A mussel-mucin composite gel and a preparation method and use thereof

Abstract

The present application relates to a kind of mussel mucin composite gel and its preparation method and purposes, including mussel mucin, bioactive ingredient, material B, biological lubricating material, crosslinking agent, analgesic drug and solvent;The mass ratio of mussel mucin and material B is 3:2~4:1, and material B is polyanion material or amphiphilic polyelectrolyte material.The mussel mucin composite gel of the present application is used as skin filling material injection, and the skin filling material is used for filling and supporting shaping in dermis middle layer, subcutaneous layer or periosteum upper level.The mussel mucin composite gel of the present application has excellent anti-degradation performance, high tissue adhesion performance, low water absorption swelling performance, and stronger tissue combination, can effectively avoid the displacement of gel after injection, can long-term maintain filling volume in dermis middle layer, subcutaneous or periosteum surface, with excellent volume stability.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a mussel adhesive protein composite gel, its preparation method, and its application. Background Technology

[0002] Minimally invasive injections to improve skin condition have rapidly gained popularity in the consumer market. With their advantages of ease of operation, rapid results, and short recovery time, they have become an important technique for facial rejuvenation and contouring. Currently, the mainstream injectable filler materials on the market are based on natural polymers, with injectable gel products featuring hyaluronic acid and collagen as core ingredients dominating the market.

[0003] To prolong the duration of action of injectable fillers in vivo and improve their mechanical support properties, existing technologies typically employ chemical cross-linking strategies to modify the aforementioned natural polymers. Commonly used cross-linking agents include exogenous small molecule compounds such as 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS). However, if these chemical cross-linking agents are not thoroughly purified after the cross-linking reaction, their residues may remain at the injection site for a long time, causing cytotoxic reactions in local tissues and even inducing granuloma formation, seriously affecting the biocompatibility and long-term safety of the product. Furthermore, gel materials, such as hyaluronic acid, are rich in hydrophilic groups such as hydroxyl and carboxyl groups on their molecular chains, making them prone to swelling due to absorption of surrounding tissue fluid in vivo. This uncontrolled volume expansion may not only lead to significant facial edema or "swelling" in the injection area in the short term, but may also affect microcirculation due to increased local tissue pressure, further exacerbating tissue reactions. This problem is particularly prominent in high-concentration or highly cross-linked injectable materials, severely affecting their application in delicate areas such as the periorbital region and lips.

[0004] At the same time, existing gel-based fillers generally suffer from insufficient resistance to displacement. When subjected to forces such as muscle movement, gravity, or external pressure, the material is prone to displacement or diffusion, leading to deformation of the filled area's contour, blurred boundaries, and even nodules, unevenness, etc., which seriously affect aesthetic results and patient satisfaction.

[0005] Although injectable gel materials based on hyaluronic acid and collagen are widely used in clinical practice, these materials still have many shortcomings in terms of biocompatibility, volume stability, anti-displacement properties, and long-term tissue compatibility. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention provides a mussel adhesive protein composite gel, its preparation method, and its uses. The mussel adhesive protein composite gel prepared by this invention possesses excellent anti-degradation properties, high tissue adhesion properties, and low water absorption and swelling properties. It is used as a dermal filler injection material, primarily for filling and supporting shaping in the mid-dermis, subcutaneous layer, and supraperiosteal layer, including but not limited to: mid-dermal filling in areas such as the infraorbital region and neck; subcutaneous filling in areas such as the midface and nasolabial folds; and deep subcutaneous and supraperiosteal shaping in areas such as the mandible, midface, and nasolabial folds.

[0007] On one hand, the present invention provides a method for preparing mussel adhesive protein composite gel, comprising the following steps: 1) Dissolve the bioactive ingredient in a solvent to obtain a bioactive solution with a mass concentration of 0.1~1wt%, and then divide it into bioactive solution A and bioactive solution B according to a volume ratio of 1:1~1:10; 2) Dissolve mussel adhesive protein and material B in the above-mentioned bioactive solution A and bioactive solution B respectively to obtain solution C and solution D. In solution C, the mass ratio of bioactive component to mussel adhesive protein is 1:4 to 1:30; in solution D, the mass ratio of bioactive component to material B is 1:1 to 1:2, wherein material B is a polyanionic material or an amphiphilic polyelectrolyte material. 3) Add solution C dropwise to solution D, stir, centrifuge, and discard the supernatant to obtain a composite condensed phase; 4) After homogenizing and crushing the composite condensed phase, a crosslinking agent is added, and the mixture is allowed to stand at room temperature for crosslinking to obtain a gel; 5) Add biological lubricant and analgesic drug to the gel, and sterilize to obtain the mussel adhesive protein composite gel.

[0008] Furthermore, in step 1), the solvent is any one of purified water, phosphate buffer, or acetate buffer with a pH of 6-8.

[0009] Furthermore, the bioactive component is any one or a combination of elastin, collagen, dipotassium glycyrrhizate, polydeoxyribonucleotide (PDRN), polynucleotide (PN), L-proline, glycine, lysine, and aspartic acid.

