Crosslinked recombinant collagen gel as well as preparation method and application thereof

Abstract

The invention discloses cross-linked recombinant collagen gel as well as a preparation method and application thereof. According to the method, recombinant collagen is taken as a raw material, gel is prepared through catalysis of a coupling agent DMTMM, and the cross-linked recombinant collagen gel is obtained through dialysis, homogenization and sterilization processes. The preparation method disclosed by the invention has the advantages of high reaction speed, high condensation efficiency, mild reaction conditions and the like, exogenous toxicity is not introduced, and the prepared cross-linked recombinant collagen gel has excellent viscoelasticity, cohesiveness and degradation resistance, is low in safety risk for injection and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials and relates to a cross-linked recombinant collagen gel and a preparation method and application thereof. Background Art

[0002] Collagen is the main structural protein in the human body and plays an important role in cell attachment, migration, proliferation, differentiation and survival. Collagen accounts for 70% to 80% of the facial dermis. With age, dermal collagen is lost or structurally changed, and the transformation of intact type I collagen to fibrotic type III collagen produces fine lines and wrinkles. One of the advantages of collagen fillers over hyaluronic acid is that they have a lower viscosity and are more effective in correcting fine lines and wrinkles because they are more evenly and naturally filled into the treatment area when injected superficially. Another advantage of collagen fillers is the hemostatic properties of collagen, and the incidence of edema and hematoma after injection is lower than other types of products on the market. Collagen fillers have the advantages of high safety and no virus risks. At the same time, they also have the characteristics of large-scale industrial production, and have broad application prospects in the field of medical beauty.

[0003] As the "new favorite" in the medical beauty industry, recombinant collagen has good biocompatibility, and its degradation products are only polypeptides or amino acid fragments. However, recombinant collagen has low viscosity, and its viscoelasticity, cohesion and degradation resistance are poor and cannot meet the tension that needs to be resisted when implanted in the dermis. Recombinant collagen has active groups such as amino, carboxyl, and hydroxyl groups in its structure. Its mechanical properties can be improved through modified cross-linking to meet the needs of dermal tissue filling. Commonly used cross-linking agents include 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 1,4-butanediol diglycidyl ether (BDDE), etc. Such cross-linking agents need to react under harsh acidic or alkaline conditions and have certain toxicity, which increases the safety risk of the product. Summary of the invention

[0004] The present invention provides a cross-linked recombinant collagen gel and a preparation method and application thereof. The present invention uses recombinant collagen as a raw material, activates the side chain carboxyl groups of the collagen molecules by a "zero-length" coupling agent 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) under a neutral environment, and then undergoes an acylation reaction with the side chain amino groups to form peptide bonds, thereby cross-linking the collagen side chains into a network structure, and obtaining a cross-linked recombinant collagen gel with excellent viscoelasticity, cohesion and anti-degradation properties.

[0005] The technical solution of the present invention is as follows:

[0006] The preparation method of cross-linked recombinant collagen gel specifically comprises the following steps:

[0007] (1) fully dissolving the recombinant collagen in a solvent to obtain a recombinant collagen solution, wherein the solvent is purified water, physiological saline or a phosphate buffer solution with a pH of 7.2 to 7.4;

[0008] (2) DMTMM is added to the recombinant collagen solution, and the reaction is carried out at 0-60° C. for 12-72 hours. The lower the reaction temperature, the longer the required reaction time. After the reaction is carried out until a stable gel is formed, the solution is dialyzed, homogenized and sterilized to obtain a cross-linked recombinant collagen gel. The molar ratio of the recombinant collagen side chain carboxyl group to DMTMM is 0.1:1 to 1:1.

[0009] Further, in step (1), the recombinant collagen is selected from one or more of recombinant type I collagen and recombinant type III collagen, and the molecular weight is 1000 to 1000000 Da. Preferably, the recombinant type III collagen is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No.5021, and the recombinant type I collagen is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No.16461. In a specific embodiment of the present invention, the recombinant type III collagen produced by fermentation of Pichia pastoris with a deposit number of CGMCC No.5021 is taken as an example.

