Method for reducing immunogenicity of animal-derived collagen implant based on electrostatic self-assembly and application

Abstract

The invention provides an animal-derived collagen implant immunogenicity reduction method based on electrostatic self-assembly, and belongs to the field of biomedical materials.The animal-derived collagen implant immunogenicity reduction method comprises the following steps that animal-derived collagen is pretreated; dissolving the pretreated animal source collagen in an acid solution to obtain a collagen solution; adding a modified molecule solution with opposite charges into the collagen solution, and carrying out self-assembly reaction under the electrostatic action; and separating, washing and drying the self-assembled product to obtain the animal-derived collagen implant with reduced immunogenicity. Impurities and non-immunogenic components in the collagen are removed through systematic pretreatment, electrostatic self-assembly and post-treatment processes, modification molecules and the collagen are tightly combined through electrostatic interaction, the surface structure of the collagen is changed, antigen epitopes are shielded, the immunogenicity of the collagen is remarkably reduced through the synergistic effect of a plurality of links, and the immunogenicity of the collagen is remarkably improved. The immunological rejection of a human body on the implant is effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a method and application for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly. Background Technology

[0002] In the field of biomedical materials, animal-derived collagen is widely used in tissue repair and medical device coatings due to its excellent biocompatibility, biodegradability, and biomimetic structure. However, its naturally occurring immunogenicity (such as telopeptides and antigenic epitopes in the triple helix structure) can easily trigger immune rejection in the human body, leading to implant material failure or inflammatory reactions. Existing methods for reducing immunogenicity (such as chemical cross-linking, enzymatic hydrolysis, and physical modification) have many drawbacks: chemical cross-linking may introduce toxic residues, enzymatic hydrolysis easily damages the three-dimensional structure of collagen, and physical modification is difficult to achieve a deep reduction in immunogenicity. Therefore, developing a method that can efficiently reduce immunogenicity while preserving the natural properties of collagen has become a key technical challenge in this field.

[0003] To address this, a method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly is proposed. Summary of the Invention

[0004] The present invention aims to solve the problems mentioned in the background art by providing a method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly.

[0005] The specific technical solution is as follows:

[0006] A method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly includes the following steps:

[0007] Animal-derived collagen is pretreated to remove impurities and non-collagen components;

[0008] Pretreated animal-derived collagen was dissolved in an acidic solution to obtain a collagen solution.

[0009] A solution of modifying molecules with opposite charges is added to the collagen solution, and a self-assembly reaction is carried out under electrostatic action. The modifying molecules are one of alginate, succinylated dextran, and hyaluronic acid. The self-assembly product is separated, washed, and dried to obtain animal-derived collagen implants with reduced immunogenicity.

[0010] The above-mentioned method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly includes the following steps for pretreating the animal-derived collagen: immersing the animal-derived collagen raw material in a trypsin solution with a mass fraction of 0.1% to 1%, enzymatically hydrolyzing it at 30-37°C for 1 to 3 hours, and then removing precipitated impurities by centrifugation.

[0011] In the above-mentioned method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, the acidic solution is an acetic acid solution with a mass fraction of 0.5% to 2%, and the concentration of the collagen solution is 1 to 5 mg / mL.

[0012] In the above-mentioned method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, the concentration of the modified molecular solution is 0.5-3 mg / mL, and the volume ratio of the collagen solution to the modified molecular solution is (2-5):1.

[0013] The above-mentioned method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, wherein the conditions for the self-assembly reaction are: temperature 20-30℃, pH value 4-6, reaction time 2-6 hours, and continuous stirring during the reaction at a stirring speed of 100-300 rpm.

[0014] The above-mentioned method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, wherein the method for separating the self-assembly products is as follows: using ultracentrifugation, centrifuging at a speed of 10,000-15,000 rpm for 15-30 minutes, and collecting the precipitate.

[0015] The above-mentioned method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly includes the following steps for washing the self-assembled product: repeatedly washing the precipitate with deionized water 3 to 5 times, centrifuging at 8000-12000 rpm for 10 to 20 minutes after each wash, and removing the supernatant.

[0016] The above-mentioned method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, wherein the drying process is freeze-drying, in which the washed precipitate is pre-frozen at -50 to -30°C for 2 to 4 hours, and then sublimated and dried under a vacuum of 10 to 50 Pa for 12 to 24 hours.

[0017] This invention also provides the application of animal-derived collagen implants with reduced immunogenicity in the preparation of tissue repair materials.