[0010] Furthermore, in step 2), the mussel adhesive protein is selected from naturally extracted or biosynthesized mussel adhesive proteins, including but not limited to Mfp-1, Mfp-2, Mfp-3, Mfp-4, Mfp-5, Mfp-6 and fusion proteins between the aforementioned types such as Mfp-151 and Mfp-361, as well as activity-enhanced fusion recombinant mussel adhesive proteins such as Mfp-1-RGD, which incorporate natural ECM components such as collagen, fibronectin, hyalin, laminin and other functional peptide segments.

[0011] Furthermore, in step 2), material B is a polyanionic material, which is any one or a combination of hyaluronic acid salt, polyglutamate, polyaspartate, alginate, chondroitin sulfate, heparin salt, and heparin sulfate.

[0012] Furthermore, in step 2), material B is an amphiphilic polyelectrolyte material, which is any one or a combination of hydroxyethyl chitosan, polylysine-polyglutamic acid copolymer, polylysine-polyaspartic acid copolymer, amphiphilic modified gelatin, amphiphilic modified collagen, and amphiphilic modified decellularized ECM material.

[0013] Further, in step 3), mixture C is added dropwise to mixture D at a rate of 0.01 mL / min to 5 mL / min; the stirring rate is 100 r / min to 500 r / min; after all mixture C is added dropwise to mixture D, stirring continues for 30 min to 60 min, and then centrifugation is performed, wherein the centrifugation rate is 2000 r / min to 10000 r / min, and the centrifugation time is 10 min to 30 min.

[0014] Further, the specific process of step 4) is as follows: the composite condensed phase is homogenized and pulverized into uniform gel particles of 10μm~500μm at a frequency of 0.1Hz~15Hz using a homogenizer for 10min~30min; then a crosslinking agent is added, and after standing at room temperature for crosslinking for 30min~2880min, the residual crosslinking agent is removed to obtain a gel; in the gel, the mass ratio of the crosslinking agent to the composite condensed phase is 0.2:1~1:2.

[0015] Further, the crosslinking agent is any one of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide (EDC / NHS), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1,1'-carbonyldiimidazole (CDI), and N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate (CMC).

[0016] Furthermore, the bio-lubricating material is any one or a combination of hyaluronic acid salt, carboxymethyl cellulose, hydroxyethyl cellulose, alginate, chondroitin sulfate, hydroxyethyl chitosan, and decellularized ECM material.

[0017] Furthermore, the analgesic drug includes any one or a combination of lidocaine hydrochloride, bupivacaine hydrochloride, procaine hydrochloride, and tetracaine hydrochloride.

[0018] On the other hand, the present invention provides a mussel adhesive protein composite gel, which is prepared by any of the preparation methods described above, and includes mussel adhesive protein, bioactive components, material B, biolubricating material, crosslinking agent, analgesic drug and solvent.

[0019] Furthermore, the mass ratio of mussel adhesive protein to material B is 3:2 to 4:1.

[0020] Furthermore, in step 4), the mass ratio of the gel, the biological lubricant, and the analgesic drug is 1000:1:1 to 100:3:5.

[0021] Furthermore, the mass fraction of the bio-lubricating material is 0.01wt%~3wt%.

[0022] Furthermore, the analgesic drug has a mass fraction of 0.005wt% to 1wt%.

[0023] Furthermore, material B is a polyanionic material or an amphiphilic polyelectrolyte material.

[0024] Furthermore, the solvent is any one of purified water, phosphate buffer, or acetate buffer with a pH of 6-8.

[0025] In another aspect, the present invention provides a use of mussel adhesive protein composite gel, wherein the mussel adhesive protein composite gel is any of the mussel adhesive protein composite gels described above, or a mussel adhesive protein composite gel prepared by any of the methods described above, and the mussel adhesive protein composite gel is used as a skin filler material for injection.

[0026] Furthermore, the skin filler material is used for filling and supporting the shaping of the middle dermis, subcutaneous layer, or upper periosteum.

[0027] Furthermore, in the uses described in this invention, the skin filling material is mainly used for filling and supporting shaping of the middle dermis, subcutaneous layer and periosteum, including but not limited to: filling of the middle dermis such as the infraorbital region and neck; subcutaneous filling of the midface and nasolabial folds; and deep shaping of the subcutaneous and periosteal layers of the mandible, midface, and nasolabial folds.

[0028] Compared with the prior art, the present invention has the following beneficial technical effects: The mussel adhesive protein composite gel prepared in this invention forms a composite condensed phase through electrostatic interaction between mussel adhesive protein and material B (a polyanionic material or an amphiphilic polyelectrolyte material). This structure effectively reduces the exposure of hydrophilic groups within the system. The subsequent addition of a cross-linking agent further densifies the gel network structure. Swelling rate tests show that the gel's swelling rate is 20%–32%, significantly lower than the comparative example, indicating low water absorption and swelling performance. In vitro enzymatic degradation experiments show that the gel has a degradation residue rate of 53–88%, exhibiting good resistance to enzymatic degradation and maintaining a relatively long degradation time in vivo. Adhesion strength tests show that the gel has high adhesion to tissues (33–61 kPa), reducing the risk of displacement after injection.

