A highly stable biogel matrix and preparation method thereof

Abstract

The present invention belongs to the field of medical materials, and in particular to a highly stable bio-glue matrix and a preparation method thereof. The bio-glue matrix includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 42-91wt% in the bio-glue matrix. The present invention obtains a highly stable bio-glue matrix with a suitable gel time, good biodegradability, biocompatibility, and biosafety, and has a low swelling rate. At the same time, it has a gain effect on tissue repair, and can solve the problems of existing medical adhesives being toxic, risky, and high swelling.

Description

Technical Field

[0001] The invention belongs to the field of medical materials, and in particular relates to a highly stable bio-glue matrix and a preparation method thereof. Background Art

[0002] Bioadhesives are one of the hottest materials to replace sutures and fixation needles. Such adhesives usually have certain hemostatic and healing functions. Currently, there are three types of commercialized bioadhesives: acrylates, fibrins, and hydrogels. Among them, biohydrogel adhesives have attracted widespread attention due to their excellent biocompatibility.

[0003] Patent No. CN114712550B provides an injectable rapid hemostatic hydrogel adhesive and its preparation method and application. It uses polypropylene and chitosan to react with sodium alginate and calcium chloride. The obtained product can effectively stop bleeding at a defect with a diameter of 2 mm within 10 seconds. Patent No. CN110484184B discloses a hydrogel adhesive and its preparation method and application. It uses polyethylene glycol derivatives and endogenous organic dopamine to react to improve the adhesive's adhesion in a water environment. However, the bonding stability in the above technical solution still does not meet expectations, and the above adhesive cannot achieve 100% degradation. Summary of the invention

[0004] In order to solve the above problems, the first aspect of the present invention provides a highly stable bio-gel matrix, wherein the bio-gel matrix includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 42-91 wt % in the bio-gel matrix.

[0005] Preferably, in the biogel matrix, the cross-linked acellular matrix extract accounts for 49-82 wt %.

[0006] Preferably, in the biogel matrix, the cross-linked acellular matrix extract accounts for 53-77 wt %.

[0007] Preferably, in the biogel matrix, the cross-linked acellular matrix extract accounts for 55-77 wt %.

[0008] Preferably, in the biogel matrix, the cross-linked acellular matrix extract accounts for 58-75 wt %.

[0009] The second aspect of the present invention provides a method for preparing a highly stable bio-gel matrix, the preparation method is as follows: S1. Preparation of a protein hydrolysate solution; S2. Preparation of an amidated activated product solution; S3. Blending the protein hydrolysate solution and the amidated activated product solution.

[0010] As a preferred embodiment, step S1 specifically comprises dispersing the decellularized matrix extract in a phosphate buffer solution to obtain a protein hydrolysate solution, so that the concentration of the decellularized matrix extract in the protein hydrolysate solution is 0.09-0.25 g / mL.

[0011] Preferably, step S1 specifically comprises dispersing the decellularized matrix extract in a phosphate buffer solution to obtain a protein hydrolysate solution, so that the concentration of the decellularized matrix extract in the protein hydrolysate solution is 0.09-0.19 g / mL.

[0012] As a preferred embodiment, the molecular weight of the decellularized matrix extract is ≥10 kDa.

[0013] As a preferred embodiment, the decellularized matrix extract contains peptide bonds, and the decellularized matrix extract is a protein or a polypeptide.

[0014] Preferably, the decellularized matrix extract is a polypeptide.

[0015] Further preferably, the preparation method of the decellularized matrix extract is specifically as follows: 0.05-0.8 mmol / mL of a protease aqueous solution is adjusted to an acidic pH, and particles of physically crushed decellularized matrix are added so that the weight proportion of the decellularized matrix particles in the protease aqueous solution is 1-20 wt %, and the decellularized matrix particles are filtered after standing, and the filtrate is taken, and the filtrate is dialyzed to obtain a substance with a molecular weight ≥ 10 kDa, and freeze-dried to obtain a decellularized matrix extract.

[0016] Preferably, the decellularized matrix is ​​selected from one or more of small intestinal submucosa, porcine bladder basement membrane, peritoneum, pericardium, dermis and the like.

[0017] As a preferred embodiment, step S2 specifically comprises dissolving the amidated activated substance in a phosphate buffer solution to obtain an amidated activated substance solution, so that the concentration of the amidated activated substance in the amidated activated substance solution is 0.09-0.25 g / mL.

