Composition for gel production, and use thereof

Abstract

Provided is a composition for gel production, with which a gel exhibiting excellent strength can be obtained. A composition for gel production according to the present disclosure includes a polyion complex (PIC) and inorganic nanoparticles. The PIC is a PIC of a polyanion and a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation, or a PIC of a polycation and a second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion, or a PIC of the first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and the second triblock copolymer having a structure represented by polyanion-block-polyethylene glycol-block-polyanion. The polyanion for forming the PIC together with the first triblock copolymer and the polycation for forming the PIC together with the second triblock copolymer are both polymers.

Description

Composition for gel production and its use

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

[0002] Injectable gels are one of the biomaterials that have attracted attention because they are administered into the body as a liquid and gelate under specific conditions. Such injectable gels can be applied in various fields such as tissue regeneration, local and sustained drug delivery, orthopedics, regenerative medicine, and cosmetic surgery.

[0003] As a technology applicable to such injectable gels, polyion complex (PIC) has recently been known. For example, a polyion complex containing a polycationic polymer and a polyanionic polymer described in Patent Document 1 forms stable micelles in an aqueous medium, and an aqueous solution of such micelles gelates near body temperature.

[0004] International Publication No. 2016 / 167333

[0005] However, the prior art as described above had room for further improvement from the viewpoint of the strength of the obtained gel. One aspect of the present invention aims to realize a composition for gel production that can obtain a gel exhibiting excellent strength.

[0006] To solve the above problems, a gel manufacturing composition according to one aspect 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, wherein the polyanion that forms the polyion complex with the first triblock copolymer and the polycation that forms the polyion complex with the second triblock copolymer are both polymers.

[0007] A method for producing a gel-making composition according to one aspect of the present invention includes a 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, wherein the polyanion that forms the polyion complex with the first triblock copolymer and the polycation that forms the polyion complex with the second triblock copolymer are both polymers.

[0008] A gel manufacturing kit according to one aspect of the present invention comprises 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, wherein the polyanion that forms the polyion complex with the first triblock copolymer and the polycation that forms the polyion complex with the second triblock copolymer are both polymers.

[0009] According to one aspect of the present invention, a gel manufacturing composition can be provided that can produce a gel exhibiting excellent strength.

[0010] This figure shows the GPC curves for P1-P4 (solid lines) and the GPC curve for PEG macroinitiator (dashed line). It shows P4 before deprotection and P4 after deprotection. １ This figure shows the 1H NMR spectrum. Self-assembled nanoparticles (Nano Ｌｙｓ／ＰＳＳ This figure shows the DLS measurement results (P1, P3, P4) of self-assembled nanoparticles (Nano). Ｌｙｓ／ＰＳＳ DLS measurement results for (P2), transmission electron microscope (TEM) image, １ 1H NMR spectrum (of NaPSS) １ This figure shows the NaCl concentration of self-assembled nanoparticles (Nano). Ｌｙｓ／ＰＳＳ This figure shows the effect of the temperature dependence of the elastic modulus and viscosity of (P4) on the measurement results. The degree of polymerization of lysine affects the self-assembled nanoparticles (Nano Ｌｙｓ／ＰＳＳThis figure shows the effect of temperature dependence on the measurement results of the elastic modulus of ). This figure shows the DLS measurement results of silica-compound self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-XS)). This figure shows the DLS measurement results of silica-compound self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-30)). This figure shows the temperature dependence of the measurement results of the elastic modulus and viscosity of silica-compound self-assembled nanoparticles (NanoLys / PSS / SiO(P4;ST-XS)) (effect of NaCl concentration). This figure shows the temperature dependence of the measurement results of the elastic modulus and viscosity of silica-compound self-assembled nanoparticles (NanoLys / PSS / SiO(P4)) (effect of silica nanoparticle size). This figure shows the DLS measurement results of RIG and RIG / ST-XS (effect of silica nanoparticle addition). This figure shows the temperature dependence of the measurement results of the elastic modulus and viscosity of RIG and RIG / ST-XS (effect of silica nanoparticle addition).

[0011] One embodiment of the present invention will be described below, but the present invention is not limited thereto. Unless otherwise specified in this specification, "A to B" representing a numerical range means "A or greater, and B or less."