[0029] Furthermore, this invention employs a zero-length crosslinking agent, which can be completely removed through neutralization dialysis. Cytotoxicity experiments show a relative cell proliferation rate of 98%–105%, confirming the gel's excellent biocompatibility. The preparation process for this product is simple, and all materials used possess good biocompatibility and tissue compatibility. Attached Figure Description

[0030] Figure 1 SEM images of the mussel adhesive protein composite gels prepared in Examples 1, 1, 2 and 5 of the present invention. Figure 2 This is a microscopic tissue image of the mussel adhesive protein composite gel prepared in the control group, Example 1, and Comparative Example 4 of the present invention, after subcutaneous implantation in rats for 4 weeks. Detailed Implementation

[0031] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific embodiments are now described in detail with reference to the accompanying drawings. The described embodiments are merely some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the detailed embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.

[0032] In the following description, when referring to the accompanying drawings, the same numbers in different drawings denote the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure.

[0033] The present invention will be further described in detail below with reference to specific embodiments. Example 1

[0034] The present invention discloses a method for preparing a mussel adhesive protein composite gel, comprising the following steps: 1) Elastin and collagen were dissolved in phosphate buffer at pH 6 to obtain a bioactive solution, wherein the mass concentration of elastin in the bioactive solution was 0.1 wt% and the mass concentration of collagen was 0.1 wt%. The bioactive solution was then divided into bioactive solution A and bioactive solution B at a volume ratio of 1:5. 2) Mfp-1 type mussel adhesive protein and material B (sodium hyaluronate) were dissolved in bioactive solution A and bioactive solution B, respectively, to obtain solution C and solution D; wherein, in solution C, the mass ratio of elastin, collagen and Mfp-1 type mussel adhesive protein was 1:1:20; and in solution D, the mass ratio of elastin, collagen and sodium hyaluronate was 1:1:2. 3) Add solution C dropwise to solution D at a rate of 2 mL / min and stir at a rate of 300 r / min until all solution C has been added to solution D. Continue stirring for 40 min, centrifuge at 5000 r / min for 10 min and discard the supernatant to obtain a composite condensed phase, wherein the mass ratio of mussel adhesive protein to material B is 2:1. 4) The composite condensed phase was homogenized and pulverized into uniform gel particles of 10μm~500μm at a frequency of 5Hz using a homogenizer for 20min; then a crosslinking agent (EDC / NHS) was added, and the mixture was allowed to stand at room temperature for 24h to crosslink, and the residual crosslinking agent in the crosslinked product was removed to obtain a gel. The mass ratio of the crosslinking agent to the composite condensed phase was 1:1. 5) Add the obtained gel with a bio-lubricating material (0.05 wt% sodium hyaluronate) and analgesic (0.01 wt% bupivacaine hydrochloride), mix thoroughly, and sterilize to obtain the mussel adhesive protein composite gel of Example 1. The SEM image of the mussel adhesive protein composite gel is shown below. Figure 1 As shown; the mass ratio of gel to biological lubricant and analgesic drug is 100:2:1. Example 2

[0035] The present invention discloses a method for preparing a mussel adhesive protein composite gel, comprising the following steps: 1) Glycine and PDRN were dissolved in acetate buffer at pH 6 to obtain a bioactive solution, wherein the mass concentration of glycine and PDRN in the bioactive solution was 0.05 wt%, and the bioactive solution was divided into bioactive solution A and bioactive solution B at a volume ratio of 1:10. 2) Mfp-3 type mussel adhesive protein and material B (sodium alginate and hydroxyethyl chitosan) were dissolved in bioactive solution A and bioactive solution B, respectively, to obtain solution C and solution D. In solution C, the mass ratio of glycine, PDRN and Mfp-3 type mussel adhesive protein was 1:1:60; in solution D, the mass ratio of glycine, PDRN and sodium alginate and hydroxyethyl chitosan was 1:1:2:2. 3) Add solution C dropwise to solution D at a rate of 0.01 mL / min and stir at a rate of 100 r / min until all solution C has been added to solution D. Continue stirring for 30 min, centrifuge at 2000 r / min for 10 min and discard the supernatant to obtain a composite condensed phase, wherein the mass ratio of mussel adhesive protein to material B is 3:2. 4) The composite condensed phase was homogenized and pulverized into uniform gel particles of 10μm~500μm at a frequency of 0.1Hz using a homogenizer for 10min. Then, a crosslinking agent (DMTMM) was added and allowed to stand at room temperature for 30min to crosslink. The residual crosslinking agent in the crosslinked product was removed to obtain a gel. The mass ratio of crosslinking agent to composite condensed phase was 1:2. 5) Add the obtained gel with bio-lubricating material (0.005 wt% carboxymethyl cellulose and 0.005 wt% sodium chondroitin sulfate) and analgesic drug (0.002 wt% procaine hydrochloride and 0.003 wt% lidocaine hydrochloride) and mix evenly. After sterilization, the mussel adhesive protein composite gel of this Example 2 is obtained, wherein the mass ratio of the gel obtained in step 4) to the bio-lubricating material and the analgesic drug is 1000:1:1. Example 3