[0018] As a preferred embodiment, the amidated activator is selected from one or more of polyethylene glycol succinimidyl carbonate (PEG-SC), polyethylene glycol succinimidyl glutarate (PEG-SG), polyethylene glycol succinimidyl butyrate (PEG-SBA), polyethylene glycol succinimidyl succinate (PEG-SS), and polyethylene glycol disuccinimidyl propionate (PEG-SPA).

[0019] Preferably, the amidation activator is selected from one or more of polyethylene glycol succinimidyl carbonate (PEG-SC), polyethylene glycol succinimidyl butyrate (PEG-SBA), polyethylene glycol succinimidyl succinate (PEG-SS), and polyethylene glycol disuccinimidyl propionate (PEG-SPA).

[0020] As a preferred embodiment, the amidation activated product contains a plurality of succinimide groups.

[0021] Preferably, the molecular weight of the amidated activated species is 1-5 kDa.

[0022] Preferably, the amidation activated substance is multi-arm polyethylene glycol succinic acid succinimidyl ester (PEG-SS).

[0023] Further preferably, the multi-arm polyethylene glycol succinimidyl succinate (PEG-SS) is a four-arm PEG-SS.

[0024] As a preferred embodiment, in step S3, when the protein hydrolysate solution and the amidated activator solution are mixed, the volume ratio of the protein hydrolysate solution to the amidated activator solution is 1:(0.85-1.45).

[0025] Preferably, in step S3, when the protein hydrolysate solution and the amidated activator solution are mixed, the volume ratio of the protein hydrolysate solution to the amidated activator solution is 1:(0.9-1.25).

[0026] More preferably, the volume ratio of the protein hydrolysate solution to the amidated activated product solution is 1:1.

[0027] The reaction of the protein hydrolysate solution and the amidated activated solution in the present invention is as follows:

[0028]

[0029] Formula I

[0030] In practice, existing technologies use 4-arm PEG-SG and collagen derivatives to form cross-linked substances as therapeutic agents for tissue formation. For example, the patent No. CN101420991A discloses this technology. At the same time, the text also discloses that in this technical solution, anti-inflammatory drugs such as antibiotics need to be added as fibrosis inhibitors to inhibit scar formation. However, the addition of such fibrosis inhibitors may cause damage to the liver organ of the human body, stimulate the gastrointestinal tract after entering the human circulation, and long-term use may cause drug resistance in the human body. In order to overcome this problem, the inventor's technical concept is to design a material without anti-inflammatory drugs as an adhesive for human wounds. Through a lot of experiments and thinking, the inventor found that when preparing the bio-glue matrix of the present invention, the molecular weight of the decellularized matrix extract is controlled to be ≥10kDa, and at the same time, the molecular weight of the amidated activated product is controlled to be 1k-5kDa, which can affect scar tissue formation and help inhibit scar formation. The inventors speculate that since wound healing is a dynamic tissue regeneration, proliferation is a very important stage in this process. During the proliferation stage, if the healing material has a specific molecular weight, the proliferation rate and process can be effectively controlled. If the molecular weight is too small, small fragments may cause excessive and continuous fibrous tissue formation. If the molecular weight is too large, the proliferation rate may slow down, thereby affecting tissue regeneration. Beneficial Effects

[0031] The highly stable bioadhesive matrix obtained by the invention has good biodegradability, biocompatibility, biosafety, low swelling rate, and has a beneficial effect on tissue repair, and can solve the problems of existing medical adhesives such as toxicity, high risk, and high swelling.

[0032] The highly stable bio-glue matrix is ​​easy to use. When in use, it only needs to mix the protein hydrolysate solution and the amidation activation solution, and can be used on wounds at any position or in any shape. Before the wound is fully recovered, it exists as an embedding material between tissues or between tissues and non-tissues. During the repair process, it can be absorbed by the tissue as a nutrient to promote tissue repair.

[0033] In the process of preparing the bio-gel matrix, an amidated activated substance containing multiple succinimide groups is used, and the volume ratio of the protein hydrolyzate solution to the amidated activated substance solution is 1: (0.85-1.45). The prepared bio-gel matrix includes 42-91wt% of the cross-linked decellularized matrix extract, which can effectively reduce the swelling rate of the bio-gel matrix and improve the stability of its swelling rate. The mass expansion rate of the bio-gel matrix in physiological saline for 48 hours is only 25%.

[0034] The highly stable biogel matrix obtained by the present invention has a suitable gel time. If the gel time is too long, the operability of the material during application will be reduced, increasing the risk of surgical exposure. If the gel time is too short, it means that the degree of solidification is high, which will make the obtained gel more brittle and harder, thereby exerting pressure on the tissue, which is not conducive to the recovery of the wound.