[0018] This invention also provides the application of animal-derived collagen implants with reduced immunogenicity in the preparation of coating materials for medical devices.

[0019] The present invention has the following beneficial effects:

[0020] 1. Highly effective reduction of immunogenicity: Through systematic pretreatment, electrostatic self-assembly and post-treatment processes, impurities and non-immunogenic components in collagen are removed. Electrostatic action is used to make the modified molecules bind tightly to the collagen, change its surface structure, and mask the antigen epitopes. Through the synergistic effect of multiple links, the immunogenicity of collagen is significantly reduced, effectively reducing the immune rejection response of the human body to the graft.

[0021] 2. Ensuring material performance: By using a specific acetic acid solution to dissolve collagen and precisely controlling the self-assembly reaction conditions, the natural structure and functional properties of collagen can be maintained while reducing immunogenicity. At the same time, by introducing modified molecules, additional properties such as good hydrophilicity and mechanical strength can be endowed to the implant, thereby improving the overall quality of the material.

[0022] 3. Improve product purity: From enzymatic hydrolysis and centrifugation to remove impurities during raw material pretreatment, to ultracentrifugation, multiple washing and freeze-drying of self-assembled products, multiple purification processes gradually remove impurities and unreacted substances, greatly improving the purity of the implants, avoiding immune reactions caused by residual substances, and ensuring product quality and safety.

[0023] 4. Strong process controllability: The parameters of each step are clear and adjustable. By adjusting the type of modifying molecules, solution concentration, reaction conditions, etc., the performance of the implant can be flexibly controlled to achieve diversified product customization and meet the needs of different application scenarios such as tissue repair and medical device coating.

[0024] 5. Expanding Application Prospects: Animal-derived collagen grafts with reduced immunogenicity and superior performance after processing can better integrate with human tissues in the field of tissue repair, promoting repair and regeneration; in the field of medical device coatings, they can improve the biocompatibility of devices and reduce the risk of complications, significantly expanding the application scope and value of animal-derived collagen in the biomedical field. Attached Figure Description

[0025] Figure 1 A flowchart of a method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, provided in an embodiment of the present invention.

[0026] Figure 2 An image showing the immunogenicity reduction effect of an animal-derived collagen implant based on electrostatic self-assembly provided in an embodiment of the present invention.

[0027] Figure 3 A biocompatibility evaluation diagram of an animal-derived collagen implant based on electrostatic self-assembly provided in an embodiment of the present invention;

[0028] Figure 4 A diagram illustrating the improved material properties of an animal-derived collagen implant based on electrostatic self-assembly, as provided in an embodiment of the present invention.

[0029] Figure 5 A diagram illustrating the improved hydrophilicity of an animal-derived collagen implant based on electrostatic self-assembly, provided in an embodiment of the present invention.

[0030] Figure 6 This is a stability test diagram of an animal-derived collagen implant based on electrostatic self-assembly, provided in an embodiment of the present invention. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual images. They should not be construed as limiting the scope of this patent. To better illustrate the embodiments of the present invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0033] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "inner," and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0034] In the description of this invention, unless otherwise explicitly specified and limited, the term "connection" or similar designation indicating a connection between components should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral part; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can refer to the internal communication between two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0035] This specific embodiment provides a method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, such as... Figures 1-6 As shown, it includes the following steps:

[0036] S1: Pre-treat animal-derived collagen to remove impurities and non-collagen components;

[0037] S2: Dissolve the pretreated animal-derived collagen in an acidic solution to obtain a collagen solution;

[0038] S3: Add a solution of modified molecules with opposite charges to the collagen solution, and a self-assembly reaction occurs under electrostatic interaction. The modified molecules are one of alginate, succinylated dextran, and hyaluronic acid. The self-assembly reaction satisfies the following electrostatic self-assembly strength equation:

[0039]

[0040] In the formula:

[0041] S e The electrostatic self-assembly strength (dimensionless) is used to quantify the degree of reaction and the potential for immunogenicity reduction. When S e When the value is ≥0.7, the immunogenicity reduction effect is significant;

[0042] C c The concentration of collagen solution (mg / mL) ranges from 1 to 5 mg / mL.

[0043] C m The concentration of the modified molecule solution (mg / mL) ranges from 0.5 to 3 mg / mL;

[0044] R v The volume ratio of collagen solution to modified molecule solution ranges from 2:1 to 5:1 (i.e., R...). v =V c / V m V c and V m (These are the volumes of the collagen solution and the modified molecule solution, respectively);

[0045] t represents the reaction time (in hours), ranging from 2 to 6 hours;

[0046] pH refers to the pH value of the reaction system, which ranges from 4 to 6.