[0036] The present invention discloses a method for preparing a mussel adhesive protein composite gel, comprising the following steps: 1) Dissolve dipotassium glycyrrhizate in phosphate buffer solution at pH 8 to obtain a bioactive solution, wherein the mass concentration of dipotassium glycyrrhizate in the bioactive solution is 1 wt%, and the bioactive solution is divided into bioactive solution A and bioactive solution B at a volume ratio of 1:1. 2) Mfp-6 mussel adhesive protein and material B (sodium polyglutamate) were dissolved in bioactive solution A and bioactive solution B, respectively, to obtain solution C and solution D. In solution C, the mass ratio of dipotassium glycyrrhizate to Mfp-6 mussel adhesive protein was 1:4; in solution D, the mass ratio of dipotassium glycyrrhizate to sodium polyglutamate was 1:1. 3) Add solution C dropwise to solution D at a rate of 5 mL / min and stir at a rate of 500 r / min until all solution C has been added to solution D. Continue stirring for 60 min, centrifuge at 10000 r / min for 30 min and discard the supernatant to obtain a composite condensed phase, wherein the mass ratio of mussel adhesive protein to material B is 4:1. 4) The composite condensed phase was homogenized and pulverized into uniform gel particles of 10μm~500μm at a frequency of 15Hz using a homogenizer for 30min. Then, a crosslinking agent (DCC) was added and allowed to stand at room temperature for 48h to crosslink. The residual crosslinking agent in the crosslinked product was removed to obtain a gel. The mass ratio of crosslinking agent to composite condensed phase was 1:5. 5) Add the obtained gel to the biological lubricant (sodium alginate with a mass fraction of 3 wt%) and the analgesic drug (tetracaine hydrochloride with a mass concentration of 1 wt%) and mix evenly. After sterilization, the mussel adhesive protein composite gel of this Example 3 is obtained. The mass ratio of the gel obtained in step 4) to the biological lubricant and the analgesic drug is 100:3:5. Example 4

[0037] The present invention discloses a method for preparing a mussel adhesive protein composite gel, comprising the following steps: 1) Aspartic acid, L-proline, and PN were dissolved in purified water at pH 7 to obtain a bioactive solution. The bioactive solution contained 0.1 wt% aspartic acid, 0.1 wt% L-proline, and 0.5 wt% PN. The bioactive solution was then divided into bioactive solution A and bioactive solution B at a volume ratio of 1:6. 2) Mfp-2 type mussel adhesive protein and material B (polylysine-polyaspartic acid copolymer and polylysine-polyglutamic acid copolymer) were dissolved in bioactive solution A and bioactive solution B, respectively, to obtain solution C and solution D. In solution C, the mass ratio of aspartic acid, L-proline, PN and Mfp-2 type mussel adhesive protein was 1:1:5:50; in solution D, the mass ratio of aspartic acid, L-proline, PN and polylysine-polyaspartic acid copolymer and polylysine-polyglutamic acid copolymer was 3:3:15:5:5. 3) Add solution C dropwise to solution D at a rate of 3 mL / min and stir at a rate of 300 r / min until all solution C has been added to solution D. Continue stirring for 50 min, centrifuge at 4000 r / min for 15 min and discard the supernatant to obtain a composite condensed phase, wherein the mass ratio of mussel adhesive protein to material B is 5:2. 4) The composite condensed phase was homogenized and pulverized into uniform gel particles of 10μm~500μm at a frequency of 10Hz using a homogenizer for 15min. Then, a crosslinking agent (CMC) was added and allowed to stand at room temperature for 10h to crosslink. The residual crosslinking agent in the crosslinked product was removed to obtain a gel. The mass ratio of crosslinking agent to composite condensed phase was 3:5. 5) Add biological lubricant (1 wt% decellularized ECM and sodium hyaluronate) and analgesic (0.5 wt% lidocaine hydrochloride) to the obtained gel and mix evenly. After sterilization, the mussel adhesive protein composite gel of this Example 4 is obtained, wherein the mass ratio of the gel obtained in step 4) to the biological lubricant and analgesic is 1000:20:5.

[0038] Comparative Example 1: The only difference between Comparative Example 1 and Example 1 is that the mass ratio of crosslinking agent to composite condensed phase is 6:1, resulting in a composite gel. Everything else is the same as in Example 1.

[0039] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that in step (3), solution C is directly mixed with solution D to obtain a composite gel. Everything else is the same as in Example 1.

[0040] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that in step (2), the mass ratio of elastin, collagen and Mfp-1 mussel adhesive protein in solution C is 1:1:60; the mass ratio of mussel adhesive protein to material B is 6:1, and a composite gel is finally obtained. All other aspects are the same as in Example 1.

[0041] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that in step (2), the mass ratio of elastin, collagen, and Mfp-1 type mussel adhesive protein in solution C is 1:1:10; the mass ratio of mussel adhesive protein to material B is 1:100, and a composite gel is finally obtained. The SEM image of the composite gel is shown below. Figure 1 As shown, everything else is the same as in Example 1.

