Laminin or fragments thereof, complexes formed with non-covalently adsorbed immunological factors and their use in biomaterials

Abstract

The present application relates to a kind of laminin or its specific fragment polypeptide, the complex formed by non-covalent adsorption immunological factor with it and its application in biomaterials.38 polypeptides screened by the present application. One or more of the complete laminin containing the polypeptide screened by the present application, or complete fibronectin is used as carrier protein, and is mixed with biomaterial base, immunological factor etc. to make complex, because there is non-covalent adsorption between carrier protein and immunological factor, immunological factor can be biomimetic adsorption carried on the material combined with carrier protein. The product containing the complex of the present application can automatically dissociate and release immunological factor in neutral environment and maintain the slow release for several days.Moreover, the product made of the complex of the present application can avoid activating the immune response of the implant, solve the problem that transgenic expression vector enters the patient's body and easily induces intense immune response, difficult to realize secondary administration etc.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a laminin or a fragment thereof, a complex formed with it and a non-covalently adsorbed immune factor, and its application in biomaterials. Background Technology

[0002] Local delivery and sustained-release systems for immune factors are crucial in regenerative medicine and tumor immunotherapy. Their core objectives are to avoid the side effects of systemic injection, reduce the total dosage, and prolong the duration of action of immune factors at the lesion site. Traditional strategies for achieving local sustained release of immune factors fall into two main categories: one is based on gene therapy, which involves locally injecting specific transgenic expression vectors (such as lentiviruses or adenoviruses) to introduce the gene of the target immune factor into local tissue and achieve its expression; the other utilizes materials science techniques to directly couple immune factors to the surface of a carrier material via covalent chemical bonds.

[0003] For gene therapy, the viral vectors used carry the risk of restoring replication capacity and causing pathogenic infection. Lentiviral vectors, in particular, may induce insertional mutations through random integration into the host genome, leading to unintended changes in gene function. Furthermore, viral vectors readily induce strong host immune responses, making secondary administration difficult, and their in vivo half-life is relatively short. Since the actual expression level of the target immune factor after gene transfection is regulated by factors such as individual immune status, tissue microenvironment, and protein molecular weight, it is difficult to precisely control, thus affecting the safety and efficacy of the treatment.

[0004] The second method, which directly couples immune factors to the surface of a carrier material via covalent chemical bonds, avoids the biosafety issues associated with viral vectors. However, because the immune factors are covalently immobilized on the material surface, they lose their ability to migrate within the in vivo microenvironment, thus hindering their release. This immobilized state also prevents the immune factors from being taken up by endocytosis through receptor-mediated internalization after binding to cell surface receptors on the contacting material. Ultimately, this results in incomplete activation of downstream signaling pathways and weakens the biological function of the immune factors.

[0005] Therefore, solving the above problems and achieving safe, controllable, and fully functional local delivery of immune factors is crucial for tumor immunotherapy. Summary of the Invention

[0006] To address the aforementioned challenges, this invention provides a laminin or a fragment thereof, a complex formed with non-covalently adsorbed immune factors, and its application in biomaterials. This invention is the first to demonstrate that laminin LN, fibronectin FN, or laminin LN fragment polypeptides specifically and non-interleukin-type immune factors exhibit non-covalent binding.

[0007] To address the aforementioned challenges, the present invention employs the following technical solution: A polypeptide with non-covalent adsorption capacity for immune factors, wherein the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NO.1 to SEQ ID NO.38.

[0008] The present invention also provides an application of the polypeptide in the preparation of biomaterial composites.

[0009] The present invention also provides a biomimetic non-covalently adsorbed sustained-release immune factor biomaterial composite, comprising a two-dimensional or three-dimensional biomaterial substrate 1, a carrier protein 2 for non-covalently adsorbed immune factors, and immune factors 3.

[0010] Preferably, the carrier protein for non-covalently adsorbed immune factors is one or more of the following: complete recombinant proteins of various subtypes of laminin LN containing the polypeptides that have non-covalent adsorption capacity for immune factors, and complete recombinant fibronectin FN.

[0011] The complete recombinant proteins of various subtypes of laminin LN described in this invention specifically include laminins of type LN111, LN211, LN332, LN411, LN421, LN511, and LN521.

[0012] Preferably, the immune factor is an interleukin-type immune factor or a non-interleukin-type immune factor.

[0013] In this invention, interleukin-type immune factors specifically include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17A, IL-17F, IL-19, IL-20, IL-21, etc., while non-interleukin-type immune factors specifically include one or more of IFN-γ, TNF-α, TNF-β, GM-CSF, M-CSF, BAFF, and MIP-α.

[0014] The present invention also provides a method for preparing the biomaterial composite, comprising the following steps: S1. The carrier protein that non-covalently adsorbs immune factors is covalently or non-covalently bound to a two-dimensional or three-dimensional biomaterial substrate to obtain a substrate modified with the carrier protein. S2. Immerse or infiltrate the substrate modified with carrier protein obtained in step S2 into immune factors and incubate at room temperature to obtain the final product.

[0015] In preparing the composite, the two-dimensional materials used in this invention are generally bioglass, silica gel, polycaprolactone (PCL), poly(methyl methacrylate) (PMMA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), and polystyrene (PS). The three-dimensional materials are mainly various common hydrogels, including polyethylene glycol hydrogel, polyacrylamide hydrogel, polyvinyl alcohol hydrogel, sodium alginate hydrogel, collagen hydrogel, chitosan hydrogel, etc.

[0016] This invention involves chemically binding carrier proteins covalently or non-covalently to the surface of two-dimensional or three-dimensional biomaterials. The biomaterials containing the carrier proteins laminin (LN), fibronectin (FN), or LN fragment peptides are then immersed or infiltrated into an immune factor solution and incubated at room temperature for 1-2 hours to fully adsorb specific immune factors. Once the adsorption of the immune factors is complete, the material will automatically and slowly release the bound immune factors in a neutral environment.