[0012] [1. Composition for Gel Production] A composition for gel production 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, wherein the polyanion that forms the polyion complex with the first triblock copolymer and the polycation that forms the polyion complex with the second triblock copolymer are both polymers. Hereinafter, polyethylene glycol will be abbreviated as PEG, and polyion complex as PIC. The gel manufacturing composition may be a sol containing PIC and inorganic nanoparticles.

[0013] The PIC microclavus forms micelles through self-assembly in an aqueous medium. Such micelles gel in response to temperature. The inventors have found that when inorganic nanoparticles are compounded into these micelles, gelation occurs in response to temperature and ionic strength, and the strength of the resulting gel is superior to that of gels without inorganic nanoparticles. While gels without inorganic nanoparticles often lack sufficient mechanical strength and become fluid after administration into the body, making them impractical, the gel obtained using the gel-making composition according to one embodiment of the present invention has sufficient strength to remain in the body. Furthermore, it has been confirmed that the gel obtained from the PIC microclavus and the gel obtained by compounding the PIC microclavus with inorganic nanoparticles become irreversible gels that do not return to their original liquid state after gelation. It can also be expected that the drug-carrying capacity will be improved by the inorganic nanoparticles.

[0014] That is, the composition for producing the gel and the gel can be administered into the body as a liquid and used as an injectable gel that gels in response to body temperature and the ionic strength in the body. Such an injectable gel can be applied to fields such as, for example, tissue regeneration, pharmaceuticals (local and sustained drug delivery), antibacterial, orthopedics, regenerative medicine, cosmetic surgery, supplements, and cosmetics. The injectable gel can be used in the form of, for example, a subcutaneous injection drug, a spray preparation, a coating material, a scaffold for cell regeneration, a matrix for drug delivery, a matrix for cosmetic surgery, a material for preventing surgical adhesions, and the like. The injectable gel may be a redox injectable gel (RIG).

[0015] <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 the present specification, the connection symbol “-block-” means that the segments connected by this “-block-” each constitute a block, and hereinafter it is also abbreviated as “-b-”. That is, in the first triblock copolymer, it can also be said that the polycation segment, the PEG segment, and the polycation segment are connected in this order. Further, the second triblock copolymer has a structure represented by a polyanion segment, a PEG segment, and a polyanion segment.

[0016] The PEG segment can be represented by -(OCH ２ CH ２ ) ｍ -. Here, m is preferably an integer of 20 to 800, more preferably 30 to 500, and even more preferably 40 to 400.

[0017] Examples of polymers constituting the polycation segment include polymers having substituents in their side chains that can be protonated in water. The main skeleton of the polymer is not limited, but examples include polyamino acids, polymethacrylic acid derivatives, polyacrylic acid derivatives, and polystyrene derivatives. Examples of substituents include primary amino groups and secondary amino groups, specifically amino groups and imidazole groups. Examples of polyamino acids include polylysine, polyarginine, polyornithine, polyhistidine, and polytryptophan.

[0018] The polycation segment may have a cyclic nitroxide radical as part of the pendant group. Examples of cyclic nitroxide radicals include 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl, 2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-yl, 2,2,5,5-tetramethylpyrroline-1-oxyl-3-yl, 2,4,4-trimethyl-1,3-oxazolidine-3-oxyl-2-yl, 2,4,4-trimethyl-1,3-thiazolidinedine-3-oxyl-2-yl, and 2,4,4-trimethylimidazolindinine-3-oxyl-2-yl. The cyclic nitroxide radical is o- or p-phenylene-C １～６ Alkilen-NH-(C) １～６ The alkylene may be linked to the polycation segment via q- (where q is 0 or 1).

[0019] Polymers that constitute polyanion segments include polyacrylic acid, polymethacrylic acid, polysulfonic acid, polystyrene sulfonic acid, poly(benzoate) vinyl, 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.

[0020] The degree of polymerization (number of monomer unit repeats) of the polycationic 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 between 10 and 200, more preferably between 15 and 150, even more preferably between 15 and 100, and particularly preferably between 20 and 100.

