Method for preparing hyaluronic acid hydrogel with slow decomposition rate

Abstract

Disclosed is a method for preparing a hyaluronic acid hydrogel, comprising the steps of: (a) mixing a basic aqueous solution, hyaluronic acid or a salt thereof, and a crosslinking agent; (b) molding the mixture of step (a) into a sheet-shaped intermediate; (c) crosslinking the sheet-shaped intermediate; and (d) purifying and pulverizing the crosslinked intermediate of step (c), thereby obtaining a hyaluronic acid hydrogel.

Description

Method for manufacturing a hyaluronic acid hydrogel with a slow degradation rate

[0001] This invention relates to a method for manufacturing a hyaluronic acid hydrogel in which the degradation rate is controlled slowly by forming a sheet-like structure prior to crosslinking.

[0002] Hyaluronic acid (HA) is a term referring to any known hyaluronic acid raw material, such as hyaluronic acid, its salts, or its precursors, either alone or in mixtures. It is a natural polymer material widely used in various fields, such as cosmetic or medical purposes, due to its excellent biocompatibility. In its natural state, hyaluronic acid is primarily found in the extracellular matrix and plays a role in stabilizing tissue structures by forming a matrix solution when mixed with fibrous tissue and collagen. As a substance found in the tissues of all vertebrates, it is an ideal chemical substance for use as a component for tissue repair purposes due to its lack of immunogenicity.

[0003] Hyaluronic acid is a substance widely applied in the fields of cosmetics, food, medical devices, and pharmaceuticals. In particular, hyaluronic acid with a large molecular weight is used as a filler that is injected into the body in a cross-linked gel form.

[0004] Fillers are medical devices in the form of disposable syringes that inject safe hyaluronic acid into the dermal layer to replenish skin tissue, such as improving wrinkles and increasing volume for aesthetic purposes. They are used to fill in sagging areas of the face, such as nosolabial folds, a low forehead, and a low nose, which cannot be improved with botulinum toxin.

[0005] Hyaluronic acid crosslinks in the form of water-insoluble hyaluronic acid gels used as fillers have the advantage of being easy to control in terms of physical properties and extending the duration of hyaluronic acid's survival in vivo from one week to several months, or even up to one year. Due to these characteristics, hyaluronic acid crosslinks are utilized for various purposes, such as biomaterials for tissue repair requiring long-term survival in vivo, scaffolds for drug delivery, or treatments for osteoarthritis.

[0006] Meanwhile, fillers that are currently being developed as filler products and are hydrophobic and biodegradable polymers, such as polylactic acid (or poly lactide, PLA), polyglycolic acid (or polyglycolide, PLGA), and poly-ε caprolactone (poly-ε), have a duration of more than 2 years in the body and possess the characteristic of inducing collagen production within skin tissue.

[0007] However, fillers composed mainly of microparticles of such hydrophobic biodegradable polymers are distributed in freeze-dried formulations, which require a reconstitution process involving the addition of physiological saline or distilled water before use, and take time to stabilize, resulting in low convenience of use. In addition, it is known that aggregation occurs due to hydrophobic interactions between the microparticles of the biodegradable polymers.

[0008] Therefore, attempts to improve hyaluronic acid hydrogels, which account for more than 80% of the existing filler market, are continuing. In particular, given that patient satisfaction with the procedure tends to be higher as the duration of the filler's effect increases, there is a demand for a new method of manufacturing hyaluronic acid hydrogels that can extend the duration of the filler's effect in the body by slowing down the degradation rate of the hyaluronic acid hydrogel.

[0009] The present invention provides a method for manufacturing a hyaluronic acid hydrogel that can slow down the rate of filler degradation in the body by improving the existing hyaluronic acid hydrogel manufacturing method and controlling the cross-linking temperature and the cross-linking form.

[0010] According to one aspect, a method for preparing a hyaluronic acid hydrogel is provided, comprising: (a) mixing a basic aqueous solution, hyaluronic acid or a salt thereof, and a crosslinking agent; (b) forming the mixture of step (a) into a sheet-shaped intermediate; (c) crosslinking the intermediate formed into a sheet-shaped intermediate; and (d) purifying and grinding the crosslinked intermediate of step (c) to obtain a hyaluronic acid hydrogel.

