In-situ cross-linked polypeptide gel as well as preparation method and application thereof

Abstract

The invention relates to in-situ cross-linked polypeptide gel and a preparation method and application thereof, and the preparation method comprises the following steps: dissolving modified polypeptide in a first solvent to obtain a component A; dissolving an amino high-molecular polymer in a second solvent to obtain a component B; mixing the component A and the component B to obtain in-situ cross-linked polypeptide gel; the modified polypeptide is mainly obtained by reacting a compound containing an NHS group with polypeptide under the action of a catalyst. The preparation method of the in-situ cross-linked polypeptide gel is simple in condition, easy to operate, mild in reaction condition, simple and easily available in raw materials, safe and good in biocompatibility, and has a wide application prospect. The prepared hydrogel containing the polypeptide chain is good in toughness, good in viscosity, good in biocompatibility, high in biological safety, biodegradable, good in plasticity, non-toxic and harmless.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to an in-situ cross-linked polypeptide gel and a preparation method and application thereof. Background Art

[0002] Hydrogel is a type of extremely hydrophilic three-dimensional network structure gel that can swell in water but not dissolve, and will not degrade in a short period of time. Due to the presence of a cross-linked network, hydrogel can swell and retain a large amount of water, and the amount of water absorbed is closely related to the degree of cross-linking. The higher the degree of cross-linking, the lower the water absorption. This property is very similar to a soft tissue. The water content in the hydrogel can be as low as a few percent or as high as 99%. The aggregated state of the gel is both a non-complete solid and a non-complete liquid. The behavior of a solid is that it can maintain a certain shape and volume under certain conditions, and the behavior of a liquid is that solutes can diffuse or penetrate from the hydrogel.

[0003] Nowadays, a lot of research has been done on the biomedical, pharmaceutical and environmental applications of hydrogels. This material has super absorption properties, which can be controlled by changing the concentration, temperature, pH, etc. of the external stimulus environment. Due to the good moisture retention, biocompatibility and degradability of hydrogels, their application and development in the biomedical field have been promoted; super-intelligent hydrogels have aroused great interest among researchers in various application fields such as tissue engineering, drug delivery, diagnosis, imaging and optics. In view of this, the present invention provides an in situ cross-linked polypeptide gel and its preparation method and application. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an in-situ cross-linked polypeptide gel and a preparation method and application thereof.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In a first aspect, a method for preparing an in situ cross-linked polypeptide gel comprises the following steps:

[0007] Dissolving the modified polypeptide in a first solvent to obtain component A; dissolving the amino polymer in a second solvent to obtain component B; mixing the component A and the component B to obtain an in-situ cross-linked polypeptide gel;

[0008] The modified polypeptide is mainly obtained by reacting a compound containing an NHS group and a polypeptide under the action of a catalyst.

[0009] The structure of the in situ cross-linked polypeptide gel is as shown in formula (I):

[0010]

[0011] In formula (I), R1 and R2 in the modified polypeptide structural formula vary according to the different polypeptides used; and R1 and R2 in the amino polymer structural formula vary according to the different materials used.

[0012] The invention has the following beneficial effects: the preparation method of the in situ cross-linked polypeptide gel of the invention has simple conditions, is easy to operate, has mild reaction conditions, has simple and easy-to-obtain raw materials, is safe, has good biocompatibility, and has broad application prospects. The polypeptide chain-containing hydrogel prepared by the invention has good toughness, good viscosity, good biocompatibility, high biosafety, is biodegradable, has good plasticity, and is non-toxic and harmless.

[0013] Based on the above technical solution, the present invention can also be improved as follows.

[0014] Furthermore, the amino polymer includes at least one of gelatin, polypeptide polymer, chitosan, carboxymethyl chitosan, polyethyleneimine, and branched polyethyleneimine;

[0015] The compound containing the NHS group includes at least one of N-hydroxysuccinimide, disuccinimidyl glutarate, disuccinimidyl suberate, (+) biotin-N-succinimidyl ester, lauric acid N-hydroxysuccinimide ester, N-hydroxysuccinimide acetoacetate, caprylic acid-N-succinimide ester, caproic acid-N-succinimide ester, N-hydroxysuccinimide acetate, and di(N-succinimide) sebacate;

[0016] The catalyst includes N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI solid);

[0017] The polypeptide comprises at least one of a plant-derived polypeptide and an animal-derived polypeptide;

[0018] The first solvent includes at least one of deionized water, double distilled water, ultrapure water, sodium carbonate-sodium bicarbonate buffer solution, PBS buffer solution, and phosphate buffer solution; the second solvent includes at least one of deionized water, double distilled water, ultrapure water, sodium carbonate-sodium bicarbonate buffer solution, PBS buffer solution, and phosphate buffer solution.

