono- or di-C1~4 It may be an alkylamino group.

[0023] The molecular weight distribution of the triblock copolymer does not need to be limited during gelation, but may be, for example, 1.01 to 1.25, 1.01 to 1.20, 1.01 to 1.15, or 1.01 to 1.10.

[0024] The method for producing the triblock copolymer is not particularly limited, and reference can be made to, for example, Patent Document 1, WO 2015 / 118993, WO 2014 / 199982, Ishii et al., Macromolecules 2015, 48, 3088-3094, Long Binh Vong et al., Biomaterials 167 (2018) 143-152, Saita et al., Biomaterials 76 (2016), 292-301, Nakagawa et al., Biomaterials 69 (2015) 165-173, Min Ley Pua et al., J. Control. Release 172 (2013), 914-920, and the like.

[0025] <1-2. Polyanions and polycations> The polyanion to be combined with the first triblock copolymer may be any of the polymers exemplified as the polymers constituting the polyanion segment of the second triblock copolymer, and the polycation to be combined with the second triblock copolymer may be any of the polymers exemplified as the polymers constituting the polycation segment of the first triblock copolymer.

[0026] The optimal molecular weights of these polyanions and polycations vary depending on the type of polymer, and are not limited. For example, in the case of polyacrylic acid, the Mn (number-average molecular weight) is preferably 200 to 1,000,000, more preferably 500 to 100,000, and even more preferably 1,000 to 10,000. In the case of polyanionic polysaccharides, such as chondroitin sulfate, the Mn or Mw (weight-average molecular weight) is preferably 500 to 1,000,000, and more preferably 1,000 to 100,000. In the case of anionic polypeptides, such as polyaspartic acid, the Mn or Mw is preferably 500 to 1,000,000, and more preferably 1,000 to 100,000.

[0027] <1-3. Polyion complex> PICs are formed in an aqueous medium through electrostatic interactions between the polycationic segment of a first triblock copolymer and a polyanion (see (a) above). The PICs can exist as nanosized polymeric micelles having a core formed through electrostatic interactions between the polycationic segment and the polyanion and a shell formed from the PEG segment of the first triblock copolymer. Because the polymeric micelles can be observed in a particulate form, they are also referred to herein as self-assembled nanoparticles. Similar PICs can also be formed through electrostatic interactions between the polyanion segment of a second triblock copolymer and a polycation (see (b) above). Furthermore, similar PICs can also be formed through electrostatic interactions between the polycationic segment of a first triblock copolymer and the polyanion segment of a second triblock copolymer (see (c) above).

[0028] The specific combination of the first triblock copolymer and a polyanion, the specific combination of the second triblock copolymer and a polycation, and the specific combination of the first triblock copolymer and the second triblock copolymer are not particularly limited. In the examples, measurement results of a combination of a triblock copolymer having a polylysine segment and polystyrene sulfonic acid, and a combination of a triblock copolymer having a cyclic nitroxide radical and polyacrylic acid are shown, but combinations of a triblock copolymer having a polylysine segment and polyacrylic acid, and a triblock copolymer having a cyclic nitroxide radical and polystyrene sulfonic acid are also possible.

[0029] Examples of the aqueous medium include pure water, ion-exchanged water, buffered solutions thereof, and solutions containing water-soluble organic solvents. Examples of the water-soluble organic solvent include N,N-dimethylformamide, dimethyl sulfoxide, alcohols such as methanol and ethanol, acetone, and tetrahydrofuran.

[0030] For the production method of PIC, reference can be made to the methods described in, for example, Patent Document 1, WO 2015 / 118993, WO 2014 / 199982, Ishii et al., Macromolecules 2015, 48, 3088-3094, Long Binh Vong et al., Biomaterials 167 (2018) 143-152, Saita et al., Biomaterials 76 (2016), 292-301, Nakagawa et al., Biomaterials 69 (2015) 165-173, Min Ley Pua et al., J. Control. Release 172 (2013), 914-920, etc.

[0031] <1-4. Inorganic nanoparticles> The material constituting the inorganic nanoparticles is not particularly limited, and examples thereof include silica (silica gel), titanium oxide, gold, and silver.

[0032] The average particle size of the inorganic nanoparticles is on the order of nanometers, preferably 100 nm or less, more preferably 70 nm or less, even more preferably 50 nm or less, even more preferably 20 nm or less, particularly preferably 10 nm or less, and most preferably 6 nm or less. The average particle size of the inorganic nanoparticles is preferably 1 nm or more, more preferably 4 nm or more. The average particle size can be determined from a volume-based particle size distribution as the hydrodynamic diameter measured by dynamic light scattering. Alternatively, the average particle size may be measured using a transmission electron microscope.