[0047] T represents the reaction temperature (°C), ranging from 20 to 30°C.

[0048] pH0 is the optimal pH reference value, fixed at 5.0 (based on the charge complementarity of collagen and modified molecules);

[0049] γ is the pH sensitivity coefficient, fixed at 0.2 (determined experimentally to reflect the effect of pH deviation from the optimal value);

[0050] α is a nonlinear exponent, fixed at 0.25 (determined experimentally to capture the synergistic effect of the parameters);

[0051] k is a proportionality constant, fixed at 1.2 (calibrated experimentally to standardize the output);

[0052] S4: The self-assembled product is separated, washed, and dried to obtain animal-derived collagen implants with reduced immunogenicity.

[0053] Pre-treating animal-derived collagen to remove impurities and non-collagen components can reduce immune responses triggered by unnecessary substances. Dissolving collagen in an acidic solution and subjecting it to electrostatic self-assembly with a solution of modifier molecules of opposite charge utilizes electrostatic interactions to bind the modifier molecules to the collagen, altering the collagen's surface structure and effectively reducing its immunogenicity. Subsequent separation, washing, and drying processes further purify the product, ensuring that the final animal-derived collagen implants have reduced immunogenicity and are suitable for the biomedical field.

[0054] The pretreatment steps for animal-derived collagen include: soaking the animal-derived collagen raw material in a trypsin solution with a mass fraction of 0.1% to 1%, enzymatically hydrolyzing it at 30-37°C for 1 to 3 hours, and then removing precipitated impurities by centrifugation.

[0055] Animal-derived collagen raw materials are pretreated with trypsin solution. Trypsin can specifically decompose impurities and non-collagen components, and centrifugation can accurately remove precipitated impurities, ensuring the purity of collagen raw materials. This provides a purer foundation for subsequent immunogenicity reduction operations, allowing subsequent self-assembly and other steps to better act on collagen and improve the immunogenicity reduction effect.

[0056] The acidic solution is an acetic acid solution with a mass fraction of 0.5% to 2%, and the collagen solution has a concentration of 1 to 5 mg / mL.

[0057] By selecting a specific acetic acid solution as the acidic solution for dissolving collagen, the acetic acid solution can fully dissolve collagen under suitable conditions, forming a stable collagen solution. This provides a uniform reaction system for the subsequent self-assembly reaction with the modified molecules, which helps the modified molecules to fully bind with collagen, thereby effectively reducing immunogenicity. The appropriate collagen solution concentration ensures that collagen molecules have adequate space and concentration for self-assembly, improving reaction efficiency and reducing immunogenicity.

[0058] The concentration of the modified molecule solution is 0.5–3 mg / mL, and the volume ratio of collagen solution to modified molecule solution is (2–5):1.

[0059] By setting appropriate concentrations of the modified molecule solution and the volume ratio of collagen solution to modified molecule solution, it is possible to ensure that the modified molecules and collagen molecules combine in an appropriate ratio during the electrostatic self-assembly reaction. This avoids both waste and potential adverse effects caused by excessive modified molecules and insufficient modified molecules that fail to effectively reduce immunogenicity. The self-assembly reaction is carried out at the optimal ratio, resulting in a significant reduction in immunogenicity.

[0060] The conditions for the self-assembly reaction are: temperature 20-30℃, pH value 4-6, reaction time 2-6 hours, and continuous stirring during the reaction at a speed of 100-300 rpm.

[0061] By limiting the temperature, pH, reaction time, and stirring speed of the self-assembly reaction, suitable temperature and pH provide an appropriate environment for the electrostatic self-assembly reaction, ensuring that the modified molecules and collagen can bind in the optimal chemical environment; reasonable reaction time ensures that the reaction proceeds fully, allowing as many modified molecules as possible to bind to the collagen surface to reduce immunogenicity; continuous stirring ensures that the reaction system is uniform, avoiding insufficient or excessive local reactions, so that the collagen in the entire reaction system can effectively self-assemble, improving the uniformity and effectiveness of reducing immunogenicity.

[0062] The method for separating the self-assembled products is as follows: use ultracentrifugation, centrifuge at 10,000-15,000 rpm for 15-30 minutes, and collect the precipitate.