[0019] The beneficial effect of adopting the above further scheme is that the amino high molecular polymer can be cross-linked with the modified polypeptide to form a three-dimensional network structure.

[0020] Furthermore, the plant-derived polypeptide includes at least one of soybean peptide, peanut peptide, corn peptide, pea peptide, rice peptide, and flaxseed peptide, and the animal-derived polypeptide includes at least one of fish collagen peptide, bovine collagen peptide, and bone collagen peptide.

[0021] The beneficial effect of adopting the above further scheme is that the adopted polypeptide has good water solubility and is biodegradable.

[0022] Furthermore, the solid content of the in-situ cross-linked polypeptide gel is 1wt%-30wt%. The hydrogel is a three-dimensional network structure formed by cross-linking the modified polypeptide and the amino polymer.

[0023] The beneficial effect of adopting the above further scheme is that the three-dimensional spatial network structure formed by the hydrogel within this range has good continuity and adhesion.

[0024] Furthermore, the mass concentration of the modified polypeptide in the component A is 1%-30%, the mass concentration of the amino polymer in the component B is 0.01%-10%; the volume ratio of the component A to the component B is 1:(0.1-10);

[0025] Preferably, the mass concentration of the modified polypeptide in component A is 2%-25%, and the mass concentration of the amino polymer in component B is 0.1%-10%; the volume ratio of component A to component B is 1:(0.1-5).

[0026] The beneficial effect of adopting the above further scheme is that the hydrogel formed by using component A and component B within the above range has good continuity and adhesion of the three-dimensional spatial network structure.

[0027] Furthermore, the modified polypeptide comprises the following specific steps: dissolving the polypeptide in a third solvent until it is clear and transparent, adding a catalyst until it is completely dissolved, and then adding a compound containing an NHS group to react to obtain a post-reaction solution; dropping the post-reaction solution into an alcohol solution, filtering, and drying to obtain a modified polypeptide.

[0028] The beneficial effect of adopting the above further scheme is that the reaction time of the polypeptide modified by the above NHS group-containing compound with the amino high molecular polymer is short and the conditions are mild.

[0029] Furthermore, the third solvent includes at least one of deionized water, double distilled water, ultrapure water, sodium carbonate-sodium bicarbonate buffer solution, PBS buffer solution and phosphate buffer solution; and the alcohol solution includes at least one of methanol and ethanol.

[0030] Further, the mass ratio of the polypeptide to the third solvent is 1:(2-10);

[0031] The molar ratio of the polypeptide, the catalyst and the compound containing the NHS group is 1:(0.1-50):(0.1-50);

[0032] Reaction parameters: reaction temperature is 0°C-50°C, reaction time is 1h-24h;

[0033] The volume ratio of the reaction solution to the alcohol solution is 1:(1-50); the reaction solution is added dropwise to the alcohol solution and stirred for 10-120 minutes;

[0034] The drying method includes vacuum freeze drying and vacuum low-temperature drying; the drying temperature of the vacuum low-temperature drying is 15°C-50°C.

[0035] The beneficial effect of adopting the above further scheme is that the modified polypeptide obtained under the reaction ratio and post-treatment conditions has better performance.

[0036] In a second aspect, an in situ cross-linked polypeptide gel is prepared by the preparation method.

[0037] In a third aspect, an application of an in situ cross-linked polypeptide gel is used to prepare a product for hemostasis, adhesion sealing, or anti-adhesion (eg, tendon, abdominal cavity, uterine cavity).

[0038] The beneficial effects of the above scheme of the present invention are: the hydrogel prepared by the present invention has good toughness and good viscosity, and can be used for hemostasis, or adhesion sealing, or anti-adhesion (such as tendons, abdominal cavity, uterine cavity) related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The modified fish collagen prepared in Example 1 of the present invention 1 H NMR spectrum.

[0040] Figure 2 This is a picture of the hydrogel prepared in Example 3 of the present invention; wherein, the upper picture is a top view of the hydrogel, and the lower picture is a front view of the hydrogel.