[0017] The substrate material and the carrier proteins, such as laminin LN, fibronectin FN, or laminin LN fragment polypeptides, are generally connected by covalent or non-covalent bonds. There is no covalent binding between the carrier protein and the immune factor; only non-covalent binding exists. The immune factor is adsorbed onto the two-dimensional surface or three-dimensional structure of the substrate material through the non-covalent binding between the carrier protein and the immune factor.

[0018] The present invention also provides the application of the polypeptide in the preparation of an anti-inflammatory hydrogel dressing for treating diabetic wounds.

[0019] The present invention also provides an anti-inflammatory hydrogel dressing for diabetic wounds, wherein the anti-inflammatory hydrogel dressing for diabetic wounds is formed by cross-linking a hydrogel precursor solution through a copolymerization reaction, wherein the hydrogel precursor solution includes a hydrogel material, a carrier protein that can be copolymerized into the hydrogel material after functionalization, an immune factor that is non-covalently adsorbed with the carrier protein, a cross-linking agent, and an aqueous medium.

[0020] Preferably, the hydrogel material is one or more of acryloyl polyethylene glycol, maleyl polyethylene glycol, and diazo-based polyethylene glycol; the carrier protein that can be copolymerized into the hydrogel material after functionalization is generated by reacting one or two of the complete recombinant protein of each subtype of laminin LN containing the polypeptide with non-covalent adsorption capacity for immune factors, and complete recombinant fibronectin FN with acryloyl-succinimide valerate, maleyl-succinimide valerate, or diazo-succinimide valerate.

[0021] Preferably, the immune factor that is non-covalently adsorbed with the carrier protein is an interleukin-type immune factor or a non-interleukin-type immune factor; the crosslinking agent is p-dithioglycol, and the dispersion concentration in the hydrogel precursor solution is 0.1~100 mg / mL; the aqueous medium is deionized water, physiological saline or phosphate buffer solution.

[0022] The present invention also provides the application of recombinant laminin intact protein or intact recombinant fibronectin containing the polypeptide having non-covalent adsorption capacity for immune factors in the preparation of immune factor adsorption and sustained release biomaterials.

[0023] The immune factors adsorbed by the polypeptides of this invention exhibit similar fluidity to those in the natural in vivo microenvironment, allowing for slow detachment and release in a neutral environment. This invention offers advantages such as broad compatibility with immune factors, adjustable immune loading levels, biomimetic release of immune factors, no restriction on cellular internalization and endocytosis for complete signal transduction, and gentle release. It effectively solves the problems of existing sustained-release immune factor systems, where the type and dosage of immune factors cannot be adjusted after processing, and immune factors cannot be released freely. It also addresses the issues in existing gene therapy for expressing immune factors, such as the risk of mutation in viral vectors, uncontrollable expression levels of immune factors, and the potential for viral vectors to trigger immune responses, making secondary administration impossible.

[0024] Because all viral vectors used in transgenic therapy have the potential to mutate and regain spontaneous replication and infectivity, and lentiviral vectors, in particular, pose certain off-target and carcinogenic risks when integrating with the host gene, the expression level of immune factors transfected by transgenic expression vectors is affected by individual immune levels, target tissue location, and the molecular weight of the immune factors, making precise dosage control difficult. It is challenging to precisely and continuously control the amount of immune factors induced in tissue expression by controlling the amount of injected vector; the induced immune factor expression level spikes to its peak within 24-48 hours. Based on this, the present invention has produced a series of low-biological-risk biomaterial composites, hydrogels, and other products. These products do not carry the risks associated with transgenic expression vectors; the release dosage of immune factors is more controllable, and stable release of immune factors can be maintained for more than five days.

[0025] Compared with the prior art, the present invention has the following advantages: (1) The adsorbent carrier in this invention is universally applicable to immune factors and does not require the design of chemical binding methods with materials for specific immune factors; (2) The base material-adsorption carrier protein complex in this invention can adjust the types and dosages of immune factors before adsorbing and carrying immune factors, rather than being unchangeable from the factory and requiring no customization from scratch, making it more flexible in personalized medicine and precision medicine. (3) In this invention, there is only non-covalent adsorption between the immune factor and the adsorbed carrier protein, and there is no covalent chemical bond connection. In a neutral environment, the immune factor can spontaneously and slowly dissociate and release. The immune factor has similar fluidity to the microenvironment in the body. After being captured by the cell receptor, it can complete receptor-mediated endocytosis and thus fully activate the corresponding signaling pathway. Attached Figure Description

[0027] Figure 1 This is a structural diagram of the biomaterial composite of the present invention; Figure 2 This is a graph showing the results of the immune factor loading amount measured by ELISA after the two-dimensional material surface in Embodiment 1 of the present invention was non-covalently bound with different laminin LN subtypes and fibronectin FN as carrier proteins, with bovine serum albumin BSA as a control, and after adsorbing immune factors. Figure 3 This is a diagram showing the immune factor loading results measured by ELISA when the surface of the two-dimensional material in Embodiment 1 of the present invention uses different laminin LN subtypes and fibronectin FN as carrier proteins through non-covalent binding, and the heparin binding domain of the carrier proteins is blocked with excess heparin. Figure 4 This is a time-release curve of IL-10 after covalent doping of the layer adhesion protein LN111 in the three-dimensional hydrogel material of Example 2 of the present invention. Figure 5 This is a schematic diagram of the process for screening immune factor fragments adsorbed by laminin LN in Example 3 of the present invention using combined molecular docking and molecular non-covalent bonding analysis. Figure 6 This is a graph showing the immune factor loading results measured by ELISA after the polypeptides screened in Example 3 were non-covalently bound to the surface of the two-dimensional material in Example 4, with bovine serum albumin (BSA) as a control, and the immune factors were adsorbed. Figure 7 This is a graph showing the binding and release curves of three immune factors measured by surface plasmon resonance (SPR) after covalently binding laminin LN332 to the surface of the two-dimensional material in Example 5. Figure 8 This is a graph showing the binding and release curves of three immune factors or two growth factors measured by surface plasmon resonance (SPR) after covalently binding the laminin fragment SEQ ID NO.30 selected in Example 5 to the surface of the two-dimensional material in Example 6. Figure 9 This is a diagram showing the healing effect of a mouse diabetic wound after treatment with IL-10 using a hydrogel dressing containing covalently doped laminin LN111 and IL-10. (Example 7 of this invention) Figure 10This is a statistical chart showing the time healing rate curve and the final healing rate ranking of the wounds after treating diabetic mice with IL-10 using a hydrogel dressing containing covalently doped laminin LN111 and IL-10. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments. These embodiments are provided to facilitate understanding of the invention and are not intended to limit the invention. Those skilled in the art can make various modifications based on the basic idea of ​​the invention, but all modifications are within the scope of the invention as long as they do not depart from its fundamental spirit.