[0063] Ultracentrifugation is used to separate self-assembled products. The powerful centrifugal force generated by ultracentrifugation can effectively separate the larger particles formed by self-assembly from the solution, accurately collect the precipitate, which is the desired self-assembled product, remove excess solution and unreacted small molecules, improve the purity of the product, provide a purer basis for subsequent processing, and help to further reduce immunogenicity and ensure product quality.

[0064] The washing steps for the self-assembled product are as follows: the precipitate is repeatedly washed with deionized water 3-5 times, and after each wash, it is centrifuged at 8000-12000 rpm for 10-20 minutes to remove the supernatant. Repeated washing of the self-assembled product with deionized water and centrifugation to remove the supernatant effectively removes residual impurities, unreacted modified molecules, and other small molecules from the product surface, further improving product purity. Multiple washing and centrifugation operations more thoroughly remove impurities, preventing these residual substances from triggering an immune response, thus better ensuring the immunogenicity reduction effect of the animal-derived collagen implants.

[0065] The drying process involves freeze drying, in which the washed precipitate is pre-frozen at -50 to -30°C for 2 to 4 hours, and then sublimated and dried under a vacuum of 10 to 50 Pa for 12 to 24 hours.

[0066] Freeze-drying, through pre-freezing followed by sublimation drying, preserves the structure and properties of self-assembled products to the maximum extent under low temperature and vacuum conditions. This avoids the damage to collagen structure caused by high temperatures and other factors in traditional drying methods, which can affect its performance and immunogenicity. At the same time, freeze-drying effectively removes moisture, bringing the product to a suitable degree of dryness for easy storage and subsequent use. This ensures that animal-derived collagen implants maintain good physical and chemical stability while reducing immunogenicity.

[0067] The immunogenicity-reduced animal-derived collagen grafts prepared using the above-described method based on electrostatic self-assembly for reducing immunogenicity can be applied in the preparation of tissue repair materials. Because the animal-derived collagen grafts prepared using the aforementioned method have reduced immunogenicity, their application in the preparation of tissue repair materials can reduce the body's immune rejection response to the implanted material, allowing the material to better integrate with human tissue, promoting tissue repair and regeneration, improving the success rate and effectiveness of tissue repair, and providing patients with a safer and more effective treatment method.

[0068] The immunogenicity-reduced animal-derived collagen grafts prepared using the above-mentioned method based on electrostatic self-assembly for reducing immunogenicity can be applied in the preparation of coating materials for medical devices. Using immunogenic animal-derived collagen grafts in the preparation of medical device coating materials can reduce the risk of immune reactions when the human body comes into contact with medical devices, improve the biocompatibility of medical devices, reduce complications caused by immune reactions, extend the service life of medical devices, and improve the safety of use, providing more reliable protection for the medical process.

[0069] In addition, this specific implementation also provides the following three embodiments:

[0070] Example 1

[0071] Preparation steps

[0072] 1. Pretreatment of animal-derived collagen: The animal-derived collagen raw material is soaked in a 0.1% trypsin solution and enzymatically hydrolyzed at 30°C for 1 hour. Then, it is centrifuged at 3000 rpm for 10 minutes to remove precipitated impurities.

[0073] 2. Dissolve the pretreated animal-derived collagen in a 0.5% acetic acid solution to prepare a collagen solution with a concentration of 1 mg / mL.

[0074] 3. Prepare a sodium alginate solution (with negatively charged modified molecules) with a concentration of 1.5 mg / mL;

[0075] Mix collagen solution and sodium alginate solution in a volume ratio of 3:1 (e.g., 30 mL collagen solution + 10 mL sodium alginate solution);

[0076] Electrostatic self-assembly reaction was carried out for 4 hours with continuous stirring at 150 rpm under conditions of 20°C and pH 5.0.

[0077] 4. Using ultracentrifugation, centrifuge at 10,000 rpm for 15 minutes, collect the precipitate, wash the precipitate repeatedly with deionized water 3 times, centrifuge at 8,000 rpm for 10 minutes after each wash, remove the supernatant, and finally pre-freeze the washed precipitate at -50℃ for 2 hours, and then sublimate and dry it under a vacuum of 10 Pa for 12 hours to obtain animal-derived collagen implants with reduced immunogenicity.

[0078] Technical effect

[0079] This embodiment achieves effective electrostatic self-assembly of alginate and collagen by precisely controlling the conditions of each step, under mild pretreatment and low concentration solution system. While preserving the original structure and properties of collagen to the greatest extent, it significantly reduces its immunogenicity. Alginate tightly wraps collagen fibers through electrostatic interaction, masking antigenic epitopes (such as the α-chain immunogenic region). ELISA detection shows a significant reduction in antibody binding, and the mild operating conditions are conducive to large-scale production.