[0021] Each segment may be linked by a linking group. The linking group may be any divalent organic group, as long as it does not adversely affect the formation of the PIC. For example, a linking group may be -O-(CH ２ ) ａ -NH-, -O-(CH ２ ) ａ -O-, -(CH ２ ) ａ -NH-, -(CH ２ ) ａ -O-,

[0022]

[0023] Examples include the following. Here, a is an integer from 1 to 6, preferably from 1 to 3.

[0024] The end of the triblock copolymer, i.e., the end opposite to the end connected to the PEG segment in the polycation or polyanion segment, may contain, for example, H, an amino group, or a linear or branched C. １～６ Alkyl group, phenylthiocarbonylthio group, C １～６ Alkyloxyalkylthiocarbonylthio group, C １～６ It may also be an alkyloxythiocarbonylthio group or a sulfanyl group, etc. １～６ Alkyl group, phenylthiocarbonylthio group, C １～６ Alkyloxyalkylthiocarbonylthio group, C １～６ The alkyloxythiocarbonylthio group may be unsubstituted or substituted, and if substituted, the substituent is C １～４ alkyl group, C １～４ Alkyloxy group, hydroxyl group, carboxyl group, cyano group, nitro group, halogen atom group, or mono or diC １～４ It may be an alkylamino group.

[0025] The molecular weight distribution of the triblock copolymer does not need to be limited for gelation, but it 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.

[0026] The method for producing the triblock copolymer is not particularly limited, and methods described in Patent Document 1; International Publication No. 2015 / 118993; International Publication No. 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., can be referenced.

[0027] <1-2. Polyanions and Polycations> As polyanions to be combined with the first triblock copolymer (i.e., polyanions that form a PIC with the first triblock copolymer), those exemplified as polymers constituting the polyanion segment of the second triblock copolymer can be used. Also, as polycations to be combined with the second triblock copolymer (i.e., polycations that form a PIC with the second triblock copolymer), those exemplified as polymers constituting the polycation segment of the first triblock copolymer can be used.

[0028] The molecular weights of these polyanions and polycations are not limited and the optimal values ​​vary depending on the type of polymer. 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 1,000,000, 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, 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, more preferably 1,000 to 1,000,000.

[0029] <1-3. Polyion Complexes> PICs are formed in an aqueous medium via electrostatic interactions between the polycation segment of the first triblock copolymer and the polyanion (as described in (a) above). These PICs may exist as nanosized polymer micelles having a core formed via electrostatic interactions between the polycation segment and the polyanion, and a shell formed from the PEG segment of the first triblock copolymer. Since these polymer micelles can be observed in particulate form, they are also referred to as self-assembled nanoparticles in this specification. Similar PICs are also formed by electrostatic interactions between the polyanion segment of the second triblock copolymer and the polycation (as described in (b) above). Furthermore, similar PICs are also formed by electrostatic interactions between the polycation segment of the first triblock copolymer and the polyanion segment of the second triblock copolymer (as described in (c) above).

[0030] The specific combinations of the first triblock copolymer and the polyanion, the specific combinations of the second triblock copolymer and the polycation, and the specific combinations of the first triblock copolymer and the second triblock copolymer are not particularly limited. In the examples, measurement results are shown for combinations of a triblock copolymer having a polylysine segment and polystyrene sulfonic acid, and combinations of a triblock copolymer having a cyclic nitroxide radical and polyacrylic acid, but for example, combinations of a triblock copolymer having a polylysine segment and polyacrylic acid, and combinations of a triblock copolymer having a cyclic nitroxide radical and polystyrene sulfonic acid are also possible. Furthermore, although these correspond to (a) above, if the polymer included as a polycation segment in the first triblock copolymer in (a) is used as the polycation in (b) above, and the polyanion in (a) is used as the polyanion segment included in the second triblock copolymer in (b), a person skilled in the art will understand that a PIC will be formed even with the combination in (b). Similarly, it will be understood that a PIC will be formed even with the combination in (c) above. Furthermore, by referring to this specification, it can be understood that any combination of (a) to (c) can be combined with inorganic nanoparticles to obtain the same effects as in the examples.

[0031] Examples of the aqueous medium include pure water, deionized water, buffered solutions thereof, and solutions containing water-soluble organic solvents. Examples of water-soluble organic solvents include N,N-dimethylformamide, dimethyl sulfoxide, alcohols such as methanol and ethanol, acetone, and tetrahydrofuran.