[0042] Comparative Example 5: The difference between Comparative Example 5 and Example 1 is that in step (4), the composite condensed phase is directly added to the crosslinking agent (EDC / NHS), and crosslinked at room temperature for 24 hours to remove residual crosslinking agent from the crosslinked product, thus obtaining a gel. Finally, a composite gel is obtained, and the SEM image of the composite gel is shown below. Figure 1 As shown, everything else is the same as in Example 1.

[0043] The composite gels of Examples 1-4 and Comparative Examples 1-5 were subjected to dynamic rheological tests, in vitro enzymatic degradation performance tests, adhesion strength tests, in vitro release performance determination, cytotoxicity experiments, swelling rate tests, and rat subcutaneous implantation experiments. The specific tests are as follows: (1) Dynamic rheological testing Dynamic rheological tests were performed on the composite gels prepared in Examples 1-4 and Comparative Examples 1-5 using a rheometer. A parallel plate clamp with a diameter of 20 mm was used. The sample was placed in the center of the plate, and the upper clamp was slowly adjusted to the set gap. Excess sample at the edges was scraped off, and an anti-evaporation cover was applied to prevent moisture loss. Dynamic oscillating shear tests were conducted at 25°C and a shear strain of 1% at a frequency of 5 Hz. The storage modulus (G') and loss modulus (G'') of the samples were recorded, and the results are shown in Table 1.

[0044] Table 1. Energy storage modulus (G') and loss modulus (G'') data for Examples 1-4 and Comparative Examples 1-5

[0045] Table 1 shows the rheological property test results. The storage modulus of Examples 1-4 ranges from 2694 to 5687 Pa, and the loss tangent is between 0.20 and 0.25. All samples exhibit an elastic-dominant characteristic where G' is significantly higher than G''. These results indicate that the mussel adhesive protein composite gels prepared in Examples 1-4 possess moderate mechanical strength (G' = 2694-5687 Pa) and suitable viscoelastic balance (tanδ = 0.20-0.25). This characteristic is beneficial for the material to maintain morphological stability after injection, while also possessing a certain degree of deformation compliance to adapt to tissue movement.

[0046] Comparative Example 1 (with excessive crosslinking agent) had a storage modulus as high as 8961 Pa, which was 146% higher than that of Example 1, and the tanδ decreased to 0.09. This result indicates that excessive crosslinking agent leads to over-crosslinking, resulting in excessively rigid gel, which is not conducive to mechanical matching with soft tissue and may affect injection performance.

[0047] The energy storage modulus of Comparative Example 2 (direct mixing) decreased by 59% compared to Example 1, and the tanδ was 0.27. This indicates that the direct mixing process used in Comparative Example 2 may lead to uneven electrostatic bonding, insufficient mechanical support strength of the prepared gel material, and decreased morphological stability.

[0048] Comparative Example 3 (with excessive addition of mussel adhesive protein) had a storage modulus of 1397 Pa, which was 62% lower than that of Example 1, and the tanδ increased to 0.38. This indicates that the mechanical strength of the gel product prepared by Comparative Example 3 was significantly reduced, the elastic response was weakened, the viscosity characteristics were relatively enhanced (tanδ increased), and the overall structural strength was reduced. In applications, it may have problems such as insufficient support and poor morphological stability.

[0049] Comparative Example 4 (with a low proportion of mussel adhesive protein) has a storage modulus similar to that of Example 2, but its tanδ is 0.3, indicating that the gel network structure prepared in Comparative Example 4 is loose and highly viscous, and may have poor volume stability after use.

[0050] Comparative Example 5 did not homogenize and break down the composite condensed phase in step (4). Its storage modulus was 2841 Pa, which was 22% lower than that of Example 1; its tanδ was 0.29, which was 16% higher than that of Example 1. This indicates that the lack of homogenization treatment led to an increase in gel particle size and a decrease in mesh continuity. The prepared gel product may have certain effects on injectability, mechanical support, morphological stability and long-term filling effect, making it difficult to meet the comprehensive requirements of clinical applications for material performance.

[0051] (2) In vitro enzyme degradation performance test Weigh 1g of the composite gels prepared in Examples 1-4 and Comparative Examples 1-5, place them in a centrifuge tube, add 3mL of protease solution with a concentration of 20U / mL, shake thoroughly to mix, and incubate in a constant temperature shaker at 37℃ and 150r / min for 0.5, 1, 2, 4, 7, 14, and 28 days, respectively. After incubation, heat the sample at 100℃ for 10min to terminate the enzymatic reaction.

[0052] After the reaction was terminated, the sample was centrifuged, the supernatant was discarded, and the residual moisture on the inner wall of the centrifuge tube was gently blotted dry with filter paper. The wet weight of the gel after enzymatic hydrolysis and the total weight of the centrifuge tube were accurately weighed. 1g of the same batch of mussel adhesive protein composite gel was taken as a blank control group. The centrifuge tubes were placed open in an oven to dry to constant weight. The dried gel samples were collected, and the dry weight of the gel was accurately weighed. Simultaneously, the weight of the empty centrifuge tubes was accurately weighed for mass calibration. The data are shown in Table 2.