[0080] Experimental data

[0081] The immunogenicity of the graft was detected by enzyme-linked immunosorbent assay (ELISA), with an untreated animal-derived collagen graft as a control. The antibody binding amount of the control group was 1.2 OD, while the antibody binding amount of the graft prepared in Example 1 was 0.3 OD, indicating a 75% reduction in immunogenicity. The cell viability was detected by cytotoxicity assay (MTT method), and the cell viability reached 95%, indicating that the graft has good biocompatibility.

[0082] Example 2

[0083] Preparation steps

[0084] 1. Pretreatment of animal-derived collagen: The animal-derived collagen raw material is soaked in a 0.5% trypsin solution and enzymatically hydrolyzed at 35°C for 2 hours. Then, it is centrifuged at 4000 rpm for 15 minutes to separate and remove precipitated impurities.

[0085] 2. Dissolve the pretreated collagen in a 1% acetic acid solution to prepare a collagen solution with a concentration of 3 mg / mL.

[0086] 3. Prepare a succinylated dextran solution with a concentration of 2.0 mg / mL (degree of substitution ≥ 0.8, ensuring high negative charge density) as the modification molecule solution. Mix the collagen solution and the modification molecule solution at a volume ratio of 4:1 (e.g., 40 mL collagen solution + 10 mL Su-Dex solution). Stir the mixture at 200 rpm for 5 hours at 25 °C and pH 5 to complete electrostatic self-assembly.

[0087] 4. Using ultracentrifugation, centrifuge at 12,000 rpm for 20 minutes to collect the precipitate. Wash the precipitate four times with deionized water. After each wash, centrifuge at 10,000 rpm for 15 minutes to remove the supernatant. Then, pre-freeze the precipitate at -40℃ for 3 hours and sublimate and dry it under a vacuum of 30 Pa for 18 hours to obtain animal-derived collagen implants.

[0088] Technical effect

[0089] This embodiment adjusts the pretreatment enzymatic hydrolysis conditions, solution concentration, and reaction parameters to ensure that succinylated dextran fully binds to collagen. The carboxyl groups (-COO-) of succinylated dextran efficiently mask collagen antigen epitopes through electrostatic interaction. ELISA detection shows a significant reduction in antibody binding. While effectively reducing immunogenicity, the dense network structure formed by electrostatic self-assembly enhances the mechanical strength and stability of the graft, improving its overall performance and making it suitable for applications with high material performance requirements.

[0090] Experimental data

[0091] ELISA testing showed that the antibody binding amount of the untreated animal-derived collagen implant was 1.2 OD, while that of the implant in this embodiment was 0.25 OD, indicating a 79.2% reduction in immunogenicity. Tensile testing showed that the tensile strength of the implant reached 15 MPa, which was 40% higher than that of the untreated collagen implant. In the cytotoxicity test, the cell survival rate was 96%, demonstrating good biocompatibility and excellent material properties.

[0092] Example 3

[0093] Preparation steps

[0094] 1. Pretreatment of animal-derived collagen: Soak the raw material in a 1% trypsin solution and enzymatically hydrolyze it at 37°C for 3 hours. Then, centrifuge it at 5000 rpm for 20 minutes to remove precipitates and impurities.

[0095] 2. Dissolve the pretreated collagen in a 2% acetic acid solution to prepare a collagen solution with a concentration of 5 mg / mL.

[0096] 3. Prepare a hyaluronic acid solution with a concentration of 3 mg / mL as the modification molecule solution. Mix the collagen solution and the modification molecule solution at a volume ratio of 5:1. Stir the mixture at 300 rpm for 6 hours at 30℃ and pH 6 to carry out electrostatic self-assembly.

[0097] 4. Use ultracentrifugation to collect the precipitate by centrifuging at 15,000 rpm for 30 minutes. Wash the precipitate 5 times with deionized water. After each wash, centrifuge at 12,000 rpm for 20 minutes to remove the supernatant. Finally, pre-freeze the precipitate at -30℃ for 4 hours and sublimate and dry it under a vacuum of 50 Pa for 24 hours to obtain animal-derived collagen implants.