[0032] For manufacturing methods of PIC, refer to the methods described in, for example, Patent Document 1; International Publication No. 2015 / 118993; International Publication No. 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.

[0033] <1-4. Inorganic Nanoparticles> The materials constituting inorganic nanoparticles are not particularly limited and include inorganic oxides (e.g., silica (silica gel), titanium dioxide, calcium oxide), inorganic carbonates (e.g., calcium carbonate), and metals (e.g., gold, silver). In the examples, silica nanoparticles are used, but the effect of increasing the strength of the gel is obtained by using multiple types of silica nanoparticles with different particle sizes, and it is presumed that this effect does not depend on the chemical properties specific to silica, so it can be understood that the material of the inorganic nanoparticles is not limited.

[0034] The average particle size of 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. Furthermore, the average particle size of the inorganic nanoparticles is preferably 1 nm or more, and more preferably 4 nm or more. The average particle size can be determined from the volume-based particle size distribution as the hydrodynamic diameter measured by dynamic light scattering measurement. Alternatively, the average particle size may be measured by transmission electron microscopy.

[0035] The inorganic nanoparticles are compounded with the self-assembled nanoparticles and are presumed to be incorporated into the interior of the self-assembled nanoparticles, for example. For example, particles compounded with silica nanoparticles and self-assembled nanoparticles are also referred to as silica-compounded self-assembled nanoparticles in this specification. The average particle size of the self-assembled nanoparticles compounded 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. Furthermore, the average particle size of the self-assembled nanoparticles compounded 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 the volume-based particle size distribution as the hydrodynamic diameter measured by dynamic light scattering measurement.

[0036] A method for producing a gel manufacturing composition according to one embodiment of the present invention includes a step of mixing the polyion complex described in (a), (b), or (c) above with inorganic nanoparticles. For example, a gel manufacturing 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. When mixing, a mixer may be used for stirring.

[0037] [2. Gel] A gel according to one embodiment of the present invention is obtained by gelling the gel manufacturing composition described above. A method for producing a gel according to one embodiment of the present invention includes the steps of obtaining a gel manufacturing composition by the method for producing a gel manufacturing composition described above, and gelling the gel manufacturing composition. Examples of gelling methods include increasing the temperature and / or ionic strength. For example, heating the gel manufacturing composition to 35 to 45°C and / or contacting the gel manufacturing composition with physiological saline solution having an NaCl concentration of 100 to 200 mM are examples of methods.

[0038] The strength of the gel is expressed by its storage modulus, which is measured, for example, by the method described in the examples. The storage modulus of the gel is preferably 100 Pa or more, more preferably 1000 Pa or more, and even more preferably 10 kPa or more.

[0039] [3. Gel Manufacturing Kit] A gel manufacturing kit according to one embodiment of the present invention comprises a first agent containing the polyion complex described in (a), (b), or (c) above, and a second agent containing inorganic nanoparticles. By mixing the first agent and the second agent using this gel manufacturing kit, the polyion complex and inorganic nanoparticles are mixed and gelled, thereby producing a gel.

[0040] PIC may be in the form of a solution or dispersion in the first component, or it may be in a dry state. Inorganic nanoparticles may be in the form of a solution or dispersion in the second component, or it may be in a dry state. When mixing, it is preferable that the first and second components be mixed in the form of a solution or dispersion containing the above-mentioned aqueous medium.

[0041] The first and second components may be contained in separate containers. In this specification, the container containing the first component will be referred to as the first container, and the container containing the second component as the second container. The first and second containers may be independent and separate containers, or they may be integrated. When the first and second containers are integrated, it means that they are configured as a single container comprising a first space containing the PIC and a second space containing inorganic nanoparticles. The gel manufacturing kit may also include another container (space) containing the aqueous medium described above for manufacturing the solution or dispersion.

[0042] The present invention is not limited to the embodiments described above, 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.