[0053] , In the formula: W t — Total mass of gel and centrifuge tube after enzymatic hydrolysis (g); W tube — Mass of empty centrifuge tubes (g); W dry — Initial dry weight (g) of gel in the blank control group.

[0054] (3) Adhesion strength test Fresh pig skin was collected, fascia and subcutaneous fat were removed, and the sample was rinsed clean with physiological saline. A rectangular sample measuring 2cm × 5cm was prepared and placed in a 37℃ constant temperature incubator for 30 minutes to maintain humidity. One end of the tissue sample was then flattened and fixed to the lower clamp of the dissection jig using medical adhesive, ensuring the tissue was free of wrinkles and tension. 0.5g of pre-filled test sample (the composite gel prepared in Examples 1-4 and Comparative Examples 1-5) was evenly applied to the marked area (1cm × 2cm) on the sample surface, ensuring clear boundaries and uniform thickness. The sample was allowed to stand at room temperature for 5 minutes to allow the filler to fully adhere to the tissue interface. Subsequently, medical adhesive was used to fix the surface of the test sample to the upper clamp of the jig, ensuring the adhesive only acted on the surface of the test sample and avoiding penetration into the tissue-test sample interface.

[0055] A 180° peel test was conducted using a universal testing machine. The tensile rate was set to 5 mm / min, and the load-displacement curves were continuously recorded during the peel process until the test sample was completely separated from the pigskin tissue. Fresh tissue samples were used for each group of experiments, and the tests were performed in parallel for 5 times. The average value was taken, and the adhesion strength was calculated based on the interface width. The data are shown in Table 2.

[0056] The formula for calculating adhesion strength is as follows: , In the formula: F max The maximum load (N) during the peeling process; A represents the actual adhesion area (m²) between the test sample and the tissue.

[0057] (4) In vitro release performance determination A standard curve for the release components was constructed using the Coomassie Brilliant Blue method. A 1 mg / mL standard stock solution of the release components was prepared, serially diluted, and then Coomassie Brilliant Blue chromogenic solution was added. The absorbance was measured at 595 nm to establish a concentration-absorbance standard curve. Mussel adhesive protein composite gels were placed in 5 mL of PBS (pH 7.4) and incubated at 37°C with constant shaking at 50 rpm. 1 mL samples were taken at 6, 12, 24, 72, 120, and 168 h, and the same volume of fresh buffer was added. The absorbance of the samples (composite gels prepared in Examples 1-4 and Comparative Examples 1-5) was measured at 595 nm after chromogenic development. The release concentration was calculated by substituting the values ​​into the standard curve, and the cumulative release rate was calculated using the volume correction method. The release curve was then plotted. The cumulative release rate data are shown in Table 2.

[0058] The cumulative release rate of the released components was determined using the following formula: , , In the formula M t The cumulative release amount (mg) at time point t; The initial total mass (mg) of the released components in the M0 mussel adhesive protein composite gel sample. C t The mass concentration (mg / mL) of the released component in the released solution taken at time point t. V0 Initial volume (mL) of the in vitro release system; C i The mass concentration (mg / mL) of the released component in the release solution during the i-th sampling. V S Volume (mL) of each sample taken and buffer replenishment.

[0059] (5) Cytotoxicity test L929 cells were cultured in H-DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2, and saturated humidity. When cell confluence reached 80%–90%, the cells were digested with trypsin and the concentration was adjusted to 2 × 10⁻⁶. 5 cells / mL available for later use.

[0060] Cells were seeded at 1×10³ cells / well in 96-well plates and cultured for 24 h. Then, serially diluted extracts of the composite gels prepared in Examples 1-4 and Comparative Examples 1-5 were added, with complete culture medium as a blank control. Cells were cultured for 24, 48, and 72 h, respectively.

[0061] At each time point, discard the culture medium, add 50 μL MTT solution to each well, and incubate at 37°C for 2 h; discard the supernatant, add 100 μL isopropanol to fully dissolve the crystals. Measure the absorbance at 570 nm using a microplate reader, and calculate the relative cell proliferation rate. The relative cell proliferation rate is shown in Table 2.

[0062] Relative cell growth rate (RGR) is calculated using the following formula: .

[0063] (6) Swelling rate test Take a 500-mesh stainless steel sieve (8cm×8cm) and dry it at 80℃ to constant weight. Record the weight as m0. Accurately weigh 0.2-0.5g of the composite gels prepared in Examples 1-4 and Comparative Examples 1-5, place them on the sieve, put the sieve into an evaporating dish, and add 30mL of 0.9% sodium chloride solution at once to completely wet the gel. Let it stand and swell for at least 30min. After swelling, remove the sieve, blot the free liquid at the bottom and edges of the sieve with filter paper until there are no wet marks on the filter paper, and weigh it immediately. Record the weight as m1. Then dry the sieve at 80℃ to constant weight and record the weight as m2. The swelling rate data are shown in Table 2.

[0064] , m0 — Constant weight of empty sieve (g) m1 — Wet weight (g) of swollen gel + sieve m2 — Constant weight (g) of gel after drying and sieve.