[0011] In one embodiment, the basic aqueous solution may include one or more selected from the group consisting of NaOH and KOH.

[0012] In one embodiment, the OH- concentration of the basic aqueous solution may be 0.05 to 1 M.

[0013] In one embodiment, the crosslinking agent may be one or more selected from the group consisting of BDDE and divinylsulfone.

[0014] In one embodiment, the concentration of the crosslinking agent may be 1 to 20 mol%.

[0015] In one embodiment, the thickness of the intermediate formed into a sheet shape in step (b) may be 1 to 10 mm.

[0016] In one embodiment, step (c) may be performed at 10 to 30°C for 6 to 340 hours.

[0017] In one embodiment, after step (d), the method may further include step (e) sterilizing the hyaluronic acid hydrogel in an autoclave.

[0018] In one embodiment, the weight ratio of the hyaluronic acid or its salt to the crosslinking agent may be 10:1 to 300:1.

[0019] A hyaluronic acid hydrogel prepared according to the method for preparing a hyaluronic acid hydrogel according to one aspect of the present invention maintains a low degradation rate in the body, allowing for long-term treatment effects.

[0020] The effects of one aspect of this specification are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configurations described in the detailed description or claims of this specification.

[0021] Figure 1 is a graph of the residual ratio of hyaluronic acid hydrogels in Example 1 and Comparative Example 1.

[0022] Hereinafter, one aspect of the present specification will be described in detail. However, the details described in the present specification may be implemented in various different forms and are therefore not limited to the embodiments described herein.

[0023] Throughout the specification, when a part is described as "including" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather allows for the inclusion of additional components.

[0024] When a range of numerical values ​​is described in this specification, unless a specific range is otherwise described, the value has the precision of significant figures provided according to the standard rules in chemistry for significant figures. For example, 10 includes a range of 5.0 to 14.9, and the number 10.0 includes a range of 9.50 to 10.49.

[0025] Method for manufacturing hyaluronic acid hydrogel

[0026] A method for preparing a hyaluronic acid hydrogel according to one aspect comprises: (a) mixing a basic aqueous solution, hyaluronic acid or a salt thereof, and a crosslinking agent; (b) forming the mixture of step (a) into a sheet-shaped intermediate; (c) crosslinking the intermediate formed into a sheet-shaped intermediate; and (d) purifying and grinding the crosslinked intermediate of step (c) to obtain a hyaluronic acid hydrogel.

[0027] In conventional methods for manufacturing hyaluronic acid hydrogels, a mixture containing hyaluronic acid and a crosslinking agent is cast into a cylindrical shape at a high temperature, and crosslinking proceeds rapidly within a short period of time. Consequently, there were problems such as the hyaluronic acid included in the composition discoloring after manufacturing, or the need to add a large amount of crosslinking agent to reach the target elasticity value.

[0028] To solve these problems, the inventors discovered that in the process of crosslinking hyaluronic acid, if the heat transfer efficiency is increased by controlling the crosslinking shape to a thin sheet form, crosslinking is possible even at a lower temperature compared to the existing process, and furthermore, the target elasticity value can be reached even with a low concentration of crosslinking agent, and thus completed the present invention.

[0029] (a) Step

[0030] (a) Step is a step of mixing a basic aqueous solution, hyaluronic acid or a salt thereof, and a crosslinking agent, and a step of creating a basic environment to uniformly mix them in order to form crosslinks on the hyaluronic acid.

[0031] The above basic aqueous solution may include one or more selected from the group consisting of NaOH and KOH. In addition, since the hydrogen ion concentration (pH) of the mixture containing hyaluronic acid must be 12 or higher in order to convert the hyaluronic acid into a hyaluronic acid crosslink, the OH- concentration of the above basic aqueous solution may be 0.05 to 1 M, preferably the OH- concentration of the above basic aqueous solution may be 0.1 to 0.7 M, and most preferably the OH- concentration of the above basic aqueous solution may be 0.15 to 0.5 M, but is not limited thereto.

