Crosslinked poly-gamma-glutamic acid hydrogel having excellent elasticity, composition for preparing same, and method for preparing same

Abstract

The present invention relates to a crosslinked poly-gamma-glutamic acid hydrogel having excellent elasticity, a composition for preparing same, and a method for preparing same. More specifically, the present invention relates to: a poly-gamma-glutamic acid hydrogel composition containing 1-10 wt% of a polyglutamic acid salt and 1-10 wt% of polyethylene glycol on the basis of the total weight of the composition, with the remainder comprising water; a method for preparing a crosslinked poly-gamma-glutamic acid hydrogel, the method comprising a step for irradiating the poly-gamma-glutamic acid hydrogel composition with an electron beam; and a crosslinked poly-gamma-glutamic acid hydrogel, which is a crosslinked product obtained by electron beam irradiation of the poly-gamma-glutamic acid hydrogel composition.

Description

Cross-linked polygamma-glutamic acid hydrogel with excellent elasticity, composition for manufacturing the same, and method for manufacturing the same

[0001] The present invention relates to a cross-linked polygamma-glutamic acid hydrogel with excellent elasticity, a composition for manufacturing the same, and a method for manufacturing the same. More specifically, the invention relates to a polygamma-glutamic acid hydrogel that is easy to inject and has excellent viscoelasticity, thereby improving persistence in vivo, a polygamma-glutamic acid hydrogel composition capable of manufacturing the same, and a method for manufacturing a cross-linked polygamma-glutamic acid hydrogel.

[0002] Hyaluronic acid fillers, which are currently the most widely used fillers, account for more than 90% of the global filler market. However, they have a very low duration with a half-life of only 1 to 3 days and are rapidly reabsorbed into the body. Consequently, products that extend the reabsorption period by using cross-linking agents with hyaluronic acid are being sold. However, problems regarding the toxicity of the cross-linking agents are also being reported with these cross-linked products.

[0003] In order to use hyaluronic acid as a filler, it must be manufactured in a form that enhances durability and possesses viscoelasticity similar to skin through chemical crosslinking methods. However, since the material used for such chemical crosslinking, such as BDDE (1,4-butanediol diglycidyl ether), is a carcinogen and toxic, residues must be removed through a dialysis process after the chemical reaction. Generally, this dialysis process takes a long time, typically around one week, which leads to problems such as increased process costs, product disposal due to microbial contamination, and product disposal due to the detection of residues.

[0004] Many tissue repair products using biodegradable polymers have also been developed. These were developed and used as filler formulations using existing biocompatible polymers, in which water-insoluble polymers were processed into micro-sized particles and dispersed through a viscous media. Formulations were used in which 20 to 50 µm polylactic acid (PLA) particles were dispersed in an aqueous solution of carboxymethylcellulose (CMC) or in which 20 to 50 µm polycaprolactone (PCL) particles were dispersed in an aqueous solution of CMC and glycerin. However, these formulations had the problem of causing inconvenience during the procedure, such as micro-particles clogging the needle during injection, and failing to produce a uniform tissue repair effect because the particles were not uniformly dispersed.

[0005] Accordingly, Korean Patent Application No. 10-2017-0139423 discloses a tissue repair product that secures functionality, physical properties, and safety suitable for tissue repair biomaterials by performing crosslinking by irradiating with radiation instead of chemical crosslinking, thereby eliminating concerns about toxicity caused by chemical crosslinking processes.

[0006] However, compared to conventional technologies that perform crosslinking by radiation irradiation without using chemical crosslinking agents, if a crosslinked polygamma-glutamic acid hydrogel with improved viscoelasticity and excellent injection power is provided, it is expected to be widely applied in related fields.

[0007] Accordingly, one aspect of the present invention is to provide a composition for manufacturing a cross-linked polygamma-glutamic acid hydrogel with excellent elasticity.

[0008] Another aspect of the present invention is to provide a method for manufacturing a cross-linked polygamma-glutamic acid hydrogel with excellent elasticity according to the present invention.

[0009] Another aspect of the present invention is to provide a cross-linked polygamma-glutamic acid hydrogel obtained by the composition for preparing a cross-linked polygamma-glutamic acid hydrogel of the present invention.