[0035] When the bioadhesive matrix obtained by the present invention is applied in a water environment, the bonding stability is significantly improved, and at the same time, 100% degradation can be achieved within three months in a physiological environment. DETAILED DESCRIPTION

[0036] The materials in the examples are all commercially available, among which PBS buffer was purchased from Thermo Fisher Scientific, and the small intestinal submucosa and porcine bladder basement membrane were both from Zhuoruan Medical Technology (Suzhou) Co., Ltd. Example 1

[0037] This example provides a highly stable bio-gel matrix, which includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 76.24 wt % in the bio-gel matrix.

[0038] This example also provides a method for preparing a highly stable bio-gel matrix, the preparation method is as follows: S1. Preparation of protein hydrolysate solution; S2. Preparation of amidated activated product solution; S3. Blending the protein hydrolysate solution and the amidated activated product solution, and drying.

[0039] Step S1 specifically comprises dispersing the decellularized matrix extract in a phosphate buffer solution to obtain a protein hydrolysate solution, so that the concentration of the decellularized matrix extract in the protein hydrolysate solution is 0.13 g / mL.

[0040] The decellularized matrix extract contains peptide bonds and has a molecular weight of ≥10kDa. The decellularized matrix extract is a polypeptide. The specific method for preparing the decellularized matrix extract is as follows: 0.31mmol / mL of the protease aqueous solution is adjusted to an acidic pH, and the physically crushed particles of the decellularized matrix (small intestinal submucosal layer) are added so that the weight proportion of the decellularized matrix particles in the protease aqueous solution is 8wt%, and the solution is filtered after standing, and the filtrate is taken, and the filtrate is dialyzed to obtain a substance with a molecular weight of ≥10kDa, and the substance is freeze-dried to obtain the decellularized matrix extract.

[0041] Step S2 specifically comprises dissolving the amidated activated product in a phosphate buffer solution to obtain an amidated activated product solution, so that the concentration of the amidated activated product in the amidated activated product solution is 0.13 g / mL.

[0042] The amidation activated product contains multiple succinimide groups. The molecular weight is 3.4 kDa. The amidation activated product is 4-arm polyethylene glycol succinic acid succinimidyl ester (PEG-SS). 4-arm polyethylene glycol succinic acid succinimidyl ester was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., model ZP-61406.

[0043] In step S3, when the protein hydrolysate solution and the amidated activated product dissolving solution are mixed, the volume ratio of the protein hydrolysate solution to the amidated activated product dissolving solution is 1:1. Example 2

[0044] This example provides a highly stable bio-gel matrix, which includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 68.55wt% in the bio-gel matrix.

[0045] This example also provides a method for preparing a highly stable bio-gel matrix, the preparation method is as follows: S1. Preparation of protein hydrolysate solution; S2. Preparation of amidated activated product solution; S3. Blending the protein hydrolysate solution and the amidated activated product solution, and drying.

[0046] Step S1 specifically comprises dispersing the decellularized matrix extract in a phosphate buffer solution to obtain a protein hydrolysate solution, so that the concentration of the decellularized matrix extract in the protein hydrolysate solution is 0.17 g / mL.

[0047] The decellularized matrix extract contains peptide bonds and has a molecular weight of ≥10kDa. The decellularized matrix extract is a polypeptide. The specific method for preparing the decellularized matrix extract is as follows: 0.42mmol / mL of the protease aqueous solution is adjusted to an acidic pH, and the particles of the physically crushed decellularized matrix are added so that the weight proportion of the decellularized matrix particles in the protease aqueous solution is 10wt%, and the solution is filtered after standing, and the filtrate is taken, and the filtrate is dialyzed to obtain a substance with a molecular weight of ≥10kDa, and the substance is freeze-dried to obtain the decellularized matrix extract.

[0048] Step S2 specifically comprises dissolving the amidated activated product in a phosphate buffer solution to obtain an amidated activated product solution, so that the concentration of the amidated activated product in the amidated activated product solution is 0.17 g / mL.

[0049] The amidation activated product contains multiple succinimide groups. The molecular weight is 5 kDa. The amidation activated product is 4-arm polyethylene glycol succinic acid succinimidyl ester (PEG-SS). 4-arm polyethylene glycol succinic acid succinimidyl ester was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., model ZP-61407.

[0050] In step S3, when the protein hydrolysate solution and the amidated activated product dissolving solution are mixed, the volume ratio of the protein hydrolysate solution to the amidated activated product dissolving solution is 1:1.