[0098] Technical effect

[0099] In this embodiment, under conditions of high solution concentration and strong reaction, hyaluronic acid and collagen are fully combined, which significantly reduces immunogenicity while giving the implant good hydrophilicity and moisturizing properties. This is beneficial for the implant to function better in the body and is suitable for applications that come into contact with moist tissues of the human body.

[0100] Experimental data

[0101] ELISA results showed that the antibody binding amount of the untreated implant was 1.2 OD, while that of the implant in this embodiment was 0.2 OD, resulting in an 83.3% reduction in immunogenicity. The contact angle test showed that the contact angle of the implant was 30°, indicating a significant improvement in hydrophilicity. After immersion in simulated body fluid for 72 hours, the mass loss rate of the implant was only 3%, demonstrating good stability. In the cytotoxicity test, the cell survival rate was 97%, indicating excellent biocompatibility.

[0102] It is worth noting that, Figure 1 A flowchart illustrating a method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly is presented. Figure 2 The figure visually compares the effects of the three embodiments in reducing immunogenicity. The percentage reduction in immunogenicity under different embodiments can be clearly seen through the difference in the height of the bars. Figure 3 The toxicity or biocompatibility of the treated collagen grafts to cells was evaluated. Higher cell survival rate indicates better biocompatibility. The differences in cell survival rate under different embodiments can be clearly seen through the bar chart. Figure 4The improved mechanical properties of the treated collagen implants are demonstrated, with the percentage increase in tensile strength reflecting the degree of improvement in material performance. The bar chart clearly shows the performance enhancement effects of different embodiments. Figure 5 The changes in contact angle in the examples demonstrate the improved hydrophilicity of the treated collagen implants. A smaller contact angle indicates better hydrophilicity. The line graph clearly shows the trend of contact angle variation with the examples. Figure 6 The stability of the treated collagen implants in a simulated bodily fluid environment is demonstrated. A lower mass loss rate indicates better stability. The line graph clearly shows the trend of mass loss rate with soaking time.

[0103] The equation for the electrostatic self-assembly strength is explained below:

[0104] This electrostatic self-assembly strength equation integrates key parameters of electrostatic self-assembly (concentration, volume ratio, pH, temperature, and time), and uses an exponential term. The power exponent α captures nonlinear synergistic effects. Where: the exponent term... Quantitative analysis of the effect of pH on charge density (when pH deviates from the optimal value of 5.0, charge complementarity weakens, reducing S...) e The power exponent α = 0.25 indicates that there is a superadditive effect between the parameters, that is, the effect of the parameters working together is better than simple addition. This is due to the physical mechanism of electrostatic self-assembly (such as the synergy of charge matching and antigen epitope masking).

[0105] Taking Example 1 as an example, calculate S e And verify the effect of reducing immunogenicity:

[0106] Parameter values:

[0107] C c =1 mg / mL, C m =0.5mg / mL, R v =2 (volume ratio), t = 2 hours, pH = 4, T = 20℃

[0108] pH0=5.0, γ=0.2, α=0.25, k=1.2.

[0109] Calculation process: Substituting the above parameter values ​​into the electrostatic self-assembly strength equation, S can be calculated. e =0.642.

[0110] Result verification: S e =0.642 < 0.7, but in Example 1, the immunogenicity decreased by 75% (antibody binding decreased from 1.20D to 0.30D), approaching the threshold. The difference stems from the nonlinearity of the equation, which can be optimized in practice by fine-tuning parameters (e.g., increasing t or adjusting pH). In Example 2 (Se ≈0.75, calculated later) and 3 (S e In ≈0.85), S e >0.7, immunogenicity decreased by 79.2% to 83.3%, which is in line with the prediction.

[0111] Optimization example: If you need to improve S e Adjustable parameters. For example, based on Example 1, if the pH is increased from 4 to 5 (closer to pH 0), then |pH - pH 0| = 0,e 0 =1, recalculate: S e ≈0.675;

[0112] Still slightly below 0.7, but increasing to 3 hours: S e ≈0.746≥0.7, at which point the reduction in immunogenicity is more significant.

[0113] The technical effect of this electrostatic self-assembly strength equation:

[0114] Decreased immunogenicity: Equation S e Directly associated with the reduction in immunogenicity (when ≥0.7, antibody binding is reduced by ≥75%). For example, Example 2 parameter: C c =3,C m =1.5,R v =3, t=4, pH=5, T=25, calculate S e ≈0.75, immunogenicity decreased by 79.2%; Example 3 parameters: C c =5,C m =3,R v =5, t=6, pH=6, T=30, S e ≈0.85, immunogenicity decreased by 83.3%.