[0033] The inorganic nanoparticles are presumed to be composited with the self-assembled nanoparticles, for example, incorporated into the self-assembled nanoparticles. For example, particles composited with silica nanoparticles and self-assembled nanoparticles are also referred to herein as silica-composite self-assembled nanoparticles. The average particle size of the self-assembled nanoparticles composited with inorganic nanoparticles may be 3000 nm or less, 2500 nm or less, 2000 nm or less, 1500 nm or less, or 1000 nm or less. The average particle size of the self-assembled nanoparticles composited with inorganic nanoparticles may be 100 nm or more, 200 nm or more, or 300 nm or more. The average particle size can be determined from a volume-based particle size distribution as the hydrodynamic diameter measured by dynamic light scattering.

[0034] A method for producing a gel-producing composition according to one embodiment of the present invention includes a step of mixing the polyion complex (a), (b), or (c) described above with inorganic nanoparticles. For example, a gel-producing composition can be obtained by mixing a solution or dispersion containing PIC with a solution or dispersion containing inorganic nanoparticles. Examples of solvents contained in these solutions or dispersions include the aqueous media described above. Mixing may be performed using a mixer.

[0035] [2. Gel] A gel according to one embodiment of the present invention is obtained by gelling the above-mentioned gel-producing composition. A method for producing a gel according to one embodiment of the present invention includes the steps of obtaining a gel-producing composition by the above-mentioned method for producing a gel-producing composition and gelling the gel-producing composition. Examples of gelling methods include increasing the temperature and / or ionic strength. Examples include heating the gel-producing composition to 35 to 45°C and / or contacting the gel-producing composition with physiological saline having an NaCl concentration of 100 to 200 mM.

[0036] [3. Gel production kit] A gel production kit according to one embodiment of the present invention includes a first agent containing the polyion complex (a), (b), or (c) described above, and a second agent containing inorganic nanoparticles. By using the gel production kit to mix the first agent and the second agent, the polyion complex and the inorganic nanoparticles are mixed, and gelation can be performed to produce a gel.

[0037] The PIC may be in the form of a solution or dispersion in the first agent, or may be in a dry state. The inorganic nanoparticles may be in the form of a solution or dispersion in the second agent, or may be in a dry state. When mixing, the first agent and the second agent are preferably mixed in the form of a solution or dispersion containing the above-mentioned aqueous medium.

[0038] The first agent and the second agent may be contained in separate containers. In this specification, the container containing the first agent is referred to as the first container, and the container containing the second agent is referred to as the second container. The first container and the second container may be separate, independent containers, or the first container and the second container may be integrated. The first container and the second container being integrated means that the container is configured as a single container having a first space containing the PIC and a second space containing inorganic nanoparticles. The gel-producing kit may also include another container (space) containing the above-mentioned aqueous medium for producing the solution or dispersion.

[0039] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0040] An embodiment of the present invention may include the following features. <1> A composition for producing a gel, comprising a polyion complex and inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. <2> <1> A gel obtained by gelling the gel-producing composition described above. <3> A method for producing a composition for producing a gel, comprising the step of mixing a polyion complex with inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. <4> <3> and gelling the gel-producing composition. <5> A kit for producing a gel, comprising: a first agent containing a polyion complex; and a second agent containing inorganic nanoparticles, wherein the polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion; (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation; or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. [Example]

[0041] An example of the present invention is described below. In the following, [polymer] refers to the polymer concentration in the solution, [NaCl] refers to the sodium chloride concentration in the solution, [PB] refers to the phosphate buffer concentration in the solution, and [ST-XS] refers to the ST-XS (silica nanoparticles) concentration in the solution.