[0029] In the embodiments, laminin LN is composed of three chains: LAMA, LAMB, and LAMC (or α, β, γ chains). Each chain has multiple subtypes (LAMA1~5, LAMB1~3, LAMC1~2). Different laminin LN subtypes are generated by different combinations of subtypes of the three subchains. The naming of laminin LN subtypes is composed of the corresponding subtype codes of its three subchain components. For example, LN332 indicates that it is composed of the three subchain subtypes LAMA3, LAMB3, and LAMC2.

[0030] Example 1: A biomimetic non-covalently adsorbed sustained-release immune factor biomaterial composite The biomaterial composite (structural diagram shown) Figure 1 It includes a two-dimensional biomaterial substrate 1, a carrier protein 2 that non-covalently adsorbs immune factors, and immune factors 3.

[0031] First, using two-dimensional polystyrene as a substrate, a surface plasma activation treatment was performed before use (the surface was treated by Corning to introduce carboxyl and hydroxyl groups, increasing the surface's non-specific adsorption capacity, polarity, and hydrophilicity). Then, the amount of immune factors adsorbed and carried by different laminin LN subtypes and recombinant fibronectin FN as carrier proteins 2 for non-covalent adsorption of immune factors was measured on the treated polystyrene surface. The specific procedure was as follows: Various immune factors 3 were dissolved in phosphate-buffered saline (PBS) (Seville Biosciences) to a concentration of 50 μg / mL. Then, 0.1 mg / mL of D-biotin-N-hydroxysuccinimide ester (Merida) dissolved in PBS was added at a molar ratio of immune factor to D-biotin-N-hydroxysuccinimide ester of 1:4. The mixture was placed in a 1.5 mL centrifuge tube (Eppendorf) and reacted at 4°C for 12 hours. At this point, immune factors 3 and biotin were coupled through the free primary amino groups (N-terminus and lysine residues) of the immune factors. Dissolve Tris-HCl (Maclean's) in PBS buffer to a concentration of 1 mg / mL. Add the same amount of Tris-HCl (Maclean's) PBS solution as the molar volume of D-biotin-N-hydroxysuccinimide ester to the aforementioned immune factor 3 reaction solution to terminate the reaction and block excess D-biotin-N-hydroxysuccinimide ester. This ensures successful biotin conjugation for each immune factor. After calculating the final molar concentration of each immune factor, store the solution at -80°C. Only one immune factor is tested at a time.

[0032] Prepare the carrier protein solution, ELISA wash buffer, and blocking buffer: Dilute 0.1 mg / mL of human recombinant laminin LN subtype stock solutions (LN111, LN211, LN332, LN411, LN421, LN511, LN521) or 1 mg / mL mouse LN111 (mLN1) to 10 nM with PBS buffer. Dissolve human recombinant fibronectin FN powder (Yo Proteins) to 10 nM with PBS buffer. Dilute eBioscience ELISA / ELISPOT diluent (5×) to 5 times its volume using ultrapure water to prepare the blocking buffer. Dilute Tween-20 to 0.05% with PBS to prepare the ELISA wash buffer (add 500 μL of Tween-20 per liter of PBS).

[0033] When testing for IL-3, IL-6, IL-12, IL-15, IL-16, IL-17A, IL-17F, IL-19, IL-20, IL-21, IFN-γ, TNF-α, TNF-β, GM-CSF, M-CSF, BAFF, and MIP-α, solutions of various carrier proteins were added to 96-well high-absorption EIA / RIA transparent flat-bottom polystyrene microplates (Corning), with 100 μL of carrier protein solution added to each well, and three replicates for each carrier protein. Bovine serum albumin (BSA) (Merck) was dissolved in PBS buffer to 0.5% w / v, and 100 μL was added to each well, in three replicates, as a non-specific binding control. The entire 96-well high-absorption EIA / RIA microplate with different carrier proteins in each well was incubated overnight at 4°C. At this point, each well of the high-absorption binding plate non-covalently binds one carrier protein. Remove the high-absorption plate coated with carrier protein that has been incubated at 4°C. Add 200 μL of Elisa washing buffer to each well and let it stand for 1 minute. Repeat this process three times. After the last incubation, remove all liquid from the wells and add 200 μL of blocking buffer to each well. Block at room temperature for 1 hour. Simultaneously, thaw the corresponding immunofactor (conjugated with biotin) and dilute it to 50 nM with PBS. After blocking at room temperature, repeat the washing steps described above once and remove all liquid from the plate. Add 100 μL of 50 nM solution of the corresponding immunofactor (conjugated with biotin) to each well of the high-absorption plate and incubate at room temperature for 2 hours. (Because the binding and adsorption are too strong at the same concentration, resulting in signal oversaturation, IFN-γ is detected at a concentration of 5 nM alone.) Simultaneously, dilute avidin-horseradish peroxidase (eBioscience) at a volume ratio of 1:1000 with blocking buffer into EP tubes. After incubation, repeat the washing steps described above three times. After a final rinse, remove all liquid from the wells and add 100 μL of avidin-horseradish peroxidase dilution to each well. Incubate at room temperature for 30 minutes. Repeat the rinsing process seven times after incubation. After a final rinse, remove all liquid from the wells and add 100 μL of TMB reaction substrate (eBioscience) to each well. Incubate at room temperature in the dark for 15 minutes. After incubation, add 100 μL of Elisa stop solution (Solepro) to each well to terminate the reaction. Immediately measure the absorbance at 450 nm using a Tecan microplate reader with 570 nm as the reference wavelength.