[0010] According to one aspect of the present invention, a polygamma-glutamic acid hydrogel composition is provided, comprising 1 to 10 weight% of polyglutamate, 1 to 10 weight% of polyethylene glycol, and the remainder being a solvent, based on the total weight of the composition.

[0011] According to another aspect of the present invention, a method for preparing a cross-linked polygamma-glutamic acid hydrogel is provided, comprising the step of obtaining a cross-linked body by irradiating an electron beam onto the polygamma-glutamic acid hydrogel composition of the present invention.

[0012] According to another aspect of the present invention, a cross-linked polygamma-glutamic acid hydrogel is provided, which is a cross-linked chain formed by electron beam irradiation of the polygamma-glutamic acid hydrogel composition of the present invention.

[0013] According to the present invention, a cross-linked polygamma-glutamic acid hydrogel having excellent viscoelasticity can be obtained without the need for the use of toxic substances for chemical cross-linking, and furthermore, the cross-linked polygamma-glutamic acid hydrogel of the present invention has excellent injectability, thereby obtaining a tissue repair material that secures functionality, physical properties, and safety that is more suitable for biomaterials for tissue repair.

[0014] Figure 1 shows the results of measuring the viscoelasticity of cross-linked polygamma-glutamic acid hydrogels prepared in the examples and comparative examples of the present invention.

[0015] Figure 2 shows the results of measuring the storage modulus (G') of cross-linked polygamma-glutamic acid hydrogels prepared in the examples and comparative examples of the present invention before and after sterilization.

[0016] Figure 3 shows the results of measuring the injection power of cross-linked polygamma-glutamic acid hydrogels prepared in the examples and comparative examples of the present invention.

[0017] Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0018] According to the present invention, a composition and a method for manufacturing a cross-linked polygamma-glutamic acid hydrogel are provided, which has excellent elasticity and simultaneously has low injectability, making injection procedures easy.

[0019] The polygamma-glutamic acid hydrogel composition of the present invention comprises a mixture of polyglutamate and polyethylene glycol, more specifically, based on the total weight of the composition, comprising 1 to 10 weight% of polyglutamate, 1 to 10 weight% of polyethylene glycol, and the remainder of a solvent, for example, comprising 2 to 8 weight% of polyglutamate, 2 to 8 weight% of polyethylene glycol, and the remainder of a solvent.

[0020] As such, the present invention, by including polyglutamate and polyethylene glycol together, improves elasticity during crosslinking through electron beam irradiation and additionally reduces injection force.

[0021] Meanwhile, the polyglutamate that can be used in the present invention preferably has a molecular weight of 10 kDa to 5000 kDa, and for example, may be 20 to 1500 kDa. If the molecular weight is less than 10 kDa, there is a problem that it is difficult to produce from microorganisms, and if it exceeds 5000 kDa, there is also a problem that commercial production is difficult, but it is not limited thereto, and it is preferable to have a molecular weight of 500 kDa or more in terms of securing excellent elasticity of the crosslinked body.

[0022] The above polyglutamate is preferably in the form of at least one salt selected from the group consisting of sodium, potassium, calcium, ammonium, and zinc salts, and, for example, sodium polyglutamate salt may be used.

[0023] Furthermore, the composition of the present invention may include other additives in an amount of 0.1 to 1 weight%, preferably 0.1 to 0.5 weight%, and more preferably 0.2 to 0.4 weight% based on the total weight of the composition, and the other additives are not limited to any component that can provide a beneficial effect to the body when the present invention is applied for tissue repair, and may be, for example, at least one selected from the group consisting of moisturizers, antioxidants, whitening agents, collagen synthesizers, anti-wrinkle agents, anti-aging agents, and UV blockers.

[0024] If the above other additive is less than 0.1 weight%, the intended effect of the other additive tends to be negligible, and if it exceeds 1 weight%, there is a problem that crosslinking does not proceed smoothly during subsequent radiation irradiation.