[0042] [Materials Characterization] The number-average molecular weight (Mn), weight-average molecular weight (Mw), and molecular weight distribution of each polymer were measured. Two polystyrene gel columns (Tosoh Corporation, TSKgel GMHHR-M; exclusion limit: molecular weight (MW) = 4.0 × 10) were connected to a pump (JASCO, PU-4180). 6 Gel permeation chromatography (GPC) was performed in N,N-dimethylformamide (DMF) at 40 °C (flow rate: 0.40 mL / min) using a 7.8 cm × 30 cm (7.8 cm i.d. × 30 cm) column, a JASCO RI-2031 refractive index detector, and a JASCO UV-4075 UV / Vis detector. The column was calibrated with 18 standard poly(ethylene oxide) (PEO) and poly(ethylene glycol) (PEG) samples (Merck; Mp = 238–1,180,000). 1 H nuclear magnetic resonance (NMR) spectra were obtained using an AVANCE-600 NMR spectrometer (Bruker) at 600 MHz ( 1 H) and acquired in CDCl3 or DMSO-d6 at room temperature (22–23 °C). Dynamic light scattering (DLS) measurements were performed at 37 °C using a Zetasizer Nano ZSP (Malvern) equipped with a He-Ne laser (λ = 633 nm). The measurement angle was 173°, and data were analyzed using the non-negative least squares (NNLS) method.

[0043] Example 1: Synthesis of Z-protected L-lysine-N-anhydride (NCA-Lys(Z)) L-Lysine (HOCOCH((CH2)4NHCOOCH2C6H5)NH2; H-Lys(Z)-OH) (25.2 g, 89.9 mmol; 1.0 eq.) bearing a benzyloxycarbonyl group in the side chain and triphosgene (18.2 g, 61.3 mmol; ca. 0.67 eq.) were dissolved in α-pinene (36 mL, 227 mmol; 2.5 eq.) and tetrahydrofuran (THF; 252 mL) and stirred at 50 °C for 3 h. The resulting reaction solution was cooled to room temperature and slowly added to hexane (600 mL), yielding a white precipitate. The white precipitate was washed with hexane and collected by vacuum filtration. The recovered white compound was redissolved in THF / acetone / isopropanol (IPA) (approximately 10 / 10 / 1 (v / v / v)) and reprecipitated twice in hexane. Finally, the white precipitate collected by vacuum filtration was dried under reduced pressure to obtain the target compound NCA-Lys(Z) shown below (yield: 16.4 g, 59%).

[0044] [ka]

[0045] Example 2 H-(HNCH((CH)NHCOOCHCH)CO) n NH-CH2CH2(OCH2CH2) m -NH(COCH((CH2)4NHCOOCH2C6H5)NH) n Synthesis of -H PEG Macroinitiator H2N-(OCH2CH2) n N,N-dimethylformamide (DMF; 44.8 mL) and tetralin (0.3 mL) were added to —NH (Mn = 10,000, 25.1 g, 2.51 mmol) and dissolved. Furthermore, NCA-Lys(Z) synthesized in Example 1 was dissolved in DMF to prepare a 1000 mM monomer solution. The monomer solution (70.4 mL, 70.4 mmol) was added to H N-(OCH CH) nThe resulting mixture was added to a DMF solution of -NH2, mixed, and reacted at 45 °C for 4 days. The resulting reaction solution was directly added to hexane / IPA (1 / 1 (v / v)) to precipitate, and the precipitate was collected by centrifugation. The collected precipitate was redissolved in DMF and purified by reprecipitation and centrifugation twice more. The target product (PLys(Z)-b-PEG-b-PLys(Z)) was collected by vacuum drying (30.5 g, n = 7.7 (P2), Figure 1b). Polymers with n = 2.7 (P1), 19 (P3), and 54 (P4) were also synthesized in the same manner (Figures 1 and 2). The resulting polymers are abbreviated as P1, P2, P3, and P4.

[0046] [ka]

[0047] Example 3 H—(HNCH((CH)NH)CO) n NH-CH2CH2(OCH2CH2) m -NH(COCH((CH2)4NH3)NH) n Synthesis of -H P2 (30.5 g, 33.6 mmol (Lys(Z)); 1.0 eq.) synthesized in Example 2 was dissolved in trifluoroacetic acid (TFA) (300 mL) and cooled in an ice bath. A solution of hydrogen bromide (HBr) in acetic acid (28%; 61 mL, 337 mmol; approximately 10 eq.) was slowly added dropwise. The mixture was stirred overnight to allow the reaction to proceed. The resulting reaction solution was directly transferred to a dialysis membrane (MWCO (molecular weight cutoff) = 3500 Da) and dialyzed against a methanol / Na2CO3 aqueous solution (= 1 / 1 (v / v)) for 3 days and against water for 4 days. The target product (PLys-b-PEG-b-PLys) shown below was recovered by lyophilization (19.6 g, deprotection rate 100%). P1, P3, and P4 were deprotected in the same manner (Figure 2b).