[0034] When testing IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-13 (due to the important physiological significance of these immune factors, the loading of carrier proteins on these immune factors was also tested to determine whether the loading of carrier proteins on these immune factors depended on the heparin-binding domain of the carrier proteins), solutions of various carrier proteins were added to 96-well high-absorption EIA / RIA transparent flat-bottom polystyrene microplates (Corning), with 100 μL of carrier protein solution added to each well, and six replicates for each carrier protein. Bovine serum albumin (BSA) (Merck) was dissolved in PBS buffer to 0.5% w / v, and 100 μL was added to each well, in three replicates, as a non-specific carrier binding control in the 96-well high-absorption EIA / RIA plates. The entire 96-well high-absorption EIA / RIA plate with different carrier proteins added to each well was incubated overnight at 4°C. At this point, each well of the high-absorption binding plate non-covalently binds one carrier protein. Remove the high-absorption plate coated with carrier proteins from the plate after incubation at 4°C. Add 200 μL of Elisa washing buffer to each well and let stand for 1 minute. Repeat three times. After the last incubation, remove all liquid from the wells and add 200 μL of blocking buffer to each well, blocking at room temperature for 1 hour. Simultaneously, thaw the corresponding test immune factor (conjugated with biotin), dilute it to 50 nM with PBS, and prepare a 10 μM heparin sodium (Solepro) solution using blocking buffer. After incubation at room temperature, repeat the washing steps once and remove all liquid from the plate. For each carrier protein in the high-absorption plate, take three replicates out of six wells and add 100 μL of 10 μM heparin sodium solution to each well. Add 100 μL of blocking buffer to each of the three replicates. For bovine serum albumin-coated three replicates, add only 100 μL of blocking buffer. Incubate at room temperature for 30 minutes, then repeat the washing steps three times. After the final shake-drying of all liquid in the wells, add 100 μL of 50 nM biotin-conjugated test immunomodulator solution to each well and incubate at room temperature for 2 hours. Simultaneously, dilute avidin-horseradish peroxidase (eBioscience) 1:1000 (v / v) with blocking buffer into the EP tubes. Repeat the washing steps three times after incubation. After the final wash-drying of all liquid in the wells, add 100 μL of avidin-horseradish peroxidase dilution to each well and incubate at room temperature for 30 minutes. Repeat the washing steps seven times after incubation. After the final shake-drying of all liquid in the wells, add 100 μL of TMB reaction substrate (Invitrogen) to each well and incubate at room temperature in the dark for 15 minutes. After incubation, add 100 μL of Elisa stop solution (Solepro) to each well to terminate the reaction, and immediately measure the absorbance at 450 nm using a Tecan microplate reader with 570 nm as the reference wavelength.

[0035] The test results are as follows Figure 2 , Figure 3 As shown. Among them Figure 2This study describes the loading of immune factors onto the surface of a two-dimensional material after non-covalent binding of different laminin LN subtypes and fibronectin FN as carrier proteins. Bovine serum albumin (BSA) was used as a non-specific adsorption control. The loading was measured by ELISA and absorbance. Figure 2It can be seen that for testing heparin competitive inhibition of immune factor 3, the replicates without heparin sodium were considered for the time being. For each immune factor, all other control conditions remained identical except for the carrier protein coating on the bottom two-dimensional surface. For each immune factor, a one-way ANOVA was performed in GraphPadPrism for statistical analysis, using BSA-coated wells as the control for all conditions. * indicates the range of P values: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Only carrier proteins with significantly higher adsorption than bovine serum albumin (BSA) for that immune factor were not labeled. Carrier proteins that were the same as, had no significant difference from, or were significantly lower than BSA were not labeled to highlight the positive correlation between adsorption amount and absorbance in the graph. Interleukin-type immune factors and a range of non-interleukin-type immune factors crucial for immune cell development / maturation, including MIP-3α, TNF-α, TNF-β, IFN-γ, BAFF, M-CSF, and GM-CSF, can be adsorbed by at least one laminin LN isoform or fibronectin FN. However, the binding pattern of laminin LN to immune factors differs significantly from the binding pattern of laminin LN to growth factor GF shown in previous studies. Most growth factor GFs show no significant difference in affinity for different laminin LN isoforms, while most immune factors are generally adsorbed in greater quantities on the LN332-coated surface than other laminin LN isoforms. This indicates that immune factors exhibit selectivity for laminin LN isoforms, revealing a difference in the mechanism by which laminin LN binds to immune factors or growth factor GF. Only IL-9, IL-16, IL-17A, and IL-21 showed low affinity for LN332, with IL-16 exhibiting no good adsorption for any LN or FN. IL-9 and IL-21 showed the highest adsorption on fibronectin-coated FN surfaces, while IL-17A showed the highest adsorption on LN411-coated surfaces. Besides LN332, IL-1α, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17A, IL-17F, IL-19, IL-20, TNF-α, GM-CSF, M-CSF, and BAFF were all adsorbed on surfaces coated with other LN isoforms. For IL-17A and IL-17F, the adsorption on LN111-coated surfaces was second only to LN332. For IL-6, the adsorption on LN411-coated surfaces was second only to LN332. For IL-3 and IL-11, only LN332 and fibronectin FN-coated surfaces showed significant adsorption. For IL-15, only LN332 and mLN1-coated surfaces showed significant adsorption. IL-5 could be significantly adsorbed by all LN-coated surfaces except LN511 and LN521.This study examined the adsorption capacity of mouse laminin LN111 (mLN1, R&D Biosystem) for all tested immune factors. The aim was to investigate changes in the adsorption capacity of laminins for immune factors during evolution. For all immune factors that could be significantly adsorbed by the human laminin LN111 coating surface, the mouse mLN1 coating surface also showed significant adsorption capacity for those immune factors. This indicates that the adsorption and binding capacity of laminins for immune factors appeared early in the evolution of primates (mice) and has important physiological significance.