[0065] (7) Subcutaneous implantation experiment in rats Forty-four rats were selected and divided into 11 groups: a blank control group and an experimental group (each group was injected with the composite gel prepared in Examples 1-4 and Comparative Examples 1-5, respectively). Each group contained four rats. The hair on both sides of the spine of the experimental rats was removed, and the injection area was thoroughly disinfected with povidone-iodine. A 5cm × 6cm injection area was marked using a tattooing method. Using a 27G needle, 0.5mL of the test sample was injected subcutaneously into the marked area, with a minimum spacing of 1cm between injection points. After injection, the injection site was disinfected again with povidone-iodine and bandaged with gauze.

[0066] Rats were euthanized by CO2 inhalation at 7, 14, and 28 days post-injection. A longitudinal incision was made along the rat's spine, and the skin was dissected layer by layer to fully expose the implantation site. The implanted sample and surrounding tissues were harvested, and tissue blocks were excised along a pre-defined dotted line. The tissue blocks were blotted dry with filter paper and immediately fixed in 10% neutral buffered formalin solution. Subsequently, the tissue blocks were dehydrated with graded alcohols, embedded in paraffin, and prepared for HE sectioning. Microscopic morphology was observed and evaluated using an optical microscope. The results are shown in Table 2. Figure 2 .

[0067] Table 2 Summary of Performance Test Data of Examples 1-4 and Comparative Examples 1-5

[0068] Based on the comprehensive performance test results, Examples 1-4 exhibited good overall performance across all indicators: degradation residue rate of 53%-88%, adhesion strength of 33-61 kPa, cumulative release rate of 32%-67% over 168 hours, relative cell proliferation rate of 98%-105%, swelling rate of 20%-32%, and no obvious inflammatory response observed histologically. This indicates that Examples 1-4 possess suitable anti-degradation ability, good tissue adhesion, controllable drug release behavior, excellent cell compatibility, and moderate swelling properties.

[0069] Comparative Example 1 suffered from excessive cross-linking due to an over-excessive cross-linking agent, resulting in an adhesion strength of only 3 kPa and a swelling rate of only 13%. Histological observation also revealed partial inflammatory cell infiltration. Combined with the aforementioned rheological results, its storage modulus was as high as 8961 Pa, and its tanδ was 0.09. These data all demonstrate that the gel prepared in Comparative Example 1 is too rigid and cannot effectively achieve tissue fixation, drug sustained release, and morphological adaptation as a filler material, and it also poses an inflammatory risk.

[0070] Comparative Example 2 suffered from an uneven network structure due to the direct mixing process. Combined with its rheological results, its storage modulus was 1503 Pa, tanδ was 0.27, and its degradation residue rate (37%) and adhesion strength (22 kPa) were lower than those of the Example Group. However, its cumulative release rate (79%) and swelling rate (38%) were higher than those of the Example Group. This indicates that Comparative Example 2 has structural defects such as local protein aggregation and uneven cross-linking density, which leads to insufficient adhesion strength, changes in drug release behavior, and increased swelling rate. It cannot meet the comprehensive requirements for mechanical support, morphological maintenance, and long-term release.

[0071] Comparative Example 3 showed that the high proportion of mussel agglutinin caused self-aggregation. Rheological results showed that its storage modulus was 1387 Pa (62% lower than Example 1) and the loss tangent was 0.38 (52% higher than Example 1), indicating that the network structure was destroyed, the mechanical strength decreased, and the viscosity increased. The degradation residue rate (69%) was similar to that of Example 1, the swelling rate (24%) was slightly lower than that of Example 1, and the adhesion strength was only 18 kPa, which was significantly lower than that of the Example group. This indicates that although the protein self-aggregation formed a certain hydrophobic region, the destruction of the electrostatic network and the decrease in electrostatic binding efficiency led to the deterioration of the overall mechanical properties of the material, and it was impossible to achieve effective tissue fixation.

[0072] Comparative Example 4 has extremely low mussel adhesive protein content and its gel structure is dominated by sodium hyaluronate. Rheological results show that its storage modulus is 2647 Pa, but its tanδ is 0.30. Combined with the degradation residue rate of 22%, adhesion strength of only 21 kPa, cumulative release rate (93%), and swelling rate (103%) of Comparative Example 4, it is indicated that the network of Comparative Example 4 is loose and lacks protein hydrophobic balance, resulting in excessively rapid drug release and excessive swelling, which cannot meet the requirements of long-term sustained release and morphology maintenance.

[0073] Comparative Example 5, lacking homogenization treatment, exhibited increased swelling ratio and decreased adhesion strength, as indicated by rheological results and these findings. This suggests that the gel particles prepared in Comparative Example 5 were coarse, with reduced network continuity, slightly faster drug release, larger volume changes after implantation, and unstable tissue adhesion, making it difficult to meet the comprehensive performance requirements for clinical applications. These results indicate that a suitable mass ratio of mussel adhesive protein to material B, an appropriate amount of cross-linking agent, and synergistic effects of the process are key conditions for obtaining gel products with both good physicochemical properties and biocompatibility, meeting the needs of tissue filling and support applications.