[0025] More specifically, the above other additives may be at least one selected from the group consisting of hyaluronic acid, laminarin, polysaccharides, saponins, collagen, polyols, amino acids, sugars, oils, natural extracts, and fermented products, but are not limited thereto.

[0026] The above solvent may be at least one selected from the group consisting of water and saline solution, preferably saline solution, for example, a PBS buffer solution with a pH of 7 to 8, preferably 7.4. Meanwhile, the "buffer solution" below may be, for example, saline solution, for example, a PBS buffer solution with a pH of 7 to 8.

[0027] Meanwhile, according to another aspect of the present invention, a method for manufacturing a cross-linked polygamma-glutamic acid hydrogel is provided, comprising the step of obtaining a cross-linked body by irradiating an electron beam onto a polygamma-glutamic acid hydrogel composition of the present invention as described above.

[0028] At this time, the electron beam is irradiated with a total dose of 30 kGy or more and less than 300 kGy, preferably with a dose of 30 to 250 kGy, and more preferably with a dose of 100 to 200 kGy.

[0029] When the above dose is less than 30 kGy, the amount of free radicals generated is insufficient, so the cross-linking in polyglutamic acid is insufficient, and the elasticity of the cross-linked material tends to decrease. When it exceeds 200 kGy, the cross-linking is excessively formed, resulting in the formation of a hard cross-linked material with very high G' and G" values, which is unsuitable for use as a tissue repair material, and there is a tendency for the gel to become rigid and fractured due to dehydration.

[0030] In this case, the above G' modulus (storage modulus) represents elasticity as the storage modulus, and the G" modulus (loss modulus) represents viscosity as the loss modulus; both use the unit Pa.

[0031] Furthermore, according to another aspect of the present invention, a tissue repair material is provided comprising a polyglutamate crosslinker obtained by irradiating the tissue repair composition of the present invention with radiation as described above.

[0032] The cross-linked polygamma-glutamic acid hydrogel of the present invention may be used after hydration, and the process may further include a step of hydrating the cross-linker under buffer solution conditions so that the hydration rate reaches 30% to 40%. In this case, the hydration process may be performed for 1 to 3 days under conditions in which an ionization solution or a buffer solution is circulated at 50 rpm to 100 rpm, and the process may be carried out with or without the use of a dialysis membrane. For example, dialysis may be performed for 1 to 3 days in an NaCl solution and / or a PBS solution, and may be performed two or more times; preferably, it may be performed within a total of 3 days, including a first dialysis step in an NaCl solution and a second dialysis step in a PBS solution.

[0033] Furthermore, the cross-linked polygamma-glutamic acid hydrogel of the present invention can be sterilized at a high temperature, for example, at a temperature of 100 to 150°C or 120 to 125°C for 3 to 30 minutes, and then cooled to room temperature, for example, 20 to 30°C. For example, the final cross-linked polygamma-glutamic acid hydrogel can be prepared by leaving it at room temperature for 24 hours or more.

[0034] The cross-linked polygamma-glutamic acid hydrogel of the present invention is a cross-linked polygamma-glutamic acid hydrogel that is a cross-linked chain formed by electron beam irradiation of the polygamma-glutamic acid hydrogel composition of the present invention described above, wherein the storage modulus (G') of the cross-linked polygamma-glutamic acid hydrogel of the present invention is 500 to 10,000 Pa, 700 to 8,000 Pa, for example, 4,000 to 6,000 Pa.

[0035] The cross-linked polygamma-glutamic acid hydrogel of the present invention has excellent elasticity and low injection force, for example, the injection force of a mixture of a mixture in which the crosslinker of the cross-linked polygamma-glutamic acid hydrogel is mixed with a buffer solution such that the content is 20 to 30 mg / ml, for example 24 mg / ml, is 5 to 15 (N).

[0036] The above-mentioned pulverized material may be obtained by placing the above-mentioned mixture into a grinder and performing the process at 200 to 2,000 rpm for 10 minutes to 3 hours, wherein a grinder equipped with blades may be used and homogenization may be performed along with the grinding.

[0037] The cross-linked polygamma-glutamic acid hydrogel of the present invention may be used for tissue repair purposes, such as cosmetic fillers, cosmetic implants, or a combination thereof, but is not limited thereto, and is not particularly limited as long as it can be applied as a biomaterial.