[0036] Figure 3 This study investigated the influence of heparin competitive inhibition on the adsorption of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-13 on the surface of two-dimensional materials after non-covalent binding of different laminin LN subtypes and fibronectin FN as carrier proteins. The results were measured using ELISA and absorbance methods. For each immune factor, multiple t-tests were used in GraphPadPrism to analyze the difference in adsorption capacity between the addition and absence of heparin to block the heparin-binding domain of the carrier protein. * indicates the significance range of P-values: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. The adsorption capacity was positively correlated with the absorbance value in the graph. For each immune factor, the adsorption capacity of laminin LN subtypes or fibronectin FN as carrier proteins on the coated surface did not decrease significantly due to the blocking of the heparin binding domain by heparin. However, in previous studies on the adsorption capacity of laminin LN or fibronectin FN for growth factor GF, the adsorption of growth factor GF by laminin LN or fibronectin FN was highly dependent on the heparin binding domain of laminin LN or fibronectin FN. When excessive heparin blocked the heparin binding domain, the adsorption capacity of the carrier protein for growth factor GF decreased significantly. This demonstrates that the adsorption of immune factors and growth factors by laminin LN or fibronectin FN mainly occurs at different sites of laminin LN or fibronectin FN.

[0037] Example 2: A three-dimensional hydrogel covalently doped with laminin and biomimetic non-covalently adsorbed with immune factors This embodiment provides a method for preparing a three-dimensional hydrogel covalently doped with laminin and biomimetically non-covalently adsorbed with immune factors, and measures the time-release ability of this hydrogel for immune factors, using a hydrogel of the same type without covalent doped laminin as a control. The specific preparation process is as follows: Step 1: Hydrogel copolymerization functionalization of laminin: 0.1 mg / mL LN111 (human recombinant laminin, Biolamina) was dialyzed for one hour in sodium bicarbonate buffer at pH 8.5 using a test tube dialysis membrane (MW=10KDA, Thermo Fisher Scientific). Then, LN111 was mixed with MAL-PEG-SVA (maleimide-polyethylene glycol-succinimide valerate, LaysanBio) at a molar ratio of 1:4 in a 1.5 mL centrifuge tube and reacted at room temperature for 2 hours. The reaction was then terminated by dialyzing in PBS buffer using a test tube dialysis membrane for one hour. After the reaction, the reaction solution was concentrated to 10-15 times the original concentration using an ultrafiltration centrifuge tube (Merck). At this point, maleimide groups were covalently bound to LN111. Step 2: Preparation of polyethylene glycol-maleimide hydrogel covalently doped with laminin and adsorbing immune factors: 4-arm-PEG-MAL (four-arm polyethylene glycol-maleimide, LaysanBio, MW 20k) was dissolved in PBS buffer to a concentration of 200 mg / mL. 7.5 μg of LN111 modified with maleimide groups obtained in Step 1 (final concentration 1.14 mg / mL, corresponding to an addition volume of 6.54 μL), 10.35 μL of four-arm polyethylene glycol-maleimide, and 2 μL of 50 μg / mL IL-10 were added to a 0.5 mL centrifuge tube. The tube was allowed to stand at room temperature for 1 hour to allow sufficient adsorption of IL-10 by LN111, resulting in experimental solution A. The blank control group solution A was replaced with an equal volume of PBS to replace the copolymerized LN111, and the other groups were prepared in the same way as in step 2. Step 3: Dissolve SH-PEG-SH (p-dimercapto-polyethylene glycol, LaysanBio) in PBS buffer to a concentration of 200 mg / mL. Take 2 μL of p-dimercapto-polyethylene glycol into an empty 0.5 mL centrifuge tube and dilute it with 2 μL of PBS to obtain solution B. Mix the above experimental group solution A or blank control group solution A with one portion of solution B (i.e., 2 μL p-dimercapto-polyethylene glycol + 2 μL PBS) and spread it on the surface of a hydrophobic sealing film to form a roughly circular disc. Incubate at 37°C for 30 minutes to form a cross-linked structure. Prepare three hydrogels for the experimental group and control group respectively according to the above steps. Step 4: Transfer the three LN111 covalently doped hydrogels and the blank control hydrogel without LN111 covalent doping to 24-well plates containing 400 μL PBS. Collect the supernatant every 24 hours and add 400 μL PBS again. Store the samples at -80℃ daily. After 5 days, use a human interleukin-10 (IL-10) kit (ELISA) (Dongge Biotechnology) to detect the IL-10 concentration of all samples.

[0038] The 5-day release curve of IL-10 after LN111 loading is shown below. Figure 4 As shown, it can be seen that the three-dimensional hydrogel material covalently doped with laminin in this invention, with the same hydrogel without LN111 as a control, shows that the hydrogel covalently doped with LN111 has a significantly smoother IL-10 release over five days compared with the hydrogel without LN111, proving that the adsorption force of LN on immune factors has a controlled and sustained release effect in the three-dimensional system.