[0051] Comparative Example 1

[0052] This example provides a highly stable biogel matrix. Unlike Example 1, the biogel matrix cannot form a gel, so there is no need to discuss the proportion of the cross-linked acellular matrix extract therein.

[0053] This example also provides a method for preparing a highly stable bio-gel matrix. The difference from Example 1 is that the decellularized matrix extract contains peptide bonds and has a molecular weight ≥5kDa. The decellularized matrix extract is a polypeptide. The specific method for preparing the decellularized matrix extract is as follows: 0.47mmol / mL of the protease aqueous solution is adjusted to an acidic pH, and the particles of the physically crushed decellularized matrix are added so that the weight proportion of the decellularized matrix particles in the protease aqueous solution is 8wt%, and the solution is filtered after standing, and the filtrate is taken, and the filtrate is dialyzed to obtain a substance with a molecular weight ≥5kDa, and the filtrate is freeze-dried to obtain a decellularized matrix extract.

[0054] Comparative Example 2

[0055] This example provides a highly stable bio-gel matrix. Different from Example 1, the bio-gel matrix includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 63.15 wt % in the bio-gel matrix.

[0056] This example also provides a method for preparing a highly stable bio-gel matrix. Unlike Example 1, the amidated activated product contains multiple succinimide groups. The molecular weight is 10 kDa. The amidated activated product is 4-arm polyethylene glycol succinic acid succinimidyl ester (PEG-SS). 4-arm polyethylene glycol succinic acid succinimidyl ester was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., model ZP-61409.

[0057] Comparative Example 3

[0058] This example provides a highly stable biogel matrix. Unlike Example 1, the biogel matrix cannot form a gel, so there is no need to discuss the proportion of the cross-linked acellular matrix extract therein.

[0059] This example also provides a method for preparing a highly stable bio-gel matrix. Unlike Example 1, the amidated activated product contains multiple succinimide groups. The molecular weight is 600 Da. The amidated activated product is 4-arm polyethylene glycol succinic acid succinimidyl ester (PEG-SS). 4-arm polyethylene glycol succinic acid succinimidyl ester was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., model ZP-61403.

[0060] Comparative Example 4

[0061] This example provides a highly stable bio-gel matrix. Different from Example 1, the bio-gel matrix includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 41.93 wt % in the bio-gel matrix.

[0062] This example also provides a method for preparing a highly stable bio-gel matrix. The difference from Example 1 is that in step S3, when the protein hydrolysate solution and the amidated activator solution are mixed, the volume ratio of the protein hydrolysate solution and the amidated activator solution is 1:2.

[0063] Comparative Example 5

[0064] This example provides a highly stable bio-gel matrix. Different from Example 2, the bio-gel matrix includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 40.17 wt % in the bio-gel matrix.

[0065] This example also provides a method for preparing a highly stable bio-gel matrix. The difference from Example 2 is that in step S3, when the protein hydrolysate solution and the amidated activator solution are blended, the volume ratio of the protein hydrolysate solution and the amidated activator solution is 1:0.7.

[0066] Test Results

[0067] 1. The content of cross-linked acellular matrix extract in the bio-gel matrix: Take the highly stable bio-gel matrix obtained in Examples 1-2 and Comparative Examples 1-5, dry it in a 37°C oven until the mass is balanced, weigh it, and record it as m0. Soak the sample in deionized water for 2, 4, 8, 12, and 24 hours, and collect and merge multiple soaking solutions. When the sample no longer has a decrease in mass after continued soaking, the bio-gel matrix is ​​taken out and fully dried in a 37°C oven. The mass at this time is recorded as m1, and the sol content = (m0-m1) / m0×100%. The above-mentioned collected and merged multiple soaking solutions are concentrated and tested for BCA protein concentration to obtain the protein content, which is recorded as m2. The mass proportion of cross-linked acellular matrix extract is calculated according to the following formula: the proportion of cross-linked acellular matrix extract = (1-(m2 / m1))×100%, and the results are shown in Table 1.

[0068] Table 1

[0069]

[0070] The biogel matrices obtained in Comparative Examples 1 and 3 cannot form gel.

[0071] 2. Gel time: In step S3 of Examples 1-2 and Comparative Examples 1-5, after the protein hydrolysate solution and the amidated activated solution were blended, the gel time t1 was determined by the vial inversion method; and the protein hydrolysate solution obtained in step S1 was allowed to stand at 25°C for 6 months, and then steps S2 and S3 were continued, and the gel time t2 was determined again by the vial inversion method according to the aforementioned method. The results are shown in Table 2.