[0038] According to the present invention, a cross-linked polygamma-glutamic acid hydrogel can be obtained that has excellent stability and elasticity without requiring chemical crosslinking, has sufficiently low injection force, and also has excellent thermal stability.

[0039] The present invention will be explained in more detail below through specific embodiments. The following embodiments are merely examples to aid in understanding the present invention and do not limit the scope of the present invention.

[0040] Examples

[0041] 1. Preparation of a polygamma-glutamic acid hydrogel composition

[0042] 8 wt% of polygamma-glutamate sodium salt powder with a molecular weight of 1,000 kDa (Vedan Co., Taiwan) and 4 wt% each of PEG powders with molecular weights of 20 kDa and 35 kD were placed in a beaker, PBS (phosphate buffered saline, pH 7.4) was added to make a total of 100 wt%, and the mixture was dissolved by stirring at 200 rpm for about 1 hour in a stirrer to prepare an aqueous solution of polygamma-glutamate sodium salt.

[0043] The case using 20 kDa PEG was designated as Example 1, the case using 35 kD PEG was designated as Example 2, and the case not using PEG was designated as Comparative Example 1, and the preparations were made as shown in Table 1 below.

[0044] [Table 1]

[0045]

[0046] 2. Preparation of cross-linked polygamma-glutamic acid hydrogel by electron beam irradiation

[0047] Polygamma-glutamic acid crosslinkers were obtained by irradiating the compositions of the comparative example and the example obtained in 1. above with electron beams of 30, 100, 150, and 200 kGy, respectively. At this time, electron beam irradiation was performed using a 10 Mev LINAC-electron accelerator (Model ELV-4, 1 Mev, Korea Atomic Energy Research Institute, Jeong-Eup, Korea). At this time, the beam current intensity was 1 mA and the electron beam energy was 10 Mev. After electron beam irradiation, the total absorbed dose was confirmed using an alanine dosimeter (Ceric cerous dosimeter, Bruker Instruments, Germany), and the error in the total absorbed dose was within 5%.

[0048] The cross-linked polygamma-glutamic acid, after the cross-linking reaction was completed, was placed in a dialysis membrane and sufficiently saturated with water for 24 to 72 hours under conditions of NaCl solution and PBS (pH 7.4) buffer solution so that the hydration rate reached 30% to 40%.

[0049] Hydrated cross-linked polygamma-glutamic acid was mixed in PBS buffer solution (pH 7.4) to a concentration of 24 mg / ml. The mixture prepared in this way was homogenized by grinding it in a blade mixer, and the storage modulus (G') was measured using a rheometer. The results are shown in Table 2 and Figure 1 below. At this time, the storage modulus (G') was measured by performing a dynamic time sweep test using a rheometer (MCR92 rheometer, Anton Paar).

[0050] At this time, the content of polygamma-glutamic acid can be obtained by measuring the content of decomposed glutamic acid (mg / ml) after acid hydrolysis, and then multiplying the purity of the raw material polygamma-glutamic acid by 70% (0.7) and a correction value of 0.88. The correction value can be obtained through the formula (molecular weight of glutamic acid residue in polygamma-glutamic acid) / (molecular weight of glutamic acid).

[0051] [Table 2]

[0052]

[0053] * Unit: Pa

[0054] As a result, as can be seen in Table 2 above, it can be confirmed that the cross-linked polygamma-glutamic acid hydrogels of Examples 1 and 2 of the present invention have excellent elasticity.

[0055] 3. Confirmation of Physical Properties of Cross-linked Polygamma-Glutamic Acid Hydrogel

[0056] (1) Thermal stability

[0057] To confirm thermal stability, the syringe filled with the homogenized sample obtained in 2 above was heated at 125°C for 8 minutes to sterilize and then cooled. After sterilization, the syringe was left at room temperature to complete the preparation of the final cross-linked polygamma-glutamic acid hydrogel. A dynamic time sweep test was performed using a rheometer (MCR92 rheometer, Anton Paar) to confirm the physical properties of G' (storage modulus), and the results are shown in Table 3 and Figure 2 below. In Table 3 below, the rate of change of G' was calculated according to the following equation (1).