[0115] Process optimization: The equations provide quantification tools by adjusting parameters (such as increasing C). m Alternatively, t) can be used to ensure Se ≥ 0.7, guaranteeing efficient self-assembly reactions and avoiding trial and error. For example, at low pH, this can be achieved by increasing R. v Or t to compensate for charge loss.

[0116] Performance Enhancement: High S e The values ​​correspond to better antigen epitope masking (e.g., the contact angle is reduced to 30° and hydrophilicity is improved in Example 3) and material stability (e.g., the mass loss rate is only 3%), which stems from the tight electrostatic binding of the modified molecules to collagen.

[0117] Universality and Customization: Equation parameters are adjustable (e.g., by changing the type of modified molecules), adapting to different applications (e.g., bone repair requires higher C). c The coating material needs to have a higher C content. mIt can achieve functional customization.

[0118] The electrostatic self-assembly strength equation S e The embedded electrostatic self-assembly working mechanism is as follows:

[0119] 1. Charge-matching mechanism: In acidic solutions, collagen carries a positive charge (amino group -NH3+), while modified molecules (such as hyaluronic acid with -COO2) are positively charged. - It carries a negative charge. The C in the equation... c C m and pH term (via Directly control the charge density to ensure maximum electrostatic attraction. Charge complementarity is strongest when the pH is close to 5.0 (pH 0 = 5.0 is the optimal value).

[0120] 2. Antigen epitope masking: In self-assembly reactions, modifying molecules coat the collagen surface through electrostatic interactions. R in the equation... v Degree of integration with t-term regulation: High R v High t values ​​increase the coverage of modified molecules, masking antigenic epitopes (such as α1(I) chain epitopes). Higher Se values ​​enhance masking efficiency, blocking immune recognition.

[0121] 3. Synergistic Effects and Structural Regulation: The power exponent α = 0.25 reflects the nonlinear synergy between parameters (such as high C). m The length t promotes the formation of nanofiber networks, and the temperature T term (denominator) ensures that the reaction takes place under mild conditions (20-30℃) to maintain the natural structure of collagen.

[0122] 4. Effect Verification: High S e A value (≥0.7) corresponds to low immunogenicity (ELISA verification) and high biocompatibility (MTT cell viability ≥95%), as shown in the examples. Post-processing (separation, washing, drying) further purified the product and fixed the self-assembled structure.

[0123] In summary, the method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly provided in this embodiment has the following advantages:

[0124] 1. Significantly reduced immunogenicity: ELISA results showed that, compared with the untreated group, the amount of antibody binding on the implant was reduced by 75% to 83.3%, effectively inhibiting humoral and cellular immune responses.

[0125] 2. Biocompatibility optimization: The cell viability rate detected by MTT assay is over 95%, and the modified molecules (such as hyaluronic acid) can endow the implant with hydrophilicity (contact angle reduced to 30°), promoting cell adhesion and proliferation.

[0126] 3. Improved material performance: The composite structure formed by electrostatic self-assembly increases the tensile strength of the implant by 40% (up to 15MPa), and the mass loss rate is only 3% after immersion in simulated body fluid for 72 hours, combining mechanical stability and controllable degradation.

[0127] 4. High process versatility: This method does not rely on toxic reagents, the reaction conditions are mild (room temperature and pressure), and the function can be customized by adjusting the type of modified molecules (alginate improves flexibility, succinylated dextran enhances antibacterial properties), making it suitable for multiple applications such as bone repair, skin regeneration, and medical device coatings.

[0128] 5. Technological Innovation: Breaking through the toxicity limitations of traditional chemical modifications, it utilizes the principle of physical self-assembly to achieve precise regulation of immunogenicity, providing a new technical path for the surface modification of biomaterials.

[0129] Working principle

[0130] This invention utilizes electrostatic self-assembly technology, leveraging the charge complementarity between collagen and modified molecules to regulate immunogenicity. The specific principle is as follows:

[0131] 1. Charge-matching mechanism: After animal-derived collagen is dissolved in an acidic solution, the amino groups (-NH3) on its molecular chain... + Under acidic conditions, they carry a positive charge, while modified molecules such as alginate, succinylated dextran, and hyaluronic acid carry a negative charge under specific pH conditions because they contain carboxyl (-COOH) or sulfonic acid groups. The two spontaneously assemble into a composite structure through electrostatic attraction.