[0072] Table 2

[0073]

[0074] 3. Overlap-shear bond strength test: Select pig skin with a structure similar to human tissue for the first overlap-shear bond strength test (F1). The test method refers to YY / T0729.1-2009. After the first test, the overlap-shear bond strength stability test is carried out. The difference from the first overlap-shear bond strength test method is that when the universal testing machine is used to test the shear, the whole material is placed in physiological saline for 24 hours before the two pieces of pig skin are pulled apart, and then continue the test according to YY / T0729.1-2009 (F2). The test results are shown in Table 3.

[0075] Table 3

[0076]

[0077] 4. Swelling rate test: Take the highly stable bio-glue matrix obtained in Examples 1-2 and Comparative Examples 1-5, and measure the swelling rate of the adhesive in physiological saline. Specifically, the initial mass is recorded as m0, and it is immersed in 37°C physiological saline. After 48 hours, it is taken out, and the excess surface water is absorbed with filter paper, and then the mass is quickly weighed and recorded as m. The swelling rate of the adhesive mass = (m-m0) / m0 × 100%. The results are shown in Table 4.

[0078] Table 4

[0079]

[0080] 5. Cell proliferation rate: After sterilization, the highly stable bio-glue matrix obtained in Example 1 and Comparative Examples 2, 4, and 5 was immersed in high-glucose DMEM supplemented with 1% (v / v) double antibody, with a solid-liquid ratio of 1 g:10 ml, and incubated in a shaker at 37°C for 1 day, 3 days, and 7 days, respectively. The supernatant after incubation was taken, 10% (v / v) serum (FBS) was added, and diluted to 3 concentrations (1×, 10×, 100×) with cell culture medium (1% double antibody, 10% FBS, 89% DMEM) to obtain adhesive extracts.

[0081] L929 cells were seeded in 24-well culture plates with a medium volume of 400 μL / well and a cell seeding density of 1×10 4 / well, and cultured in a cell culture incubator at a constant temperature for 24 hours. Replace the culture medium in each well of the 24-well plate with the above-mentioned adhesive extract diluted at different times, and continue to culture in the cell culture incubator for 24 hours. At the same time, set up a culture medium without extract as an experimental control group. The number of stock cells was tested with the help of the MTT kit, and the absorbance value (OD value) of each well was measured at 490 nm with an enzyme marker. TCP (tissue culture plate) was used as a blank control. The formula for calculating the cell proliferation rate is as follows:

[0082]

[0083] The results are shown in Table 5:

[0084] Table 5

[0085]

Claims

1. A highly stable biogel matrix, characterized in that: The bio-gel matrix includes a cross-linked acellular matrix extract, and the cross-linked acellular matrix extract accounts for 42-91 wt % in the bio-gel matrix; The method for preparing the highly stable bio-gel matrix is ​​as follows: S1. preparing a protein hydrolysate solution; S2. Preparation of amidated activated product solution; wherein the molecular weight of the amidated activated product is 1-5 kDa; S3. The protein hydrolysate solution and the amidated activated product solution are blended; Step S1 specifically comprises dispersing the decellularized matrix extract in a phosphate buffer solution to obtain a protein hydrolysate solution; the molecular weight of the decellularized matrix extract is ≥10 kDa, and the decellularized matrix extract is a polypeptide.

2. The highly stable biogel matrix according to claim 1, characterized in that: Step S1 specifically comprises dispersing the decellularized matrix extract in a phosphate buffer solution to obtain a protein hydrolysate solution, so that the concentration of the decellularized matrix extract in the protein hydrolysate solution is 0.09-0.25 g / mL.

3. The highly stable biogel matrix according to claim 1, characterized in that: Step S2 specifically comprises dissolving the amidated activated product in a phosphate buffer solution to obtain an amidated activated product solution, wherein the concentration of the amidated activated product in the amidated activated product solution is 0.09-0.25 g / mL.

4. The highly stable biogel matrix according to claim 3, characterized in that: The amidation activator is selected from one or more of polyethylene glycol succinimide carbonate, polyethylene glycol succinimide glutarate, polyethylene glycol succinimide butyrate, polyethylene glycol succinic acid succinimide ester, and polyethylene glycol disuccinimide propionate.

5. The highly stable biogel matrix according to claim 4, characterized in that: The amidated activated species contains multiple succinimide groups.

6. The highly stable biogel matrix according to claim 1, characterized in that: In step S3, when the protein hydrolysate solution and the amidated activated product dissolving solution are mixed, the volume ratio of the protein hydrolysate solution to the amidated activated product dissolving solution is 1:(0.85-1.45).

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