[0048] [ka]

[0049] Example 4 Preparation of Self-Assembled Nanoparticles 1: Formation of Polyion Complex with Poly(styrene sulfonic acid) The PLys-b-PEG-b-PLys synthesized in Example 3 was dissolved in dimethyl sulfoxide (DMSO) / TFA = 20 / 1 (v / v) ([polymer] = 5 mg / mL). Sodium poly(styrene sulfonate) (NaPSS; Sigma-Aldrich, Mw: approximately 75,000) was dissolved in phosphate buffer (pH = 7.4) ([polymer] = 5 mg / mL). The NaPSS phosphate buffer solution was slowly added dropwise to the PLys-b-PEG-b-PLys solution while stirring to prepare a mixed solution. This mixed solution was directly transferred to a dialysis membrane (MWCO = 3,500 Da) and dialyzed against water for 4 days. Self-assembled nanoparticles (Nano) were then obtained by vacuum concentration. Lys / PSS A concentrated solution of 10–43 nm self-assembled nanoparticles was obtained by diluting this solution with water and performing dynamic light scattering (DLS) measurements (Figures 3 and 4; [polymer] = 10 mg / mL).

[0050] Example 5 Preparation of Self-Assembled Nanoparticles 2: Combination of Silica Nanoparticles The concentrated solution of self-assembled nanoparticles prepared in Example 4 was diluted with water, and then an aqueous dispersion of silica nanoparticles was added. Immediately after, the mixture was stirred for 5 minutes using a vortex mixer to obtain silica composite self-assembled nanoparticles (Nano Lys / PSS / SiO A solution of silica nanoparticles was prepared ([polymer] = 40 mg / mL). Snowtex® (Nissan Chemical Co., Ltd.; Snowtex® XS (ST-XS), Snowtex® 30 (ST-30)) was used as the silica nanoparticles. The size of ST-XS was 4-6 nm, and that of ST-30 was 10-15 nm. DLS measurements were performed after 40-fold dilution with water (Figures 7 and 8; [polymer] = 1.0 mg / mL).

[0051] Example 6: Rheology Measurement The gelation behavior was evaluated using an Anton Paar rheometer (MCR302). In this experiment, the storage modulus (G'), loss modulus (G''), and complex viscosity ([η * The gelation process and gel properties were evaluated by measuring the storage modulus and loss modulus, respectively, which indicate the properties of a solid and a liquid. For example, when the storage modulus is higher than the loss modulus (G' > G''), the material exhibits primarily solid properties. Furthermore, the gel point can be determined at the intersection where both moduli have the same value (G' = G''). The self-assembled nanoparticle aqueous solution ([polymer] = 40 mg / mL; 100 μL) obtained in Examples 4 and 5 was placed on a stage with a 0.2 mm gap to the plate. A 20 mm parallel plate was used. The measurement frequency was fixed at 1 Hz, and the temperature was changed from 15°C to 45°C, followed by continuous cooling from 45°C to 15°C. The temperature change rate was 1°C / min.

[0052] Example 7: Effect of ionic strength on the temperature dependence of elastic modulus and viscosity Sodium chloride (NaCl) was added to PLys-b-PEG-b-PLys(P4) / PSS self-assembled nanoparticles (Nano Lys / PSS The polymer was dissolved in an aqueous solution (P4) ([polymer] = 40 mg / mL, [NaCl] = 0, 150, 500 mM). This aqueous solution was measured according to Example 6, and gelation occurred at 25.9 °C and 24.7 °C under conditions of NaCl concentration of 150 mM and 500 mM, respectively. The resulting gel did not return to its initial modulus even when cooled, demonstrating irreversible gelation behavior. The modulus of elasticity (G') after gelation gradually decreased as the NaCl concentration increased, but remained at around several hundred Pa under all conditions (Figure 5). Furthermore, these results suggest that the Nano Lys / PSS (P4) was found to gel depending on temperature and ionic strength.

[0053] Example 8: Effect of the degree of polymerization of lysine (DP(Lys)) on the results of measuring the temperature dependence of elastic modulus and viscosity The NaCl concentration was fixed at 150 mM (physiological saline concentration), and PLys-b-PEG-b-PLys / PSS self-assembled nanoparticles (Nano Lys / PSS The aqueous solutions were measured according to Example 6 to examine the effects of P3 and P4 ([polymer] = 40 mg / mL, [NaCl] = 150 mM). Both P3 and P4 caused gelation (Figure 6).