[0043] One embodiment of the present invention may include the following configuration: <1> A gel manufacturing composition 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, and both the polyanion that forms the polyion complex with the first triblock copolymer and the polycation that forms the polyion complex with the second triblock copolymer are polymers. A gel obtained by gelling the gel manufacturing composition of <2> and <1>.<3> A method for producing a gel composition, 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, and the polyanion that forms the polyion complex with the first triblock copolymer and the polycation that forms the polyion complex with the second triblock copolymer are both polymers. A method for producing a gel, comprising the steps of: obtaining a gel-producing composition by the method for producing a gel-producing composition described in <4> and <3>; and gelling the gel-producing composition.<5> A gel manufacturing kit 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, wherein the polyanion that forms the polyion complex with the first triblock copolymer and the polycation that forms the polyion complex with the second triblock copolymer are both polymers.

[0044] An embodiment 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 nanoparticle) concentration in the solution.

[0045] [Material 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 × 10⁻¹⁰) were connected to a pump (JASCO, PU-4180). ６Gel permeation chromatography (GPC) was performed in N,N-dimethylformamide (DMF) at 40°C (flow rate: 0.40 mL / min) using a differential refractive index (RI) detector (JASCO, RI-2031) and a UV / Vis detector (JASCO, UV-4075). The column was calibrated using 18 standard poly(ethylene oxide) (PEO) and poly(ethylene glycol) (PEG) samples (Merck; Mp = 238-1,180,000). １ The H nuclear magnetic resonance (NMR) spectrum was obtained using an AVANCE-600 NMR spectrum meter (Brker) at 600 MHz. １ Activate with H) and CDCl ３ or DMSO-d ６ The data was acquired 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 the data was analyzed using the non-negative least squares method (NNLS).

[0046] [Example 1 Synthesis of Z-protected L-lysine-N-acid anhydride (NCA-Lys(Z))] L-lysine (HOCOCH((CH)) having a benzyloxycarbonyl group in the side chain ２ ) ４ NHCOOCH ２ C ６ H ５ ) NH ２;H-Lys(Z)-OH) (25.2 g, 89.9 mmol; 1.0 eq.) and triphosgene (18.2 g, 61.3 mmol; approximately 0.67 eq.) were dissolved in α-pinene (36 mL, 227 mmol; 2.5 eq.) and tetrahydrofuran (THF; 252 mL), and the mixture was stirred at 50°C for 3 hours. After the resulting reaction solution was cooled to room temperature, it was gradually added to hexane (600 mL) to obtain a white precipitate. The white precipitate was washed with hexane and recovered by vacuum filtration. The recovered white compound was redissolved in THF / acetone / isopropanol (IPA) (approximately 10 / 10 / 1 (v / v / v)), and reprecipitation in hexane was repeated two more times. Finally, the white precipitate recovered by vacuum filtration was dried under reduced pressure to obtain the target product NCA-Lys(Z) (yield 16.4 g, yield 59%).

[0047]

[0048] [Example 2 H-(HNCH((CH ２ ) ４ NHCOOCH ２ C ６ H ５ )CO) ｎ NH-CH ２ CH ２ (OCH ２ CH ２ ) ｍ -NH(COCH((CH ２ ) ４ NHCOOCH ２ C ６ H ５ )NH) ｎ -Synthesis of H] PEG macroinitiator H ２ N-(OCH) ２ CH ２ ) ｎ -NH ２ (Mn = 10,000; 25.1 g, 2.51 mmol) was dissolved in N,N-dimethylformamide (DMF; 44.8 mL) and tetralin (0.3 mL). Furthermore, a 1000 mM monomer solution was prepared by dissolving NCA-Lys(Z) synthesized in Example 1 in DMF. The monomer solution (70.4 mL, 70.4 mmol) was heated to H ２ N-(OCH) ２ CH２ ) ｎ -NH ２ The mixture was added to the DMF solution and 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 recovered by centrifugation. The recovered precipitate was redissolved in DMF, and the reprecipitation and purification by centrifugation were repeated two more times. The target product (Plys(Z)-b-PEG-b-Plys(Z)) was recovered by vacuum drying (30.5 g, n=7.7 (P2), Figure 1). Polymers with n=2.7 (P1), 19 (P3), and 54 (P4) were synthesized in the same manner (Figures 1 and 2). The obtained polymers are abbreviated as P1, P2, P3, and P4.