[0032] For example, the OH- concentration of the above basic aqueous solution is 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.10M, 0.11M, 0.12M, 0.13M, 0.14M, 0.15M, 0.16M, 0.17M, 0.18M, ​​0.19M, 0.20M, 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0.29M, 0.30M, 0.31M, 0.32M, 0.33M, 0.34M, 0.35M, 0.36M, 0.37M, 0.38M, 0.39M, 0.40M, 0.41M, 0.42M, 0.43M, 0.44M, 0.45M, 0.46M, 0.47M, 0.48M, 0.49M, 0.50M, 0.51M, 0.52M, 0.53M, 0.54M, 0.55M, 0.56M, 0.57M, 0.58M, 0.59M, 0.60M, 0.61M, 0.62M, 0.63M, 0.64M, 0.65M, 0.66M, 0.67M, 0.68M, 0.69M, 0.70M, 0.71M, 0.72M, 0.73M, 0.74M, 0.75M, 0.76M, 0.77M, 0.78M, 0.79M, 0.80M, 0.81M, 0.82M, 0.83M, 0.84M, 0.85M, 0.86M, 0.87M, 0.88M, 0.89M, 0.90M, 0.91M, 0.92M, 0.93M, 0.94M, 0.95M, 0.96M, 0.97M, 0.98M, 0.99M, 1.00M, or a value between two of these values. If the OH- concentration of the basic aqueous solution is below the above range, the pH of the mixture containing the hyaluronic acid becomes excessively low, and crosslinking may not occur properly. If the OH- concentration of the basic aqueous solution exceeds the above range, the hyaluronic acid may decompose excessively, and as the chemical structure is altered, problems such as failure to form crosslinking or browning may occur.

[0033] The above hyaluronic acid or its salt is one or more selected from the group consisting of hyaluronic acid, sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, lithium hyaluronate, and zinc hyaluronate, and is a natural polymer having repeating units of N-acetylglucosamine and glucuronic acid, which can contain a large amount of water molecules within the molecule and thus has excellent hydrophilicity, thereby having the effect of repairing wrinkles in a filler composition injected into the human body.

[0034] The concentration of the hyaluronic acid or its salt may be 10 to 25 weight% with respect to the total weight of the basic aqueous solution containing the hyaluronic acid or its salt, preferably 12 to 23 weight%, and most preferably 15 to 20 weight%, but is not limited thereto. For example, the concentration of the hyaluronic acid or its salt may be 10 weight%, 11 weight%, 12 weight%, 13 weight%, 14 weight%, 15 weight%, 16 weight%, 17 weight%, 18 weight%, 19 weight%, 20 weight%, 21 weight%, 22 weight%, 23 weight%, 24 weight%, 25 weight%, or a value between two of these values ​​with respect to the total weight of the basic aqueous solution containing the hyaluronic acid or its salt, but is not limited thereto. If the weight of the hyaluronic acid or its salt falls outside the above range, the physical properties of the manufactured hyaluronic acid hydrogel, such as elasticity, may deteriorate, or the structure may be deformed and discolored.

[0035] The above crosslinking agent may be a bisepoxide-based or divinyl sulfone-based crosslinking agent having two or more epoxy functional groups, and specifically, the crosslinking agent may be 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, or neopentyl glycol diglycidyl ether. It may be one or more selected from the group consisting of diglycidyl ether), polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether, divinyl sulfone, polyethylene glycol, and sorbitol polyglycidyl ether, and preferably 1,4-butanediol diglycidyl ether (1,It may be 4-butanedio diglyciyl ether (BDDE), polyethylene glycol (PEG), or divinyl sulfone (DVS), preferably one or more selected from the group consisting of BDDE and divinyl sulfone, and most preferably BDDE. When BDDE is used as the crosslinking agent, the physical properties of the hyaluronic acid hydrogel produced, such as elasticity, can be improved due to excellent crosslinking stability and biocompatibility.