[0010] Furthermore, in step (1), the mass concentration of the recombinant collagen solution is 1% to 20%, preferably 10% to 16%.

[0011] Furthermore, in step (2), the reaction is carried out at room temperature for 24 hours. The room temperature in the present invention is 20-30°C.

[0012] Furthermore, in step (2), the dialysate used in dialysis is selected from one or both of purified water and physiological saline, and the dialysis time is 24 to 48 hours. Preferably, the dialysate is first dialyzed with purified water for 12 to 24 hours, and then dialyzed with physiological saline for 12 to 24 hours.

[0013] Furthermore, in step (2), the sterilization method is irradiation sterilization, moist heat sterilization or dry heat sterilization, preferably moist heat sterilization, and the sterilization condition is 121° C. sterilization for 10 min.

[0014] The present invention provides cross-linked recombinant collagen gel prepared by the above preparation method.

[0015] The present invention also provides the use of the cross-linked recombinant collagen gel as a filling material for injection.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) The present invention uses a coupling agent DMTMM to activate the side chain carboxyl groups of recombinant collagen molecules under a neutral environment, and through an acylation reaction, the side chains of the collagen are cross-linked into a three-dimensional network structure, thereby giving it excellent viscoelasticity, cohesion and anti-degradation properties.

[0018] (2) The present invention uses a "zero-length" coupling agent DMTMM to prepare a cross-linked recombinant collagen gel, which has mild reaction conditions, high grafting efficiency, few by-products and is easy to remove. During the coupling process, the collagen bonding molecules are directly combined with each other, and DMTMM will not be embedded in the final product. After the cross-linking reaction, DMTMM can be completely removed by a simple dialysis step. The safety risk for injection is low and it is suitable for large-scale production. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative purposes to illustrate and explain the present invention, and should not be construed as limiting the scope of protection of the present invention.

[0020] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The recombinant type III collagen described in the present invention is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No.5021, which has been fully disclosed in Chinese patent ZL 201110327865.5; the recombinant type I collagen is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No.16461, which has been fully disclosed in Chinese patent ZL201811589560.X, and both recombinant collagens can be purchased.

[0021] Example 1

[0022] Dissolve recombinant type III collagen in purified water to prepare a recombinant collagen solution with a mass concentration of 10%. Weigh DMTMM according to the molar ratio of the side chain carboxyl of the recombinant collagen to DMTMM of 1:1, and add it to the recombinant collagen solution, stir evenly, react at 50℃~60℃ for 24h, dialyze the obtained gel with physiological saline for 24h, then homogenize the gel block, and sterilize it with wet heat to obtain the cross-linked recombinant collagen gel.

[0023] Example 2

[0024] Recombinant type III collagen was dissolved in phosphate buffer solution to prepare a recombinant collagen solution with a mass concentration of 14%. DMTMM was weighed according to the molar ratio of the side chain carboxyl of the recombinant collagen to DMTMM of 1:1, and added to the recombinant collagen solution, stirred evenly, and reacted at room temperature for 24 hours. The obtained gel was dialyzed with purified water for 24 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0025] Example 3

[0026] Recombinant type III collagen was dissolved in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl of the recombinant collagen to DMTMM of 1:0.5, and added to the recombinant collagen solution, stirred evenly, reacted at 50°C to 60°C for 24 hours, and the obtained gel was dialyzed with physiological saline for 24 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0027] Example 4

[0028] Recombinant type III collagen was dissolved in physiological saline to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl of the recombinant collagen to DMTMM of 1:0.1, and added to the recombinant collagen solution, stirred evenly, and reacted at room temperature for 48 hours. The obtained gel was dialyzed with purified water for 24 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0029] Example 5

[0030] Recombinant type III collagen was dissolved in physiological saline to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl group of the recombinant collagen to DMTMM of 1:1, and added to the recombinant collagen solution, stirred evenly, and reacted at 0-8°C for 72 hours. The obtained gel was dialyzed with purified water for 12 hours and physiological saline for 12 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0031] Example 6

[0032] Recombinant type III collagen was dissolved in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl group of the recombinant collagen to DMTMM of 1:1, and added to the recombinant collagen solution, stirred evenly, and reacted at room temperature for 24 hours. The obtained gel was dialyzed with purified water for 24 hours and physiological saline for 24 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0033] Comparative Example 1