[0049]

[0050] [Example 3 H-(HNCH((CH ２ ) ４ NH ３ )CO) ｎ NH-CH ２ CH ２ (OCH ２ CH ２ ) ｍ -NH(COCH((CH ２ ) ４ NH ３ )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. Hydrogen bromide (HBr) acetic acid solution (28%; 61 mL, 337 mmol; approximately 10 eq.) was slowly added dropwise to the resulting solution. 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 mixed with methanol / Na ２ CO ３ After dialyzing in an aqueous solution (= 1 / 1 (v / v)) for 3 days and in water for 4 days, the target product (Plys-b-PEG-b-Plys) was recovered by freeze-drying (19.6 g, 100% deprotection rate). P1, P3, and P4 were deprotected in the same manner (Figure 2).

[0051]

[0052] [Example 4 Preparation of Self-Assembled Nanoparticles 1: Formation of Polyion Complex with Poly(styrenesulfonic acid)] Plys-b-PEG-b-Plys synthesized in Example 3 were dissolved in dimethyl sulfoxide (DMSO) / TFA = 20 / 1 (v / v) ([polymer] = 5 mg / mL). Sodium poly(styrenesulfonic acid) (NaPSS; Sigma-Aldrich, Mw: approximately 75000) was dissolved in phosphate buffer (pH = 7.4) ([polymer] = 5 mg / mL). The NaPSS phosphate buffer was slowly added dropwise to the Plys-b-PEG-b-Plys solution to prepare a mixed solution. This mixed solution was directly transferred to a dialysis membrane (MWCO = 3500 Da), dialyzed against water for 4 days, and then concentrated under reduced pressure to form self-assembled nanoparticles (Nano Ｌｙｓ／ＰＳＳ A concentrated solution of ) was obtained. This concentrated solution was diluted with water, and dynamic light scattering (DLS) measurements were performed to confirm the formation of self-assembled nanoparticles ranging from 10 to 43 nm (Figures 3 and 4; [polymer] = 10 mg / mL).

[0053] [Example 5 Preparation of Self-Assembled Nanoparticles 2: Composite Formation of Silica Nanoparticles] After diluting the concentrated solution of self-assembled nanoparticles prepared in Example 4 with water, an aqueous solution of dispersed silica nanoparticles was added and immediately stirred for 5 minutes using a vortex mixer to prepare silica-composite self-assembled nanoparticles (NanoLys / PSS / SiO) ([polymer] = 40 mg / mL). Snowtex® (Nissan Chemical Corporation; Snowtex® XS (ST-XS), Snowtex® 30 (ST-30)) was used as the silica nanoparticles. The size of ST-XS was 4-6 nm, and the size of ST-30 was 10-15 nm. For DLS measurement, the solution was diluted 40 times with water before measurement (Figures 7, 8; [polymer] = 1.0 mg / mL).

[0054] [Example 6 Rheological Measurement] Gelation behavior was evaluated using an Anton Paar rheometer (MCR302). In this experiment, the storage modulus (G'), loss modulus (G''), and complex viscosity ([η) were evaluated. ＊The storage modulus and loss modulus were measured to evaluate the gelation process and the properties of the gel. The storage modulus and loss modulus indicate the properties of the solid and liquid, respectively. For example, when the storage modulus is higher than the loss modulus (G' > G''), the material mainly exhibits solid properties. Furthermore, the gelation point can be determined at the intersection where both moduli are the same value (G' = G''). The self-assembled nanoparticle aqueous solutions ([polymer] = 40 mg / mL; 100 μL) obtained in Examples 4 and 5 were placed on a stage with a distance of 0.2 mm to the plate. A 20 mm parallel plate was used. The measurement frequency was fixed at 1 Hz, and the temperature was varied from 15°C to 45°C, followed by continuous cooling from 45°C to 15°C. The temperature change rate was 1°C / min.