[0036] The concentration of the crosslinking agent may be 1 to 20 mol% with respect to the total molar amount of the hyaluronic acid or its salt, preferably 2 to 10 mol%, and most preferably 3 to 7 mol%, but is not limited thereto. For example, the concentration of the crosslinking agent is 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3.0 mol%, 3.1 mol%, 3.2 mol%, 3.3 mol%, 3.4 mol%, 3.5 mol%, 3.6 mol%, 3.7 mol%, 3.8 mol%, 3.9 mol%, 4.0 mol%, with respect to the total molar amount of the hyaluronic acid or its salt. 4.1mol%, 4.2mol%, 4.3mol%, 4.4mol%, 4.5mol%, 4.6mol%, 4.7mol%, 4.8mol%, 4.9mol%, 5.0mol%, 5.1mol%, 5.2mol%, 5.3mol%, 5.4mol%, 5.5mol%, 5.6mol%, 5.7mol%, 5.8mol%, 5.9mol%, 6.0mol%, 6.1mol%, 6.2mol%, 6.3mol%, 6.4mol%, 6.5mol%, 6.6mol%, 6.7mol%, 6.8mol%, 6.9mol%, 7.0mol%, 7.1mol%, 7.2mol%, 7.3mol%, 7.4mol%, 7.5mol%, 7.6mol%, 7.7mol%, 7.8mol%, 7.9mol%, 8.0mol%, 8.1mol%, 8.2mol%, 8.3mol%, 8.4mol%, 8.5mol%, 8.6mol%, 8.7mol%, 8.8mol%, 8.9mol%, 9.0mol%, 9.1mol%, 9.2mol%, 9.3mol%, 9.4mol%, 9.5mol%, 9.6mol%, 9.7mol%, 9.8mol%, 9.9mol%, 10.0mol%, 10.1mol%, 10.2mol%, 10.3mol%, 10.4mol%, 10.5mol%, 10.6mol%, 10.7mol%, 10.8mol%, 10.9mol%, 11.0mol%, 11.1mol%, 11.2mol%, 11.3mol%, 11.4mol%, 11.5mol%, 11.6mol%, 11.7mol%, 11.8mol%, 11.9mol%, 12.0mol%, 12.1mol%, 12.2mol%, 12.3mol%, 12.4mol%, 12.5mol%, 12.6mol%, 12.7mol%, 12.8mol%, 12.9mol%, 13.0mol%, 13.1mol%, 13.2mol%, 13.3mol%, 13.4mol%, 13.5mol%, 13.6mol%, 13.7mol%, 13.8mol%, 13.9mol%, 14.0mol%, 14.1mol%, 14.2mol%, 14.3mol%, 14.4mol%, 14.5mol%, 14.6mol%, 14.7mol%, 14.8mol%, 14.9mol%, 15.0mol%, 15.1mol%, 15.2mol%, 15.3mol%, 15.4mol%, 15.5mol%, 15.6mol%, 15.7mol%, 15.8mol%, 15.9mol%, 16.0mol%, 16.1mol%, 16.2mol%, 16.3mol%, 16.4mol%, 16.5mol%, 16.6mol%, 16.7mol%, 16.8mol%, 16.9mol%, 17.0mol%, 17.1mol%, 17.2mol%, 17.3mol%, 17.4mol%, 17.5mol%, 17.6mol%, 17.7mol%, 17.8mol%, 17.9mol%, 18.0mol%, 18.1mol%, 18.2mol%, 18.3mol%, 18.4mol%, 18.5mol%, 18.6mol%, 18.7mol%, 18.8mol%, 18.9mol%, 19.0mol%, 19.1mol%, 19.2 mol%, 19.3 mol%, 19.4 mol%, 19.5 mol%, 19.6 mol%, 19.7 mol%, 19.8 mol%, 19.9 mol%, 20.It may be 0 mol% or a value between the two values. If the concentration of the crosslinking agent exceeds the above range, the crosslinking may be excessively formed, causing the manufactured hyaluronic acid hydrogel to swell or leaving a crosslinking agent in the manufactured hyaluronic acid hydrogel, and if the concentration of the crosslinking agent is below the above range, the crosslinking may be insufficiently formed, which may degrade physical properties such as elasticity of the manufactured hyaluronic acid hydrogel.