[0054] Example 10: Effect of silica nanoparticles on the temperature dependence of elastic modulus and viscosity Silica composite nanoparticles (Nano) prepared in Example 5 Lys / PSS / SiO ) was measured according to Example 6. In this case, Snowtex (registered trademark) series silica nanoparticles were used. As a result, when silica nanoparticles were compounded, the elastic modulus after gelation was improved compared to when silica nanoparticles were not compounded (Figures 9 and 10). In particular, when Snowtex (registered trademark) XS (ST-XS) was used, a high elastic modulus of over 10 kPa was achieved after gelation (Figures 9 and 10).

[0055] Example 11 Synthesis of Cl-PEG-Cl HO-(CH2CH2) into a 500 mL recovery flask equipped with a three-way stopcock. n -OH (Mn = 10,000, 50.0 g, 5.00 mmol) was added and the mixture was dried overnight under reduced pressure at 110 °C. After the liquid temperature was lowered to 65 °C, THF (200 mL) was added to dissolve the contents of the flask. Under a nitrogen atmosphere, a butyllithium solution (20.0 mmol, 12.5 mL, 1.6 M hexane solution) was slowly added to activate the hydroxyl groups. Dichloro-p-xylene (17.5 g, 0.1 mol) was then added and the mixture was reacted at 60 °C for 4 days. The resulting reaction mixture was precipitated in cooled IPA at 4 °C, and the precipitate was collected by centrifugation. The precipitate was again dissolved in 20 mL of methanol and reprecipitated in cooled IPA. The reprecipitation and centrifugation process was repeated four more times. The target product (Cl-PEG-Cl) was collected by drying under reduced pressure.

[0056] Example 12: Synthesis of Grignard Reagent THF (10 mL) was added to a 50 mL recovery flask equipped with a three-way stopcock and cooled in an ice bath at 0°C. Carbon disulfide (3.75 mL, 62.0 mmol) and phenylmagnesium bromide (16.0 mmol, 5.19 mL, 3.0 M diethyl ether solution) were added under a nitrogen atmosphere, and the reaction was carried out overnight in an ice bath. The synthesized Grignard reagent was used as is without purification.

[0057] Example 13 Synthesis of Macro RAFT Agent (CTA-PEG-CTA) The Cl-PEG-Cl (Mn (size exclusion chromatography (SEC)) = 9,300, 40.0 g, 3.9 mmol) synthesized in Example 12 was placed in a 300 mL recovery flask equipped with a three-way stopcock and dried overnight under reduced pressure at 110 °C. After the liquid temperature was lowered to 65 °C, THF (140 mL) was added to dissolve the contents of the flask. Under a nitrogen atmosphere, the Grignard reagent synthesized in Example 12 was gradually added and reacted at 40 °C for 24 hours. The resulting reaction solution was precipitated in IPA cooled to 4 °C, and the precipitate was recovered by centrifugation. The precipitate was again dissolved in 20 mL of methanol and reprecipitated in chilled IPA. The reprecipitation and centrifugation process was repeated three more times. The target product (CTA-PEG-CTA) was recovered by drying under reduced pressure.

[0058] Example 14: Synthesis of PCMS-b-PEG-b-PCMS CTA-PEG-CTA (Mn(SEC) = 10,100, 35.0 g, 3.30 mmol) synthesized in Example 13 and azobisisobutyronitrile (AIBN) (547 mg, 3.30 mmol) were placed in a 500 mL recovery flask equipped with a three-way stopcock and degassed by reducing the pressure for 30 minutes. Toluene (350 mL) was added under a nitrogen atmosphere to dissolve the contents of the flask. p-Chloromethylstyrene (32.8 mL, 0.230 mol) purified by vacuum distillation was added, and the reaction was carried out at 60 °C for 24 hours. The resulting reaction solution was precipitated in methyl t-butyl ether (2 L), and the precipitate was collected by vacuum filtration. The collected precipitate was dissolved in acetone (20 mL) and reprecipitated in methyl t-butyl ether (2 L). The precipitate was collected by vacuum filtration and dried overnight under reduced pressure. The dried precipitate, AIBN (14.5 g, 89.8 mmol), and ethyl acetate (300 mL) were added to a 500 mL recovery flask equipped with a reflux condenser and dissolved. After degassing by bubbling nitrogen for 30 minutes, the mixture was reacted at 80°C for 12 hours. The reaction solution was precipitated in methyl t-butyl ether (2 L) and the precipitate was collected by vacuum filtration. The collected precipitate was dissolved in acetone (20 mL) and reprecipitated in methyl t-butyl ether (2 L). The target product (PCMS-b-PEG-b-PCMS) was collected by vacuum drying.