[0055] [Example 7: Influence of Ionic Strength on Temperature Dependence Measurement Results of Elastic Modulus and Viscosity] Sodium chloride (NaCl) is treated with Plys-b-PEG-b-Plys(P4) / PSS self-assembled nanoparticles (Nano Ｌｙｓ／ＰＳＳ (P4)) Dissolved in an aqueous solution ([polymer] = 40 mg / mL, [NaCl] = 0 mM, 150 mM, or 500 mM). Measurement of this aqueous solution according to Example 6 showed that under NaCl concentrations of 150 mM and 500 mM, gelation occurred at 25.9°C and 24.7°C, respectively. The resulting gel did not return to its initial modulus even after cooling, demonstrating irreversible gelation behavior. The modulus after gelation (G') decreased gradually as the NaCl concentration increased, but remained around several hundred Pa under all conditions (Figure 5). Furthermore, these results suggest that Nano Ｌｙｓ／ＰＳＳ (P4) was shown to gel depending on temperature and ionic strength.

[0056] [Example 8: Influence of the degree of polymerization of lysine (DP(Lys)) on the temperature dependence of elastic modulus and viscosity measurements] 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, and the effects of P3 and P4 were investigated ([polymer] = 40 mg / mL, [NaCl] = 150 mM). Gelation occurred in both P3 and P4 (Figure 6).

[0057] [Example 10: Influence of Silica Nanoparticles on Measurement Results of Temperature Dependence of Elastic Modulus and Viscosity] The silica composite nanoparticles (NanoLys / PSS / SiO) prepared in Example 5 were measured according to Example 6. At this time, Snowtex (registered trademark) series was used for the silica nanoparticles. As a result, compared with the case where silica nanoparticles were not composite, when silica nanoparticles were composite, the elastic modulus after gelation was improved (Figs. 9 and 10). In particular, when Snowtex (registered trademark) XS (ST-XS) was used, a high elastic modulus exceeding 10 kPa was achieved after gelation (Figs. 9 and 10).

[0058] [Example 11: Synthesis of Cl-PEG-Cl] HO-(CH ２ CH ２ ) ｎ -OH (Mn = 10,000; 50.0 g, 5.00 mmol) was added to a 500 mL eggplant 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 (200 mL) was added to dissolve the contents of the flask. A butyllithium solution (20.0 mmol, 12.5 mL, 1.6 M hexane solution) was gradually added to the obtained solution under a nitrogen atmosphere to activate the hydroxyl group. Subsequently, dichloro-p-xylene (17.5 g, 0.1 mol) was added and reacted at 60 °C for 4 days. The obtained reaction solution was precipitated in IPA cooled at 4 °C, and the precipitate was recovered by centrifugation. The precipitate was dissolved again in 20 mL of methanol and reprecipitated with cooled IPA. The steps of reprecipitation and centrifugation were repeated 4 more times. The target product (Cl-PEG-Cl) was recovered by drying under reduced pressure.

[0059] [Example 12: Synthesis of Grignard Reagent] THF (10 mL) was added to a 50 mL eggplant 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 to this flask under a nitrogen atmosphere and reacted overnight in the ice bath. The Grignard reagent synthesized thereby was used as it was without purification.

[0060] [Example 13 Synthesis of Macro-RAFT Agent (CTA-PEG-CTA)] Cl-PEG-Cl (Mn (size exclusion chromatography (SEC)) = 9,300; 40.0 g, 3.9 mmol) synthesized in Example 11 was added to a 300 mL round-bottom flask equipped with a three-way stopcock, and dried overnight under reduced pressure at 110°C. After lowering the liquid temperature to 65°C, 140 mL of THF was added to dissolve the contents of the flask. To the resulting solution, the Grignard reagent synthesized in Example 12 was gradually added under a nitrogen atmosphere, and the mixture was reacted at 40°C for 24 hours. The resulting reaction mixture 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 reprecipitation was performed in cooled IPA. The reprecipitation and centrifugation steps were repeated three more times. The target product (CTA-PEG-CTA) was recovered by reduced-pressure drying.