[0037] (b) Step

[0038] (b) is a step of forming the mixture of step (a) into a sheet-shaped intermediate, and is a step of pre-treating the mixture of step (a) to efficiently form crosslinks into a sheet structure.

[0039] The thickness of the intermediate formed into the sheet shape above may be 1 to 10 mm, and preferably, the thickness of the intermediate formed into the sheet shape above may be 2 to 7 mm. For example, the thickness of the intermediate formed into the sheet shape above is 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2.0mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3.0mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4.0mm, 4.1mm, 4.2mm, 4.3mm, 4.4mm, 4.5mm, 4.6mm, 4.7mm, 4.8mm, 4.9mm, 5.0mm, 5.1mm, 5.2mm, 5.3mm, 5.4mm, 5.5mm, 5.6mm, 5.7mm, 5.8mm, 5.9mm, 6.0mm, 6.1mm, 6.2mm, 6.3mm, 6.4mm, 6.5mm, 6.6mm, 6.7mm, 6.8mm, 6.9mm, 7.0mm, 7.1mm, 7.2mm, 7.3mm, 7.4mm, 7.5mm, 7.6mm, 7.7mm, 7.8mm, 7.9mm, 8.0mm, 8.1mm, 8.2mm, 8.3mm, 8.4mm, 8.5mm, 8.6mm, 8.7mm, 8.8mm, 8.9mm, The thickness may be 9.0mm, 9.1mm, 9.2mm, 9.3mm, 9.4mm, 9.5mm, 9.6mm, 9.7mm, 9.8mm, 9.9mm, 10.0mm, or a value between two of these values. If the thickness of the intermediate formed into the sheet shape exceeds the above range, heat transfer to the interior during the crosslinking step may not be properly carried out, resulting in uneven crosslinking, or the time required to remove the basic aqueous solution or unreacted crosslinking agent during the purification step may increase. If the thickness is below the above range, the intermediate formed into the sheet shape may tear, and the hydrogel may be lost during the purification step.

[0040] (c) Step

[0041] (c) Step is a step of crosslinking the intermediate formed into the sheet shape above, and has the characteristic of being able to produce a hyaluronic acid hydrogel with excellent physical properties even though crosslinking is carried out at a lower temperature and at a lower concentration of crosslinking agent compared to conventional technology.

[0042] Step (c) above may be performed at 10 to 30 ℃ for 6 to 340 hours, but is not limited thereto. Step (c) above may be performed at room temperature for 24 hours or more, and accordingly, excellent crosslinking may be formed.

[0043] In particular, in the present invention, crosslinking is formed more effectively by forming a sheet-shaped intermediate in the pretreatment fixation of step (b), and accordingly, a hyaluronic acid hydrogel with excellent physical properties can be produced even at a lower concentration of crosslinking agent.

[0044] (d) Step

[0045] (d) is a step of obtaining a hydrogel by purifying and grinding the cross-linked intermediate of step (c) above, and may consist of purification and grinding processes.

[0046] The process of purifying the cross-linked intermediate of step (c) above can be carried out by using a buffer solution such as PBS (Phosphate Buffered Saline) and changing the buffer solution at regular intervals until more than 90% of the target weight of the hyaluronic acid hydrogel is reached. At this time, if purification is completed before reaching the target weight, the final physical properties and weight can be controlled based on additives, etc.

[0047] The purified hydrogel can be ground to a uniform particle size using a tool. At this stage, the average particle size of the hydrogel particles ground may be 500 μm or less, preferably 300 μm or less, and most preferably 250 μm or less, but is not limited thereto. If the average particle size of the hydrogel particles satisfies the above range, the particle size is adjusted to a size suitable for injection, thereby increasing convenience of the procedure, and the physical properties, such as elasticity, of the finally manufactured hyaluronic acid hydrogel may be excellent.