[0041] Figure 3 The hydrogel prepared in Example 6 of the present invention is attached to pig skin; wherein a is a photo of the hydrogel just injected into the surface of pig skin, b and c are photos of the gel firmly attached when the pig skin loaded with gel is folded and restored, d is a photo of the gel firmly attached during water flushing, and e is a photo of the gel after the water flushing is completed;

[0042] Figure 4 This is a diagram showing the changes in the relative mass of the hydrogels of Examples 3 and 6 of the present invention. DETAILED DESCRIPTION

[0043] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0044] The fish collagen peptide used in this patent comes from Aladdin Biochemical Technology Co., Ltd.; the soybean peptide comes from Shanghai McLean Biochemical Technology Co., Ltd.

[0045] Example 1: Preparation of modified polypeptides

[0046] This embodiment relates to a method for preparing a modified polypeptide, comprising the following steps:

[0047] Accurately weigh 5.0g of fish collagen peptide in a sample bottle, add 17.0g of deionized water, and stir to dissolve at room temperature; accurately weigh 7.67g of N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI solid) and add it to the above sample bottle in batches, and continue to stir until EDCI is completely dissolved; after EDCI is completely dissolved, accurately weigh 4.6g of N-hydroxysuccinimide (NHS), add it to the above reaction system in batches, stir to dissolve, and keep the reaction for 3 hours. The reaction solution after the reaction is added dropwise to 300mL of ethanol to precipitate, and the filter cake is obtained by filtering. The filter cake is dissolved in 20mL of deionized water, and then added dropwise to 300mL of ethanol again to precipitate. The product is filtered and the solid product is obtained by vacuum freeze-drying. The solid product finally obtained is 1 H NMR spectrum Figure 1 As shown by Figure 1 The H NMR results show that the modified fish collagen peptide contains NHS groups, indicating that the modification is successful. The simplified structure and reaction equation of the modified peptide are shown in Formula II:

[0048]

[0049] Example 2: Preparation of modified polypeptides

[0050] This embodiment relates to a method for preparing a modified polypeptide, comprising the following steps:

[0051] Accurately weigh 5.0g of soybean peptide in a sample bottle, add 20g of deionized water, and stir and dissolve at room temperature; accurately weigh 7.43g of EDCI solid and add it to the above sample bottle in batches, and continue to stir until EDCI is completely dissolved; after EDCI is completely dissolved, accurately weigh 4.46g of N-hydroxysuccinimide, add it to the above reaction system in batches, stir and dissolve, and keep reacting for 3 hours. The reaction solution after the reaction is added dropwise to 350mL of ethanol for precipitation, and the filter cake is filtered to obtain a filter cake, which is dissolved in 20mL of deionized water, and then added dropwise to 350mL of ethanol again, precipitated, filtered to obtain the product, and vacuum freeze-dried to obtain a solid product.

[0052] Example 3: Hydrogel preparation

[0053] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps:

[0054] Accurately weigh 100 mg of the modified fish collagen peptide prepared in Example 1 into a centrifuge tube, add 905 mg of deionized water to dissolve for later use, and record it as component A; use 956 mL of 0.1 M sodium carbonate-sodium bicarbonate buffer solution with a pH value of 7.6 to dissolve 50 mg of carboxymethyl chitosan, and record it as component B; take two syringes, draw 0.5 mL of component A and component B into the centrifuge tube respectively, shake well to mix, and the hydrogel can be obtained within 0.5-5 minutes at room temperature. The hydrogel prepared in this example is as follows Figure 2 As shown by Figure 2 It can be seen that the hydrogel has a complete gel morphology and good overall continuity. The hydrogel structure is shown in formula (III):

[0055]

[0056] Example 4: Hydrogel Preparation

[0057] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps:

[0058] Accurately weigh 100 mg of the modified soybean peptide prepared in Example 2 into a centrifuge tube, add 905 mg of deionized water to dissolve for later use, and record it as component A; use 956 mL of 0.1 M sodium carbonate-sodium bicarbonate buffer solution with a pH value of 7.6 to dissolve 50 mg of carboxymethyl chitosan, and record it as component B; take two syringes, draw 0.5 mL of component A and component B into the centrifuge tube respectively, shake well to mix, and the hydrogel can be obtained within 0.5-5 minutes at room temperature.