[0039] Example 3: Screening for fragments of laminin LN with adsorption capacity for immune factors using molecular docking combined with non-covalent molecular bonding analysis. Figure 5 This is a schematic diagram of the process for screening fragments of laminin (LN) capable of adsorbing immune factors through molecular docking combined with non-covalent molecular interaction analysis. Three-dimensional molecular models of different immune factors were obtained from the Protein Database Bank database. Molecular docking was performed using the HDOCK molecular docking platform with each splice variant (LAMA1-5, LAMB1-3, LAMC1-2) to obtain all possible predicted binding posture models. These binding posture models were imported into MaestroViewer software. The immune factors and laminin (LN) splice variants in the binding posture models were defined as Chain A and Chain R, respectively. Non-covalent interaction analysis was then performed on the two chains to obtain all amino acid residues on the corresponding laminin (LN) splice variant that could potentially form non-covalent bonds with the corresponding immune factor in the predicted binding posture. Fragments of approximately 20 amino acid residues in length covering the most likely non-covalent bonds with the immune factor were selected from the laminin (LN) splice variants as binding sites for the immune factor. The positions of the fragments of laminin LN that can adsorb immune factors in the amino acid sequences of each chain subtype of laminin LN are shown in Table 1.

[0040] Table 1. Fragments of laminin LN selected in this invention that can adsorb immune factors. SEQ ID NO.1 LAMA1 262-461 20 SEQ ID NO.2 LAMA1 2201-2400 20 SEQ ID NO.3 LAMA1 2501-2550 20 SEQ ID NO.4 LAMA2 1650-1700 20 SEQ ID NO.5 LAMA2 2011-2060 20 SEQ ID NO.6 LAMA2 2051-2200 20 SEQ ID NO.7 LAMA2 2310-2360 20 SEQ ID NO.8 LAMA3 47-296 20 SEQ ID NO.9 LAMA3 2471-2620 20 SEQ ID NO.10 LAMA3 2701-2800 23 SEQ ID NO.11 LAMA4 418-530 25 SEQ ID NO.12 LAMA4 901-1000 20 SEQ ID NO.13 LAMA4 1300-1350 20 SEQ ID NO.14 LAMA4 1467-1823 20 SEQ ID NO.15 LAMA5 301-400 20 SEQ ID NO.16 LAMA5 2381-2430 20 SEQ ID NO.17 LAMA5 2736-2929 22 SEQ ID NO.18 LAMB1 50-100 20 SEQ ID NO.19 LAMB1 31-270 20 SEQ ID NO.20 LAMB1 1467-1786 20 SEQ ID NO.21 LAMB1 1721-1770 20 SEQ ID NO.22 LAMB2 42-91 20 SEQ ID NO.23 LAMB2 61-110 20 SEQ ID NO.24 LAMB2 380-430 20 SEQ ID NO.25 LAMB2 1701-1798 20 SEQ ID NO.26 LAMB3 30-250 20 SEQ ID NO.27 LAMB3 35-85 20 SEQ ID NO.28 LAMB3 671-720 20 SEQ ID NO.29 LAMB3 1027-1056 20 SEQ ID NO.30 LAMC1 70-287 20 SEQ ID NO.31 LAMC1 150-180 20 SEQ ID NO.32 LAMC1 1364-1609 20 SEQ ID NO.33 LAMC1 1364-1609 20 SEQ ID NO.34 LAMC1 1451-1500 20 SEQ ID NO.35 LAMC1 1501-1609 20 SEQ ID NO.36 LAMC2 534-1111 20 SEQ ID NO.37 LAMC2 22-1111 20 SEQ ID NO.38 LAMC2 1021-1070 20 Table 2. Fragments of laminin LN that adsorb growth factors, screened in existing technologies and studies. SEQ ID NO.39 A1-04 LAMA1 2734-2745 12 SEQ ID NO.40 A1-05 LAMA1 2750-2760 12 SEQ ID NO.41 A2-05 LAMA2 2800-2811 12 SEQ ID NO.42 A3-04 LAMA3 2932-2951 20 SEQ ID NO.43 A3-05 LAMA3 3031-3043 13 SEQ ID NO.44 A3-06 LAMA3 3043-3062 20 SEQ ID NO.45 A4-04 LAMA4 1413-1432 20 SEQ ID NO.46 A4-05 LAMA4 1521-1540 20 SEQ ID NO.47 A5-04 LAMA5 3300-3330 31 SEQ ID NO.48 A5-05 LAMA5 3312-3325 14 SEQ ID NO.49 A5-06 LAMA5 3345-3356 12 SEQ ID NO.50 A5-07 LAMA5 3417-3436 20 SEQ ID NO.51 A5-08 LAMA5 3539-3550 12 Example 4: Determination of the amount of immune factors adsorbed and carried by non-covalently bound LN fragments of different laminin proteins on the surface of two-dimensional materials as carrier proteins.

[0041] Heparin-biotin sodium salt (Merck) was dissolved in PBS buffer to a final concentration of 50 nM to test the heparin adsorption capacity of the laminin LN fragment. Immune factors IL-4, IL-10, IFN-γ, and growth factors VEGF-A165 and HB-EGF (purchased from Peprotech) were dissolved in phosphate-buffered PBS (Seville Biosciences) to a concentration of 50 μg / mL. Then, 0.1 mg / mL D-biotin-N-hydroxysuccinimide ester (Merida) dissolved in PBS was added at a molar ratio of immune factor or growth factor: D-biotin-N-hydroxysuccinimide ester of 1:4. The mixture was incubated in 1.5 mL centrifuge tubes at 4°C for 12 hours. At this point, the immune factor or growth factor was coupled to biotin through its free primary amino groups (N-terminus and lysine residues). Dissolve tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, Maclean's) in PBS buffer to a final concentration of 1 mg / mL. Add an equal molar amount of Tris-HCl (Maclean's) PBS solution to the aforementioned immune factor or growth factor reaction solution to terminate the reaction, blocking any excess D-biotin-N-hydroxysuccinimide ester. Calculate the final molar concentration of each immune factor and store at -80°C. Test only one immune factor or growth factor at a time.

[0042] All amino acid sequences in Tables 1 and 2 were artificially synthesized by Genscript Biotech. Each laminin LN fragment polypeptide was dissolved in PBS buffer to a concentration of 1 μM and stored at -80°C. Blocking buffer and ELISA wash buffer were prepared according to the method in Example 1.