[0048] The step of mixing an additive into the above-mentioned crosslinked intermediate to adjust the hydrogen ion concentration (pH) to 5 to 9 may be further included.

[0049] The above additive may be one or more selected from the group consisting of acidic substances, osmotic pressure regulators, viscosity-regulating additives, and pain-relieving additives.

[0050] The above acidic substance is an aqueous solution comprising a substance that forms a pH of 6.0 or lower when dissolved in water at a concentration of 0.1 mol / L. Specifically, the acidic aqueous solution may be one or more selected from the group consisting of an aqueous solution of phosphoric acid, an aqueous solution of acetic acid, an aqueous solution of hydrochloric acid, an aqueous solution of nitric acid, an aqueous solution of sulfuric acid, an aqueous solution of boric acid, an aqueous solution of diethyl barbituric acid, an aqueous solution of sodium phosphate monobasic or sodium phosphate hydrate, a potassium phosphate monobasic or potassium phosphate hydrate, an ammonium phosphate monobasic or ammonium phosphate hydrate, and a sodium acetate or sodium acetate hydrate, but is not limited thereto. no.

[0051] The above osmotic pressure regulator may be one or more selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2) and magnesium chloride (MgCl2), but is not limited thereto.

[0052] The above pain-relieving additive may be one or more selected from the group consisting of lidocaine or its salt and bupivacaine or its salt, but is not limited thereto.

[0053] After step (d) above, (e) a step of sterilizing the hyaluronic acid hydrogel in an autoclave may be further included. Specifically, the step involves filling the hyaluronic acid hydrogel into a container and then sterilizing it in the autoclave, which is a process of sterilizing it in a sterile state for use as a filler to be injected into the human body.

[0054] The weight ratio of the hyaluronic acid or its salt to the crosslinking agent may be 10:1 to 300:1, preferably 20:1 to 150:1, and most preferably 30:1 to 100:1. For example, the above weight ratios are 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 101:1, 102:1, 103:1, 104:1, 105:1, 106:1, 107:1, 108:1, 109:1, 110:1, 111:1, 112:1, 113:1, 114:1, 115:1, 116:1, 117:1, 118:1, 119:1, 120:1, 121:1, 122:1, 123:1, 124:1, 125:1, 126:1, 127:1, 128:1, 129:1, 130:1, 131:1, 132:1, 133:1, 134:1, 135:1, 136:1, 137:1, 138:1, 139:1, 140:1, 141:1, 142:1, 143:1, 144:1, 145:1, 146:1, 147:1,148:1, 149:1, 150:1, 151:1, 152:1, 153:1, 154:1, 155:1, 156:1, 157:1, 158:1, 159:1, 160:1, 161:1, 162:1, 163:1, 164:1, 165:1, 166:1, 167:1, 168:1, 169:1, 170:1, 171:1, 172:1, 173:1, 174:1, 175:1, 176:1, 177:1, 178:1, 179:1, 180:1, 181:1, 182:1, 183:1, 184:1, 185:1, 186:1, 187:1, 188:1, 189:1, 190:1, 191:1, 192:1, 193:1, 194:1, 195:1, 196:1, 197:1, 198:1, 199:1, 200:1, 201:1, 202:1, 203:1, 204:1, 205:1, 206:1, 207:1, 208:1, 209:1, 210:1, 211:1, 212:1, 213:1, 214:1, 215:1, 216:1, 217:1, 218:1, 219:1, 220:1, 221:1, 222:1, 223:1, 224:1, 225:1, 226:1, 227:1, 228:1, 229:1, 230:1, 231:1, 232:1, 233:1, 234:1, 235:1, 236:1, 237:1, 238:1, 239:1, 240:1, 241:1, 242:1, 243:1, 244:1, 245:1, 246:1, 247:1, 248:1, 249:1, 250:1, 251:1, 252:1, 253:1, 254:1, 255:1, 256:1, 257:1, 258:1, 259:1, 260:1, 261:1, 262:1, 263:1, 264:1, 265:1, 266:1, 267:1, 268:1, 269:1, 270:1, 271:1, 272:1, 273:1, 274:1, 275:1, 276:1, 277:1, 278:1, 279:1, 280:1, 281:1, 282:1, 283:1, 284:1, 285:1, 286:1, 287:1, 288:1, 289:1, 290:1,The ratio may be 291:1, 292:1, 293:1, 294:1, 295:1, 296:1, 297:1, 298:1, 299:1, 300:1, or a ratio between two of these values. If the weight ratio falls outside the above range, the physical properties of the manufactured hyaluronic acid hydrogel, such as elasticity, may deteriorate, or discoloration may occur as the structure is deformed.