[0132] 2. Antigen epitope masking: Modified molecules coat the surface of collagen through electrostatic interactions, forming a nanoscale protective layer that effectively masks immunogenic sites in collagen molecules (such as antigenic determinants on the α1(I) and α2(I) chains), blocking the immune system's recognition.

[0133] 3. Microstructure regulation: During electrostatic self-assembly, by regulating parameters such as solution concentration, reaction temperature, and pH value, collagen and modified molecules can be induced to form an ordered nanofiber network or thin film structure. This structural optimization further reduces immunogenicity and improves material performance.

[0134] How to use

[0135] 1. Raw material pretreatment: The animal-derived collagen raw material is soaked in 0.1% to 1% trypsin solution and enzymatically hydrolyzed at 30-37℃ for 1 to 3 hours. Non-collagen impurities are removed by centrifugation to obtain high-purity collagen.

[0136] 2. Solution preparation: Dissolve the pretreated collagen in 0.5% to 2% acetic acid solution to prepare a collagen solution of 1 to 5 mg / mL; at the same time, prepare a solution of negatively charged modified molecules (alginate / succinylated dextran / hyaluronic acid, etc.) of 0.5 to 3 mg / mL.

[0137] 3. Electrostatic self-assembly: Mix collagen solution and modified molecule solution at a volume ratio of (2-5):1, stir and react for 2-6 hours at 20-30℃ and pH 4-6, and form a composite assembly through electrostatic interaction.

[0138] 4. Post-processing: The assembled products were separated by ultracentrifugation (10,000-15,000 rpm), washed with deionized water 3-5 times, and then freeze-dried (pre-frozen at -50 to -30℃, and sublimated under vacuum at 10-50 Pa) to obtain collagen implants with reduced immunogenicity.

[0139] The above are merely preferred embodiments of the present invention and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should recognize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly, characterized in that, Includes the following steps: Animal-derived collagen is pretreated to remove impurities and non-collagen components; Pretreated animal-derived collagen was dissolved in an acidic solution to obtain a collagen solution. A solution of modifying molecules with opposite charges is added to the collagen solution, and a self-assembly reaction is carried out under electrostatic action. The modifying molecules are one of alginate, succinylated dextran, and hyaluronic acid. The self-assembled products were separated, washed, and dried to obtain animal-derived collagen implants with reduced immunogenicity.

2. The method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly according to claim 1, characterized in that, The steps for pretreating animal-derived collagen include: soaking the animal-derived collagen raw material in a trypsin solution with a mass fraction of 0.1% to 1%, enzymatically hydrolyzing it at 30-37°C for 1 to 3 hours, and then removing precipitated impurities by centrifugation.

3. The method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly according to claim 1, characterized in that, The acidic solution is an acetic acid solution with a mass fraction of 0.5% to 2%, and the concentration of the collagen solution is 1 to 5 mg / mL.

4. The method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly according to claim 1, characterized in that, The concentration of the modified molecular solution is 0.5–3 mg / mL, and the volume ratio of the collagen solution to the modified molecular solution is (2–5):

1.

5. The method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly according to claim 1, characterized in that, The conditions for the self-assembly reaction are: temperature 20-30℃, pH value 4-6, reaction time 2-6 hours, and continuous stirring during the reaction at a speed of 100-300 rpm.

6. The method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly according to claim 1, characterized in that, The method for separating the self-assembled products is as follows: using ultracentrifugation, centrifuging at a speed of 10,000-15,000 rpm for 15-30 minutes, and collecting the precipitate.

7. The method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly according to claim 1, characterized in that, The steps for washing the self-assembled product are as follows: wash the precipitate repeatedly with deionized water 3 to 5 times, and centrifuge at 8000-12000 rpm for 10 to 20 minutes after each wash to remove the supernatant.

8. The method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly according to claim 1, characterized in that, The drying process is freeze drying, in which the washed precipitate is pre-frozen at -50 to -30°C for 2 to 4 hours, and then sublimated and dried under a vacuum of 10 to 50 Pa for 12 to 24 hours.

9. The application of an immunogenic animal-derived collagen graft prepared by the method for reducing the immunogenicity of animal-derived collagen grafts based on electrostatic self-assembly as described in any one of claims 1-8 in the preparation of tissue repair materials.

10. The application of an immunogenic animal-derived collagen implant prepared by the method for reducing the immunogenicity of animal-derived collagen implants based on electrostatic self-assembly as described in any one of claims 1-8 in the preparation of coating materials for medical devices.

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