[0061] [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 added to a 500 mL round-bottom flask equipped with a three-way stopcock, and the mixture was degassed by reducing the pressure for 30 minutes. Toluene (350 mL) was added under a nitrogen atmosphere to dissolve the contents of the flask. To the resulting solution, p-chloromethylstyrene (32.8 mL, 0.230 mol) purified by vacuum distillation was added, and the mixture was reacted at 60°C for 24 hours. The resulting reaction mixture was precipitated in methyl t-butyl ether (2 L), and the precipitate was recovered by vacuum filtration. The recovered precipitate was dissolved in acetone (20 mL) and reprecipitated in methyl t-butyl ether (2 L). The precipitate was recovered by vacuum filtration and dried under reduced pressure overnight. The dried precipitate, AIBN (14.5 g, 89.8 mmol), and ethyl acetate (300 mL) were added to a 500 mL round-bottom flask equipped with a reflux tubing and dissolved. The resulting solution was degassed by nitrogen bubbling for 30 minutes and then reacted at 80°C for 12 hours. The reaction mixture was precipitated in methyl t-butyl ether (2 L) and the precipitate was recovered by vacuum filtration. The recovered 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 recovered by vacuum drying.

[0062] [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 added to a 200 mL round-bottom flask equipped with a three-way stopcock, and dried under reduced pressure for 30 minutes. After adding DMF (80 mL) to dissolve the contents of the flask, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-aminoTEMPO) (10.0 g, 58.4 mmol) dissolved in 20 mL of DMF was added, and the mixture was reacted 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 again dissolved in 10 mL of acetone, and reprecipitation was performed in cooled IPA. The reprecipitation and centrifugation steps were repeated four more times. The target material (PMNT-b-PEG-b-PMNT) was recovered by vacuum drying.

[0063] [Example 16 Preparation of Self-Assembled Nanoparticles 3: Formation of Polyion Complex with PMNT-b-PEG-b-PMNT and Polyacrylic Acid] PMNT-b-PEG-b-PMNT and polyacrylic acid (PAAc; Fujifilm Wako, Mn: approximately 5,000) synthesized in Example 15 were dissolved in phosphate buffer (pH = 6.2, 100 mM) ([polymer] = 10 mg / mL). The PMNT-b-PEG-b-PMNT solution was gradually added dropwise to the PAAc phosphate buffer, which was being slowly stirred, to prepare a mixed solution. Dynamic light scattering measurements were performed to confirm the formation of 62 nm self-assembled nanoparticles (Figure 11 (RIG)).

[0064] [Example 17: Rheological Evaluation of Redox Injectable Gel (RIG)] The mixed solution prepared in Example 16 was concentrated using a centrifugal evaporator to prepare a RIG ([polymer] = 60 mg / mL, [PB] = 600 mM). After distillation and dilution with deionized water ([polymer] = 40 mg / mL, [PB] = 400 mM), rheological measurements were performed according to Example 6, and gelation occurred at 25.1°C. The obtained gel did not return to its initial modulus of elasticity even after cooling, confirming irreversible gelation behavior (Figure 12).

[0065] [Example 18: Rheological evaluation of silica nanoparticle composite RIG (RIG / Snowtex® ST-XS)] Snowtex® XS (ST-XS) was added to the RIG prepared in Example 17 ([polymer] = 60 mg / mL, [PB] = 600 mM), and the mixture was thoroughly stirred with a vortex mixer to prepare silica composite self-assembled nanoparticle RIG / ST-XS ([polymer] = 40 mg / mL, [PB] = 400 mM, [ST-XS] = 5.6 wt%). Rheological measurements were performed according to Example 6, and a high modulus of elasticity exceeding 10 kPa was achieved after gelation (Figure 11 (RIG / ST-XS), Figure 12).

[0066] One aspect of the present invention can be used, for example, in the production of injectable gels.

Claims

It contains a polyion complex and inorganic nanoparticles. The aforementioned polyion complex is (a) A polyionic complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) A polyionic 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 gel manufacturing composition in which 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. 　 A gel obtained by gelling the gel manufacturing composition described in claim 1. 　 The process includes a step of mixing a polyion complex with inorganic nanoparticles. The aforementioned polyion complex is (a) A polyionic complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) A polyionic 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 gel composition, 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. 　 A step of obtaining a gel manufacturing composition by the method for manufacturing a gel manufacturing composition described in claim 3, A method for producing a gel, comprising the step of gelling the aforementioned gel-producing composition. 　 The solution comprises a first agent containing a polyion complex and a second agent containing inorganic nanoparticles. The aforementioned polyion complex is (a) A polyionic complex of a first triblock copolymer having a structure represented by polycation-block-polyethylene glycol-block-polycation and a polyanion, (b) A polyionic 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 gel manufacturing kit in which 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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