[0034] Recombinant type III collagen was dissolved in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl group of the recombinant collagen to DMTMM of 1:10, and added to the recombinant collagen solution and stirred evenly, reacted at room temperature for 24 hours, and the obtained gel was dialyzed with purified water for 24 hours and physiological saline for 24 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0035] Comparative Example 2

[0036] Recombinant type III collagen was dissolved in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl of the recombinant collagen to DMTMM of 1:0.01, and added to the recombinant collagen solution and stirred evenly. The reaction was carried out at room temperature for 24 hours. The obtained gel was in a weakly cross-linked state. It was dialyzed with purified water for 24 hours and physiological saline for 24 hours in turn. The dialyzed gel was liquefied after wet heat sterilization.

[0037] Comparative Example 3

[0038] Dissolve recombinant type III collagen in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. Weigh DMTMM according to the molar ratio of the side chain carboxyl of the recombinant collagen to DMTMM of 1:1, and add it to the recombinant collagen solution, stir evenly, and react at 0-8°C for 24 hours. The obtained gel is in a weakly cross-linked state, and dialyzed with purified water for 24 hours and physiological saline for 24 hours in turn. The dialyzed gel is liquefied after wet heat sterilization.

[0039] Comparative Example 4

[0040] Recombinant type III collagen was dissolved in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl group of the recombinant collagen to DMTMM of 1:1, and added to the recombinant collagen solution, stirred evenly, and reacted at room temperature for 12 hours. The obtained gel was dialyzed with purified water for 24 hours and physiological saline for 24 hours in turn. The dialyzed gel blocks were homogenized and liquefied after wet heat sterilization.

[0041] Comparative Example 5

[0042] Recombinant type III collagen was dissolved in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl group of the recombinant collagen to DMTMM of 1:1, and added to the recombinant collagen solution, stirred evenly, and reacted at 50-60°C for 10 hours. The obtained gel was dialyzed with purified water for 24 hours and physiological saline for 24 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0043] Comparative Example 6

[0044] Recombinant type III collagen was dissolved in purified water to prepare a recombinant collagen solution with a mass concentration of 16%. DMTMM was weighed according to the molar ratio of the side chain carboxyl group of the recombinant collagen to DMTMM of 1:1, and added to the recombinant collagen solution, stirred evenly, and reacted at room temperature for 24 hours. The obtained gel was dialyzed with physiological saline for 10 hours, and then the gel block was homogenized and sterilized with wet heat to obtain a cross-linked recombinant collagen gel.

[0045] Performance Test 1

[0046] 1. Detection equipment: Agilent high performance liquid chromatograph, model 1260, equipped with UV detector. The chromatographic column is Xtimate C18 reverse phase column, with specifications of 4.6mm×250mm×5μm. Mobile phase: The mobile phase uses phosphate (0.005mol / L disodium hydrogen phosphate dodecahydrate, 0.02mol / L potassium dihydrogen phosphate)-methanol solution, and the molar ratio of phosphate to methanol is 85:15. Detection conditions: column temperature 30℃, detection wavelength 236nm, injection volume 50μL. Reference substance: DMTMM reference substance solution with a concentration of 0.4μg / ml.

[0047] 2. Detection method: Weigh 0.5 mg of each gel sample of Examples 1-6 and Comparative Examples 1, 5, and 6, place in a 10 mL volumetric flask, add a 2.5 g / L chlorocetylpyridinium (CPC) aqueous solution to dilute to the scale, soak at room temperature for 2 hours, and then centrifuge at a centrifuge temperature of 4°C, a centrifugal speed of 10000 rpm, and a centrifugal time of 30 min. Take the supernatant and filter to obtain the test solution for later use. Use the above-mentioned HPLC instrument and conditions to analyze the reference solution and the test solution to obtain an HPLC chromatogram. Under the given conditions, the retention time of the reference DMTMM is about 4.8 min. The formula for calculating the residual amount of DMTMM in the test sample by the external standard method is as follows:

[0048] C=(Cr*Ar / Ax)*n,

[0049] Wherein: Cr is the concentration of the reference substance, μg / ml; Ar is the peak area of ​​the reference substance; Ax is the peak area of ​​the test solution; n is the dilution factor, n=20; C is the residual amount of DMTMM in the cross-linked recombinant collagen gel, μg / ml.