[0055] The above sterilization may be carried out at 100 to 150°C for 0.1 to 1 hour, but is not limited thereto.

[0056] The storage modulus (G') of the hyaluronic acid hydrogel produced by the above manufacturing method may be 50 to 700 Pa, preferably 100 to 500 Pa, and most preferably 150 to 400 Pa. For example, the storage modulus (G') of the hyaluronic acid hydrogel produced through the above manufacturing method is 50Pa, 60Pa, 70Pa, 80Pa, 90Pa, 100Pa, 110Pa, 120Pa, 130Pa, 140Pa, 150Pa, 160Pa, 170Pa, 180Pa, 190Pa, 200Pa, 210Pa, 220Pa, 230Pa, 240Pa, 250Pa, 260Pa, 270Pa, 280Pa, 290Pa, 300Pa, 310Pa, 320Pa, 330Pa, 340Pa, 350Pa, 360Pa, 370Pa, 380Pa, 390Pa, 400Pa, 410Pa, 420Pa, 430Pa, 440Pa, 450Pa, 460Pa, 470Pa, 480Pa, 490Pa, 500Pa, 510Pa, 520Pa, 530Pa, 540Pa, 550Pa, 560Pa, 570Pa, 580Pa, 590Pa, 600Pa, 610Pa, 620Pa, 630Pa, 640Pa, 650Pa, 660Pa, 670Pa, 680Pa, 690Pa, 700Pa, or a value between two of these values. If the hyaluronic acid hydrogel satisfies the above elastic range, the degradation rate in the body can be maintained slowly.

[0057] The embodiments of this specification will be described in more detail below. However, the following experimental results represent only representative results among the above embodiments, and the scope and content of this specification should not be interpreted as being narrowed or limited by the embodiments. The respective effects of various embodiments of this specification not explicitly presented below will be described in detail in the relevant sections. Unless otherwise stated, each experiment was performed at room temperature (25°C) and atmospheric pressure (1 bar).

[0058] Examples

[0059] Sodium hyaluronate with a molecular weight of 1,000,000 g / mol is dissolved in a 0.25 M NaOH solution to achieve a HA concentration of 17 wt% in the sodium hydroxide solution. Then, 4.5 mol% of BDDE is mixed relative to the weight of the added sodium hyaluronate, and the mixture is formed into a thin sheet with a thickness of approximately 5 mm. Subsequently, the pre-treated mixture in sheet form is crosslinked at 25°C for 64 hours. Once crosslinking is complete, the HA gel is purified by changing the PBS at regular intervals until it reaches 90% of the target weight. The HA gel that has reached 90% of the target weight is ground using a 150 μm sieve, and a lidocaine-PBS solution is added and mixed so that the lidocaine content becomes 3 mg / mL at the target weight. Finally, fill 1 mL of the manufactured hyaluronic acid hydrogel into pre-filled syringes and sterilize them in an autoclave at 121°C for 15 minutes.

[0060] Comparative example

[0061] We used Restylane Define Lidocaine from Galderma, a commercially available hyaluronic acid hydrogel product (using 1,000,000 to 2,000,000 g / mol of sodium hyaluronate, with a concentration of HA in the sodium hydroxide solution of 20 wt%).