[0059] Example 5: Hydrogel Preparation

[0060] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps: accurately weighing 100 mg of the modified fish collagen peptide prepared in Example 1 into a centrifuge tube, adding 905 mg of deionized water to dissolve for later use, recorded as component A; using 956 mL of deionized water to dissolve 50 mg of a 50 wt% PEI (polyethyleneimine) aqueous solution, recorded as component B; taking two syringes, respectively drawing 0.5 mL of component A and component B into the centrifuge tube, shaking and mixing thoroughly, and obtaining the hydrogel within 0.1-5 minutes at room temperature. The hydrogel structure is as shown in formula (IV):

[0061]

[0062] Example 6

[0063] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps: The difference between this embodiment and Example 3 is that the amount of modified fish collagen peptide is increased from 100 mg to 160 mg.

[0064] Example 7

[0065] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps: The difference between this embodiment and Example 3 is that the amount of modified fish collagen peptide is increased from 100 mg to 160 mg; when mixing, 0.2 mL of component A and 0.8 mL of component B are aspirated.

[0066] Example 8

[0067] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps: The difference between this embodiment and Example 3 is that the amount of modified fish collagen peptide is increased from 100 mg to 200 mg.

[0068] Example 9

[0069] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps: The difference between this embodiment and Example 4 is that the amount of modified soybean peptide is increased from 100 mg to 160 mg.

[0070] Example 10

[0071] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps: The difference between this embodiment and embodiment 5 is that the amount of 50wt% PEI aqueous solution is increased from 50mg to 80mg.

[0072] Embodiment 11

[0073] This embodiment relates to a method for preparing an in situ cross-linked polypeptide gel, comprising the following steps: The difference between this embodiment and embodiment 5 is that the amount of 50wt% PEI aqueous solution is increased from 50mg to 100mg.

[0074] Comparative Example 1

[0075] Accurately weigh 25 mg of the modified fish collagen peptide prepared in Example 1 into a centrifuge tube, add 905 mg of deionized water to dissolve for later use, and record it as component A; use 956 mL of 0.1 M sodium carbonate-sodium bicarbonate buffer solution with a pH value of 7.6 to dissolve 50 mg of carboxymethyl chitosan, and record it as component B; take two syringes, draw 0.5 mL of component A and component B into the centrifuge tube respectively, shake and mix thoroughly, and the hydrogel can be obtained within 0.5-5 minutes at room temperature. The hydrogel prepared under this condition degrades after one day, and the degradation time is very fast.

[0076] Comparative Example 2

[0077] Accurately weigh 25 mg of unmodified fish collagen peptide in a centrifuge tube, add 905 mg of deionized water to dissolve and set aside, record as component A; use 956 mL of 0.1 M sodium carbonate-sodium bicarbonate buffer solution with a pH value of 7.6 to dissolve 50 mg of carboxymethyl chitosan, record as component B; take two syringes, draw 0.5 mL of component A and component B into the centrifuge tube respectively, shake and mix thoroughly, and the hydrogel can be obtained within 0.5-5 minutes at room temperature. The hydrogel prepared under this condition will degrade in one day.

[0078] Test example

[0079] (1) Adhesion

[0080] The hydrogel prepared in Example 6 was injected into the surface of pig skin, and then observed and flushed with water. The results were as follows: Figure 3 As shown by Figure 3 The results show that the prepared hydrogel can return to its original water state after squeezing, and the water flushing experiment found that the prepared hydrogel has good adhesion and will not fall off or slip when flushed with water.

[0081] (2) In vitro degradation

[0082] The hydrogels prepared in the above Examples 3-11 and the hydrogels prepared in Control Examples 1 and 2 were subjected to in vitro degradation, and (200±10) mg of the hydrogels were accurately weighed in a 50 mL centrifuge tube. Five parallel samples were set for each hydrogel, and 100-120 times the volume of a PBS aqueous solution with a pH value of 7.4 was added to each centrifuge tube, and each centrifuge tube was placed in a constant temperature shaking water bath at 37°C and a shaking rate of 100 rpm / min. The PBS aqueous solution was replaced every 24 hours. When weighing, first wipe the water on the surface of the hydrogel with filter paper, then weigh the mass of the hydrogel and record it. The results are shown in Table 1 below. The changes in the relative mass of the hydrogels in Example 3 and Example 6 (relative mass = mass of the hydrogel at different degradation times / initial mass of the hydrogel) were calculated, and the results are shown in Table 1 below. Figure 4 shown.

[0083] Table 1

[0084]

[0085] Depend on Figure 4 The results show that the prepared hydrogel is still not completely degraded after 8 days of degradation.