[0050] The results are shown in Table 1. The quantitative limit of this method is 0.03 μg / ml. It can be seen from Table 1 that the residual amount of DMTMM is related to the feed ratio of recombinant collagen and DMTMM and the dialysis time. The more DMTMM is fed and the shorter the dialysis time, the higher the residual amount of DMTMM. In addition, a reaction time that is too short will cause an inadequate reaction, resulting in a high residual amount of DMTMM, which can easily cause safety risks during injection.

[0051] Table 1 DMTMM residue test results

[0052]

[0053]

[0054] Performance Test 2

[0055] According to the state of the gel after cross-linking and its tolerance to moist heat sterilization, it can be seen that the amount of coupling agent added, reaction temperature, and reaction time are the main factors affecting the cross-linking strength of recombinant collagen gel. Swelling degree is the main performance indicator to characterize the degree of cross-linking of recombinant collagen gel. It changes the swelling degree of the polymer by affecting the diffusion and adsorption behavior of solvent molecules in the polymer network. According to the determination method of swelling degree of cross-linked sodium hyaluronate gel in Appendix B of YY / T 0962-2021 for plastic surgery, the test was carried out. Take a 500-mesh sieve (8cm×8cm square, folded into a 4cm×4cm×2cm square groove) and put it in a drying oven at 80°C to constant weight, recorded as m0. Weigh 0.2g to 0.5g of the gel samples of Examples 1-6 and Comparative Examples 1, 5, and 6 respectively and place them on a 500-mesh sieve. Place the sieve in an evaporating dish and add 30mL of 0.9% sodium chloride solution at one time to completely soak the sample. After the sample is fully swollen (at least 30 minutes), take out the sieve together with the sample, and use filter paper to absorb the liquid at the bottom and around the sieve until there is no wet mark on the filter paper. The weight is recorded as m1. Then put the sieve in a drying oven at 80°C until constant weight is recorded as m2. According to the formula: Where: Q is the swelling degree of the test sample; m1 is the mass of the sieve plus the test sample, in grams (g); m2 is the mass of the sieve plus the test sample after drying, in grams (g); m0 is the mass of the sieve after constant weight, in grams (g). The swelling degree Q is calculated, and the results are shown in Table 2.

[0056] Table 2 Swelling test results

[0057] Test samples Swelling degree Example 1 32.40 Example 2 15.93 Example 3 23.52 Example 4 46.87 Example 5 19.03 Example 6 16.19 Comparative Example 1 9.24 Comparative Example 5 40.01 Comparative Example 6 18.60

[0058] The swelling test results in Table 2 show that the cross-linked recombinant collagen gel prepared under conditions such as reducing the coupling agent concentration, lowering the reaction temperature, and shortening the reaction time has a lower degree of cross-linking, a higher swelling degree, and a lower tolerance to moist heat sterilization.

[0059] Performance Test 3

[0060] Osmotic pressure plays an extremely important role in various biological processes involving the diffusion of solutes or the transport of liquids through biological membranes, and in maintaining the steady-state balance of the cell's physiological environment. The osmotic pressure molar concentration of normal human blood is 285-310 mOsmol / kg. When the osmotic pressure of the extracellular fluid is too high (hypertonic stress) or too low (hypotonic stress), the cells will lose water or swell with water, thereby affecting the normal physiological function of the cells. Take the gel samples of Examples 1-6 and Comparative Examples 1, 5, and 6 and test them according to the osmotic pressure molar concentration determination method of 0632 of the Pharmacopoeia of the People's Republic of China (Part IV) (2020 Edition). The test results are shown in Table 3.