[0062] Experimental Example 1: Evaluation of the degradation rate of hyaluronic acid hydrogel

[0063] The compositions of the above examples and comparative examples were prepared, and 1 g of the sample was placed in an EP tube and centrifuged. Subsequently, to decompose the prepared hyaluronic acid hydrogel, 50 μl of 200 U / mL hyaluronidase solution (enzyme solution) was added to the centrifuged EP tube, and the residual rate of the hyaluronic acid hydrogel was measured at time intervals and recorded, as shown in Figure 1 and Table 1 below. In Table 1 below, the 50% decomposition point refers to the point when the residual rate reached 50%, and the 100% decomposition point refers to the point when the residual rate reached 0%.

[0064] Example 1 Comparative Example 150% decomposition point 65 hr 28 hr 100% decomposition point 192 hr 140 hr

[0065] From FIG. 1 and Table 1, it can be seen that Example 1 takes more than 60 hours to reach 50% degradation and more than 180 hours to reach 100% degradation. In other words, it can be seen that in Example 1, crosslinking occurs at low temperatures and the degree of crosslinking is excellent even when the concentration of the crosslinking agent is maintained at a low level, thereby delaying degradation in the body. On the other hand, Comparative Example 1 degrades relatively quickly, resulting in a short retention time in the body, which may lead to reduced user satisfaction during the procedure.

[0066] Experimental Example 2: Evaluation of the elasticity of hyaluronic acid hydrogel

[0067] Hyaluronic acid hydrogels of the examples and comparative examples were prepared, and the storage modulus (G') of the gel was measured five times using a rheometer, and the standard deviation was recorded.

[0068] As a result of the measurement, the example showed an elasticity value of 304.68 ± 6.17 Pa, and the comparative example showed an elasticity value of 286.03 ± 6 Pa. Through this, it can be seen that it exhibits elasticity similar to or greater than that of conventionally used hyaluronic acid hydrogels, making it suitable for use as a filler, and furthermore, the degradation rate is maintained slowly, which can increase satisfaction with the procedure.

[0069] The foregoing description of this specification is for illustrative purposes only, and those skilled in the art to which one aspect of this specification pertains will understand that other specific forms can be easily modified without altering the technical concept or essential features described in this specification. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0070] The scope of this specification is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of this specification.

Claims

1. (a) A step of mixing a basic aqueous solution, hyaluronic acid or a salt thereof, and a crosslinking agent; (b) a step of forming the mixture of step (a) into a sheet-shaped intermediate; (c) a step of crosslinking the intermediate formed into the sheet shape above; and (d) a step of purifying and grinding the cross-linked intermediate of step (c) above to obtain a hyaluronic acid hydrogel; comprising a method for preparing a hyaluronic acid hydrogel.

2. In Paragraph 1, A method for preparing a hyaluronic acid hydrogel, wherein the above basic aqueous solution comprises one or more selected from the group consisting of NaOH and KOH.

3. In Paragraph 1, A method for preparing a hyaluronic acid hydrogel, wherein the OH- concentration of the above basic aqueous solution is 0.05 to 1 M.

4. In Paragraph 1, A method for preparing a hyaluronic acid hydrogel, wherein the crosslinking agent is one or more selected from the group consisting of BDDE and divinylsulfone.

5. In Paragraph 1, A method for preparing a hyaluronic acid hydrogel, wherein the concentration of the crosslinking agent is 1 to 20 mol%.

6. In Paragraph 1, A method for manufacturing a hyaluronic acid hydrogel, wherein the thickness of the intermediate formed into a sheet shape in step (b) above is 1 to 10 mm.

7. In Paragraph 1, A method for preparing a hyaluronic acid hydrogel, characterized in that the above step (c) is performed at 10 to 30°C for 6 to 340 hours.

8. In Paragraph 1, A method for preparing a hyaluronic acid hydrogel, further comprising, after step (d) above, (e) sterilizing the hyaluronic acid hydrogel in an autoclave.

9. In Paragraph 1, A method for preparing a hyaluronic acid hydrogel, wherein the weight ratio of the hyaluronic acid or its salt to the crosslinking agent is 10:1 to 300:1.

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