[0074] The invention has been described through the specific embodiments described above. Those skilled in the art should understand that various modifications and equivalent substitutions can be made to the invention without departing from its scope. Parts not described in detail in this specification are well-known to those skilled in the art. Furthermore, various modifications can be made to the invention for specific situations or circumstances without departing from the scope of this application. Therefore, the invention is not limited to the specific embodiments disclosed, but should include all embodiments falling within the scope of the claims.

Claims

1. A method for preparing a mussel adhesive protein composite gel, characterized in that, Includes the following steps: 1) Dissolve the bioactive ingredient in a solvent to obtain a bioactive solution with a mass concentration of 0.1wt%~1wt%, and then divide the bioactive solution into bioactive solution A and bioactive solution B according to a volume ratio of 1:1~1:10; 2) Dissolve mussel adhesive protein and material B in bioactive solution A and bioactive solution B respectively to obtain solution C and solution D; in solution C, the mass ratio of bioactive component to mussel adhesive protein is 1:4 to 1:30; in solution D, the mass ratio of bioactive component to material B is 1:1 to 1:2; material B is a polyanionic material or an amphiphilic polyelectrolyte material; 3) Add solution C dropwise to solution D, stir, centrifuge, and discard the supernatant to obtain a composite condensed phase; 4) After homogenizing and crushing the composite condensed phase, a crosslinking agent is added, and the mixture is allowed to stand at room temperature for crosslinking to obtain a gel; 5) Add biological lubricant and analgesic drug to the gel, and sterilize to obtain the mussel adhesive protein composite gel.

2. The method for preparing the mussel adhesive protein composite gel according to claim 1, characterized in that, In step 1), the solvent is any one of purified water, phosphate buffer, and acetate buffer with a pH of 6-8; The bioactive component is any one or a combination of elastin, collagen, dipotassium glycyrrhizate, polydeoxyribonucleotide (PDRN), polynucleotide (PN), L-proline, glycine, lysine, and aspartic acid.

3. The method for preparing the mussel adhesive protein composite gel according to claim 1, characterized in that, In step 3), solution C is added dropwise to solution D at a rate of 0.01 mL / min to 5 mL / min; the stirring rate is 100 r / min to 500 r / min; after all solution C has been added to solution D, stirring continues for 30 min to 60 min, followed by centrifugation; the centrifugation rate is 2000 r / min to 10000 r / min, and the centrifugation time is 10 min to 30 min.

4. The method for preparing the mussel adhesive protein composite gel according to claim 1, characterized in that, The specific process of step 4) is as follows: the composite condensed phase is homogenized and pulverized into gel particles of 10μm~500μm at a frequency of 0.1Hz~15Hz using a homogenizer for 10min~30min; then a crosslinking agent is added, and the mixture is allowed to stand at room temperature for 30min~2880min for crosslinking, and then the residual crosslinking agent is removed to obtain a gel; in the gel, the mass ratio of crosslinking agent to composite condensed phase is 0.2:1~1:

2.

5. The method for preparing the mussel adhesive protein composite gel according to claim 1, characterized in that, In step 2), material B is a polyanionic material, which is any one or a combination of hyaluronic acid salt, polyglutamate, polyaspartate, alginate, chondroitin sulfate, heparin salt, and heparin sulfate.

6. The method for preparing the mussel adhesive protein composite gel according to claim 1, characterized in that, In step 2), material B is an amphiphilic polyelectrolyte material, which is any one or a combination of hydroxyethyl chitosan, polylysine-polyglutamic acid copolymer, polylysine-polyaspartic acid copolymer, amphiphilic modified gelatin, amphiphilic modified collagen, and amphiphilic modified decellularized ECM material.

7. The method for preparing mussel adhesive protein composite gel according to claim 1, characterized in that, The crosslinking agent is any one of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide (EDC / NHS), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1,1'-carbonyldiimidazole (CDI), and N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate (CMC). The bio-lubricating material is any one or a combination of hyaluronic acid salt, carboxymethyl cellulose, hydroxyethyl cellulose, alginate, chondroitin sulfate, hydroxyethyl chitosan, and decellularized ECM material; The analgesic drugs include any one or a combination of lidocaine hydrochloride, bupivacaine hydrochloride, procaine hydrochloride, and tetracaine hydrochloride.

8. A mussel adhesive protein composite gel, characterized in that, The mussel adhesive protein composite gel is prepared by the preparation method according to any one of claims 1 to 7, and includes mussel adhesive protein, bioactive components, material B, biolubricating material, crosslinking agent, analgesic drug and solvent; The mass ratio of mussel adhesive protein to material B is 3:2~4:1; The bio-lubricating material has a mass fraction of 0.01 wt% to 3 wt%. The analgesic drug has a mass fraction of 0.005 wt% to 1 wt%. Material B is a polyanionic material or an amphiphilic polyelectrolyte material.

9. The use of a mussel adhesive protein composite gel, characterized in that, The mussel adhesive protein composite gel is the mussel adhesive protein composite gel according to claim 8, and the mussel adhesive protein composite gel is used as a skin filler material for injection.

10. The use of the mussel adhesive protein composite gel according to claim 9, characterized in that, The skin filler material is used for filling, supporting, and shaping the middle layer of the dermis, the subcutaneous layer, or the upper layer of the periosteum.

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