[0061] Table 3 Osmotic pressure test results

[0062] Test samples Osmol value (mOsmol / Kg) Example 1 309 Example 2 16 Example 3 311 Example 4 10 Example 5 298 Example 6 302 Comparative Example 1 301 Comparative Example 5 302 Comparative Example 6 298

[0063] Performance Test 4

[0064] The cross-linked recombinant collagen gel prepared in Example 6 was extracted with culture medium at a ratio of 0.1 g gel: 1.2 mL culture medium, and the extract was used for later use. L929 cells in the logarithmic growth phase were taken and digested with trypsin, and the cell suspension was centrifuged (1000 rpm, 5 min) and the original culture was discarded, and then resuspended in culture medium to obtain 1×10 5 / mL cell suspension. The suspended cells were inoculated into a 96-well plate at 100ul per well and cultured in a cell culture incubator (37°C, 5% CO2) for 24 hours. The cell morphology was observed under a microscope. After the cells grew to form a monolayer, the original culture medium in the 96-well plate was discarded. 100μl of the test sample extract was added to the corresponding wells of the 96-well plate, and a blank control sample and a positive control sample (1% ZDEC) were set up. The 96-well plate was placed in a cell culture incubator (37°C, 5% CO2) and cultured for 24 hours. There were 5 replicate wells in each group, and the average cell survival rate was calculated. After 24 hours of culture, the 96-well plate was taken out, the cell morphology was observed under a microscope, and then the liquid was removed, 50μL MTT (1mg / mL) was added to each well, and the cells were cultured in a CO2 incubator. After 2 hours, the supernatant was removed, 100CO2 DMSO was added to each well to dissolve the crystals, and the absorbance value at a wavelength of 570nm was measured on an enzyme reader to calculate its cytotoxicity. The formula for calculating the survival rate is as follows: Where: OD 570eOD is the average optical density of 100% dilution of the test sample; 570b is the average optical density of the blank.

[0065] Table 4 Cell activity

[0066]

[0067] The results in Table 4 show that the cross-linked recombinant collagen gel prepared in Example 6 has no cytotoxicity and has excellent biocompatibility.

Claims

1. A method for preparing a cross-linked recombinant collagen gel, characterized in that: The specific steps include: (1) fully dissolving the recombinant collagen in a solvent to obtain a recombinant collagen solution, wherein the solvent is purified water, physiological saline or a phosphate buffer solution with a pH of 7.2 to 7.4; (2) DMTMM is added to the recombinant collagen solution, and the reaction is carried out at 0-60° C. for 12-72 hours. The lower the reaction temperature, the longer the required reaction time. After the reaction is carried out until a stable gel is formed, the solution is dialyzed, homogenized and sterilized to obtain a cross-linked recombinant collagen gel. The molar ratio of the side chain carboxyl group of the recombinant collagen to DMTMM is 0.1:1-1:

1.

2. The preparation method according to claim 1, characterized in that: In step (1), the recombinant collagen is selected from one or more of recombinant type I collagen and recombinant type III collagen, and has a molecular weight of 1000-1000000 Da.

3. The preparation method according to claim 2, characterized in that The recombinant type III collagen is produced by fermenting Pichia pastoris with a deposit number of CGMCC No.5021, and the recombinant type I collagen is produced by fermenting Pichia pastoris with a deposit number of CGMCC No.16461.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass concentration of the recombinant collagen solution is 1% to 20%.

5. The preparation method according to claim 1, characterized in that: In step (1), the mass concentration of the recombinant collagen solution is 10% to 16%.

6. The preparation method according to claim 1, characterized in that: In step (2), the reaction is carried out at room temperature for 24 hours.

7. The preparation method according to claim 1, characterized in that: In step (2), the dialysate used in dialysis is selected from one or both of purified water and physiological saline, and the dialysis time is 24 to 48 hours; the sterilization method is irradiation sterilization, moist heat sterilization or dry heat sterilization.

8. The preparation method according to claim 7, characterized in that: In step (2), the dialyzate is first dialyzed with purified water for 12 to 24 hours, and then dialyzed with normal saline for 12 to 24 hours; the sterilization condition for moist heat sterilization is 121° C. for 10 minutes.

9. The cross-linked recombinant collagen gel obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the cross-linked recombinant collagen gel according to claim 9 as a filling material for injection.

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