[0043] The aforementioned solutions of various laminin LN fragments were added to 96-well high-absorption EIA / RIA transparent flat-bottom polystyrene microplates (Corning), with 100 μL of laminin LN fragment solution added to each well. Each laminin LN fragment was added in triplicate. Laminin LN fragments named starting with A, belonging to all LAMA chain subtypes, were added to different wells within the same 96-well high-absorption EIA / RIA plate. Fragments named starting with B or C, belonging to all LAMB and LAMC chain subtypes, were added to different wells within a separate 96-well high-absorption EIA / RIA plate. Bovine serum albumin (BSA) was dissolved in PBS buffer to 0.5% w / v, and 100 μL of BSA solution was added to three empty wells in each high-absorption binding plate as a non-specific binding vector control. The entire 96-well high-adhesion EIA / RIA plate, with different laminin LN fragments added to each well, was incubated overnight at 4°C. Thus, each well of the high-adhesion plate was non-covalently bound to a laminin LN fragment.

[0044] For each test of an immune factor, growth factor, or heparin, remove the highly absorbent plate coated with laminin LN fragments that has been incubated at 4°C. Add 200 μL of Elisa washing buffer to each well and let it stand for 1 minute. Repeat this process three times. After the final incubation, remove all liquid from the wells and add 200 μL of blocking buffer to each well. Block at room temperature for 1 hour. Simultaneously, thaw the corresponding immune factor being tested (conjugated with biotin) and dilute it to 50 nM with PBS. After incubation at room temperature, repeat the washing steps described above once and remove all liquid from the plate. Add 100 μL of 50 nM heparin-biotin sodium solution or the corresponding tested immune factor or growth factor (conjugated with biotin) to each well of the high-adsorption plate, and incubate at room temperature for 2 hours (due to excessive binding and adsorption at the same concentration leading to signal oversaturation, IFN-γ was detected at a concentration of 5 nM alone). Simultaneously, dilute avidin-horseradish peroxidase (eBioscience) 1:1000 (v / v) in blocking buffer into the EP tubes. After incubation, repeat the washing steps three times. For the final wash, dry all liquid in the wells and add 100 μL of avidin-horseradish peroxidase dilution to each well, incubate at room temperature for 30 minutes, and repeat the washing steps seven times. Finally, dry all liquid in the wells and add 100 μL of TMB reaction substrate (Invitrogen) to each well, incubate at room temperature in the dark for 15 minutes. After incubation, add 100 μL of Elisa stop solution (Solepro) to each well to terminate the reaction, and immediately use a Tecan microplate reader to measure the absorbance at a wavelength of 450 nm with 570 nm as the reference wavelength.

[0045] After non-covalently binding of different laminin LN fragments to the surface of the two-dimensional material (same as Example 2) as carrier proteins, bovine serum albumin (BSA) was used as a non-specific adsorption control. The adsorption loads of heparin, immune factors, and growth factors were measured by ELISA and absorbance methods after adsorption of immune factors, as shown below. Figure 6 As shown. Figure 6In this example, SEQ ID NO.1~SEQ ID NO.38 are polypeptides that can non-covalently adsorb immune factors screened in Example 3; A1-04, A1-05, A2-05, A3-04, A3-05, A3-06, A4-04, A4-05, A5-04, A5-05, A5-06, A5-07, and A5-08 are fragments of laminin LN that can non-covalently adsorb growth factors screened in existing technologies and studies, and are also used as controls in this example. For each immune factor, growth factor, or heparin, statistical analysis was performed using a one-way ANOVA test in GraphPadPrism, with BSA-coated wells as controls for all conditions. The p-value range is marked with *: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. As shown in the figure, the laminin LN fragments exhibiting significant adsorption capacity for immune factors and growth factors differ considerably. The laminin LN fragments that significantly adsorbed the tested growth factors highly overlapped with those that significantly adsorbed heparin, especially A1-04, A4-04, A4-05, A5-04, and A5-07. However, only a small portion of the laminin LN fragments that significantly adsorbed the tested immune factors overlapped with the heparin-adsorbing fragments. This is consistent with the results of Example 1 and further explains the findings in Example 1. Figure 3 The heparin-binding domain of the heparin-shielded laminin LN does not affect the ability of the laminin LN coating surface to adsorb immune factors.

[0046] Example 5: Surface plasmon resonance (SPR) assay of laminin's ability to bind and release three immune factors. This embodiment describes the binding and release time curves of three immune factors measured by surface plasmon resonance (SPR) after covalently binding laminin LN332 to the surface of a two-dimensional material. The specific process is as follows: Surface plasmon resonance (SPR) measurements were conducted by Hangzhou Yanqu Information Technology Co., Ltd. A Biacore T100 SPR system (GE Healthcare) was used for SPR testing. Following the manufacturer's instructions, LN332 was immobilized on the carboxyl chip using the amine coupling method kit provided by the chip manufacturer (GE Healthcare), with immobilization amounts ranging from 1000 to 2000 resonance units (RU). IL-4 or IFN-γ was prepared at concentration gradients of 10, 20, 30, 40, 50, 75, and 100 nM in running buffer (1×PBS, pH 7.2, containing 0.05% v / v surfactant P20, hereinafter the same). Each concentration of immune factor solution was sequentially injected into the flow channel at a flow rate of 30 μL / min. After each concentration was measured, the sensor chip was regenerated using 10 mM NaOH solution to remove residual conjugates and restore the chip surface. Steady-state kinetic fitting of the experimental results was performed using BIAevaluation software.