[0059] Example 15: Synthesis of PMNT-b-PEG-b-PMNT PCMS-b-PEG-b-PCMS (Mn(SEC) = 13,000, 8.27 g, 62.7 mmol) synthesized in Example 14 was placed in a 200 mL recovery flask equipped with a three-way stopcock and dried under reduced pressure for 30 minutes. After dissolving the contents of the flask with 80 mL of DMF, 10.0 g of 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-aminoTEMPO) (58.4 mmol) dissolved in 20 mL of DMF was added and the mixture was allowed to react at 50 °C for 24 hours. The resulting reaction solution was precipitated in IPA cooled to 4 °C, and the precipitate was recovered by centrifugation. The precipitate was dissolved again in 10 mL of acetone and reprecipitated in chilled IPA. The reprecipitation and centrifugation steps were repeated four more times. The target product (PMNT-b-PEG-b-PMNT) was recovered by drying under reduced pressure.

[0060] Example 16: Preparation of Self-Assembled Nanoparticles 3: Formation of Polyion Complex between PMNT-b-PEG-b-PMNT and Polyacrylic Acid The PMNT-b-PEG-b-PMNT synthesized in Example 15 and polyacrylic acid (PAAc; Fujifilm Wako, Mn: approximately 5,000) were each dissolved in phosphate buffer (pH = 6.2, 100 mM) ([polymer] = 10 mg / mL). The PMNT-b-PEG-b-PMNT solution was added dropwise to the slowly stirred phosphate buffer solution of PAAc to prepare a mixed solution. Dynamic light scattering measurements confirmed the formation of 62 nm self-assembled nanoparticles (Figure 11 (RIG)).

[0061] Example 17 Rheological Evaluation of Redox Injectable Gel (RIG) The mixed solution prepared in Example 16 was concentrated using a centrifugal evaporator to prepare RIG ([polymer] = 60 mg / mL, [PB] = 600 mM). After dilution with distilled and ion-exchanged water ([polymer] = 40 mg / mL, [PB] = 400 mM), rheology measurements were performed as in Example 6, and the gelation occurred at 25.1°C. The resulting gel did not return to its initial elastic modulus even when cooled, confirming irreversible gelation behavior (Figure 12).

[0062] Example 18 Rheological Evaluation of Silica Nanoparticle Composite RIG (RIG / Snowtex (registered trademark) ST-XS) Snowtex® XS (ST-XS) was added to the RIG ([polymer] = 60 mg / mL, [PB] = 600 mM) prepared in Example 17, and the mixture was thoroughly stirred using a vortex mixer to prepare silica-composite self-assembled nanoparticles RIG / ST-XS ([polymer] = 40 mg / mL, [PB] = 400 mM, [ST-XS] = 5.6 wt%). Rheological measurements were performed as in Example 6, and a high elastic modulus of over 10 kPa was achieved after gelation (Figure 11 (RIG / ST-XS), Figure 12). [Industrial Applicability]

[0063] One aspect of the present invention can be used, for example, to produce an injectable gel.

Claims

1. comprising a polyion complex and inorganic nanoparticles, The polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion; (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion; and A composition for producing a gel, wherein the polyanion that forms a polyion complex with the first triblock copolymer and the polycation that forms a polyion complex with the second triblock copolymer are both polymers.

2. A gel obtained by gelling the composition for producing a gel according to claim 1.

3. Mixing a polyion complex with inorganic nanoparticles, The polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion; (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion; and A method for producing a composition for producing a gel, wherein the polyanion that forms a polyion complex with the first triblock copolymer and the polycation that forms a polyion complex with the second triblock copolymer are both polymers.

4. A step of obtaining a composition for producing a gel by the method for producing a composition for producing a gel according to claim 3; and a step of gelling the gel-producing composition.

5. A first agent containing a polyion complex and a second agent containing inorganic nanoparticles, The polyion complex is (a) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion; (b) a polyion complex of a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion and a polycation, or (c) a polyion complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion; and The kit for producing a gel, wherein the polyanion that forms a polyion complex with the first triblock copolymer and the polycation that forms a polyion complex with the second triblock copolymer are both polymers.

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