[0058] Rate of change of G' = (G' before sterilization - G' after sterilization) / G' before sterilization... Equation (1)

[0059] [Table 3]

[0060]

[0061] * unit: %

[0062] Figure 2(a) shows the results related to the sample of Comparative Example 1, where it can be seen that the storage modulus (G') itself is not high when a dose of less than 200 kGy is applied, Figure 2(b) shows the results related to the sample of Example 1, and Figure 2(c) shows the results related to the sample of Example 2, where it can be confirmed that the storage modulus (G') improves with increasing molecular weight of PEG added to PGA, and the rate of change in the range of 30 to 150 kGy is significantly reduced compared to Comparative Example 1.

[0063] (2) Injection power

[0064] To determine the ease of injection when injecting each sample prepared in the above examples and comparative examples, the injection force was compared using an extrusion force meter (Multi test 2.5-dV, Mecmesin). After inserting a pre-filled syringe containing the sample of the example or comparative example to be measured into a jig, the syringe plunger was adjusted to be centered on the fixing plate, and the injection force was measured at a speed of 12 mm / min and is shown in Table 4 and Figure 3.

[0065] [Table 4]

[0066]

[0067] * Unit: N

[0068] As can be seen in Table 4 above, it was confirmed that the cross-linked polygamma-glutamic acid hydrogels of Examples 1 and 2 had high viscoelasticity while having low injectability. In other words, the cross-linked polygamma-glutamic acid hydrogel obtained by the present invention indicates that it is possible to perform the procedure more easily by maintaining elasticity while lowering injectability.

[0069] Although embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and it will be obvious to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention as described in the claims.

Claims

1. A polygamma-glutamic acid hydrogel composition comprising 1 to 10 weight% of polyglutamate, 1 to 10 weight% of polyethylene glycol, and the remainder of a solvent, based on the total weight of the composition.

2. The polygamma-glutamic acid hydrogel composition according to claim 1, wherein the polyglutamate has a molecular weight of 10 kDa to 5000 kDa.

3. A polygamma-glutamic acid hydrogel composition according to claim 1, wherein the polyglutamate is in the form of at least one salt selected from the group consisting of sodium, potassium, calcium, ammonium, and zinc.

4. A polygamma-glutamic acid hydrogel composition according to claim 1, wherein the solvent is at least one selected from the group consisting of water and saline solution.

5. A method for preparing a cross-linked polygamma-glutamic acid hydrogel, comprising the step of obtaining a cross-linked body by irradiating an electron beam onto a polygamma-glutamic acid hydrogel composition according to any one of claims 1 to 4.

6. A method for manufacturing a cross-linked polygamma-glutamic acid hydrogel according to claim 5, wherein the electron beam is irradiated with a total dose of 30 kGy or more and less than 300 kGy.

7. A method for preparing a cross-linked polygamma-glutamic acid hydrogel according to claim 5, further comprising the step of hydrating the cross-linker under buffer solution conditions such that the hydration rate reaches 30% to 40%.

8. A cross-linked polygamma-glutamic acid hydrogel, which is a cross-linked chain formed by electron beam irradiation of a polygamma-glutamic acid hydrogel composition according to any one of claims 1 to 4.

9. A cross-linked polygamma-glutamic acid hydrogel according to claim 8, wherein the storage modulus (G') of the cross-linked polygamma-glutamic acid hydrogel is 500 to 10,000.

10. A cross-linked polygamma-glutamic acid hydrogel according to claim 8, wherein the injection force of the pulverized mixture of the crosslinker of the cross-linked polygamma-glutamic acid hydrogel mixed with a buffer solution such that the crosslinker content is 20 to 30 mg / ml is 1 to 20 (N).

11. A method for manufacturing a cross-linked polygamma-glutamic acid hydrogel according to claim 10, wherein the cross-linked polygamma-glutamic acid hydrogel is for tissue repair use, such as for use as a cosmetic filler, for use as a molding implant, or for use in combination thereof.

Images