[0086] In summary, the preparation method of the in situ cross-linked polypeptide gel of the present invention has simple conditions, is easy to operate, has mild reaction conditions, simple and easy-to-obtain raw materials, is safe, has good biocompatibility, and has broad application prospects. The polypeptide chain-containing hydrogel prepared by the present invention has good toughness, good viscosity, good biocompatibility, high biosafety, biodegradability, good plasticity, and is non-toxic and harmless.

[0087] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing an in situ cross-linked polypeptide gel, characterized in that: The steps include: Dissolving the modified polypeptide in a first solvent to obtain component A; dissolving the amino polymer in a second solvent to obtain component B; mixing the component A and the component B to obtain an in-situ cross-linked polypeptide gel; The modified polypeptide is mainly obtained by reacting a compound containing an NHS group and a polypeptide under the action of a catalyst.

2. The method for preparing an in situ cross-linked polypeptide gel according to claim 1, characterized in that: The amino polymer includes at least one of gelatin, polypeptide polymer, chitosan, carboxymethyl chitosan, polyethylene imine, and branched polyethylene imine; The compound containing the NHS group includes at least one of N-hydroxysuccinimide, disuccinimidyl glutarate, disuccinimidyl suberate, (+) biotin-N-succinimidyl ester, lauric acid N-hydroxysuccinimide ester, N-hydroxysuccinimide acetoacetate, caprylic acid-N-succinimide ester, caproic acid-N-succinimide ester, N-hydroxysuccinimide acetate, and di(N-succinimide) sebacate; The catalyst includes N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; The polypeptide comprises at least one of a plant-derived polypeptide and an animal-derived polypeptide; The first solvent includes at least one of deionized water, double distilled water, ultrapure water, sodium carbonate-sodium bicarbonate buffer solution, PBS buffer solution, and phosphate buffer solution; the second solvent includes at least one of deionized water, double distilled water, ultrapure water, sodium carbonate-sodium bicarbonate buffer solution, PBS buffer solution, and phosphate buffer solution.

3. The method for preparing an in situ cross-linked polypeptide gel according to claim 2, characterized in that: The plant-derived polypeptide includes at least one of soybean peptide, peanut peptide, corn peptide, pea peptide, rice peptide, and flaxseed peptide; and the animal-derived polypeptide includes at least one of fish collagen peptide, bovine collagen peptide, and bone collagen peptide.

4. The method for preparing an in situ cross-linked polypeptide gel according to claim 1, characterized in that: The solid content of the in-situ cross-linked polypeptide gel is 1.0 wt%-30 wt%.

5. The method for preparing an in situ cross-linked polypeptide gel according to claim 1, characterized in that: The mass concentration of the modified polypeptide in the component A is 1%-30%, the mass concentration of the amino high molecular polymer in the component B is 0.01%-10%; the volume ratio of the component A to the component B is 1:(0.1-10).

6. The method for preparing an in situ cross-linked polypeptide gel according to claim 1, characterized in that: The modified polypeptide comprises the following specific steps: dissolving the polypeptide in a third solvent until it is clear and transparent, adding a catalyst until it is completely dissolved, and then adding a compound containing an NHS group to react to obtain a post-reaction solution; and dropping the post-reaction solution into an alcohol solution, filtering, and drying to obtain the modified polypeptide.

7. The method for preparing an in situ cross-linked polypeptide gel according to claim 6, characterized in that: The third solvent includes at least one of deionized water, double distilled water, ultrapure water, sodium carbonate-sodium bicarbonate buffer solution, PBS buffer solution and phosphate buffer solution; the alcohol solution includes at least one of methanol and ethanol.

8. The method for preparing an in situ cross-linked polypeptide gel according to claim 6, characterized in that: The mass ratio of the polypeptide to the third solvent is 1:(2-10); The molar ratio of the polypeptide, the catalyst and the compound containing the NHS group is 1:(0.1-50):(0.1-50); Reaction parameters: reaction temperature is 0°C-50°C, reaction time is 1h-24h; The volume ratio of the post-reaction solution to the alcohol solution is 1:(1-50).

9. An in situ cross-linked polypeptide gel, characterized in that: Prepared by the preparation method described in any one of claims 1 to 8.

10. An application of an in situ cross-linked polypeptide gel, characterized in that: The in situ cross-linked polypeptide gel according to claim 9 is used to prepare hemostatic, adhesive sealing, or anti-adhesion products.

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