[0047] For IL-10, due to Example 4 Figure 6 The dominant adsorption fragment for IL-10 in LN332 (LAMA3+LAMB3+LAMC2) is A3-05. The lysine content is too high, resulting in excessive interference from the occupancy effect of the amine coupling method. Therefore, the inverted method must be used to test the binding and release time curve of IL-10 and LN332. According to the manufacturer's instructions, IL-10 was immobilized on the carboxyl chip using the amine coupling method kit provided by the chip manufacturer (GE Healthcare), with immobilization amounts ranging from 1000 to 2000 resonance units (RU). LN332 was prepared in the above-mentioned running buffer at concentration gradients of 6.25, 12.5, 25, 50, 100, and 200 nM. The remaining procedures were the same as for the detection of IL-4 or IFN-γ.

[0048] The binding and release time curves of three immune factors, measured by surface plasmon resonance (SPR), were obtained by covalently binding laminin LN332 to the surface of the two-dimensional material. Figure 7 As shown, the left side represents the real-time binding-dissociation signal results, and the right side represents the steady-state kinetic fitting results. Significant adsorption exists between LN332 and IL4, IFN-γ, and IL-10. The binding of LN332 with the former two is a classic protein binding pattern, characterized by slow binding and slow adsorption. The binding and dissociation of LN332 with IL-10 exhibits a fast binding and fast dissociation pattern, which is commonly seen in polyelectrolyte protein interactions, such as the binding of histone H1.0 and thymosin α. The steady-state fitting shows that the binding amount saturates with increasing concentration of ligand LN332, and the curve curves downward. This is a characteristic of specific binding in fast binding and fast adsorption. Otherwise, there is no concentration saturation phenomenon in non-specific fast binding and fast adsorption, and the binding amount is proportional to the concentration.

[0049] Example 6: Surface Plasmon Resonance (SPR) Measurement of the Binding and Release Capacity of Peptide Fragments to Immune Factors or Growth Factors In this embodiment, after binding a LAMC1 chain fragment (SEQ ID NO. 30) of the laminin protein to the surface of a two-dimensional material via biotin-streptavidin coupling, surface plasmon resonance (SPR) measurements and binding-release time curves of three immune factors or two growth factors were obtained.

[0050] As in Embodiment 4 of the present invention Figure 6 As shown, SEQ ID NO.30 is the fragment in LN111 (LAMA1+LAMB1+LAMC1) that provides the main adsorption and binding for IL-10. Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize N-terminal biotinylated and C-terminal biotinylated peptides of SEQ ID NO.30. Surface plasmon resonance (SPR) measurements were performed by Hangzhou Yanqu Information Technology Co., Ltd. using a Biacore T100 SPR system (GE Healthcare). According to the manufacturer's instructions, the N-terminal and C-terminal biotinylated SEQ ID NO.30 peptides were mixed at a molar ratio of 1:1 and loaded onto a streptavidin-modified chip according to the chip manufacturer's (GE Healthcare) instructions, with fixed amounts ranging from 1000 to 2000 resonance units (RU). Prepare IL-4, IFN-γ, IL-10, HB-EGF, or VEGF-A165 in running buffer at concentration gradients of 100, 200, 400, 600, 800, and 1000 nM, respectively. The rest of the procedure is the same as for the detection of IL-4 or IFN-γ.

[0051] Figure 8 This is a graph from Example 6 of the present invention, showing the binding and release time curves of three immune factors or two growth factors measured by surface plasmon resonance (SPR) after biotin-streptavidin conjugate of the two-dimensional material surface with the laminin LAMC1 chain fragment SEQ ID NO.30. The left side shows the real-time binding-dissociation signal results, and the right side shows the steady-state kinetic fitting results. SEQ ID NO.30 showed no significant binding with IL4, IFN-γ, VEGF-A165, or HB-EGF, but significant adsorption with IL-10, exhibiting a fast binding and fast dissociation pattern, commonly seen in polyelectrolyte protein interactions, such as the binding of histone H1.0 and thymosin α. The steady-state fitting shows that the binding amount saturates with increasing IL-10 ligand concentration, and the curve curves downwards. This is a characteristic of specific binding in fast binding and fast adsorption; otherwise, there is no saturation phenomenon in non-specific fast binding and fast adsorption, and the binding amount is proportional to the concentration. This result further illustrates that the binding sites of laminin LN with growth factor GF and the binding sites of laminin LN with immune factors are not the same.

[0052] Example 7: Treatment of diabetic wounds in mice with IL-10 using a hydrogel dressing containing covalently doped laminin LN111. First, following the method in Example 2, ten LN111 covalently doped hydrogels loaded with IL-10, IL-10 + VEGF-A165, or without any biological factors were prepared. After hydrogel copolymerization functionalization of laminin LN111, four-arm polyethylene glycol-maleimide (molecular weight 20 kg / mol, LaysanBio) was dissolved in PBS to a concentration of 200 mg / mL. 6.54 μL of LN111 modified with maleimide groups (prepared in this invention, final concentrated concentration 1.14 mg / mL), 10.35 μL of four-arm polyethylene glycol-maleimide, 2 μL of 50 μg / mL IL-10 + 2 μL of PBS, or 2 μL of 50 μg / mL IL-10 + 2 μL of 50 μg / mL VEGF-A165, or 4 μL of [unspecified ingredient] were added to a 0.5 mL centrifuge tube. PBS was used as hydrogel solution A, which was loaded with IL-10, IL-10+VEGF-A165, or no biological agents. Ten aliquots of solution A were prepared for each of the three conditions as biological replicates. The solutions were allowed to stand at room temperature for 1 hour to allow sufficient adsorption of IL-10 by LN111. 2 μL of 200 mg / mL SH-PEG-SH (p-dimercapto-polyethylene glycol, LaysanBio) dissolved in PBS was taken into an empty 0.5 mL centrifuge tube as solution B. Thirty aliquots of solution B were prepared. Each aliquot of solution A or blank control solution A was mixed with one aliquot of solution B and placed on the surface of a hydrophobic sealing film to form approximately spherical droplets. The mixture was incubated at 37 °C for 30 minutes to form a cross-linked structure. After sealing, the mixture was stored at 4 °C.

[0053] In this embodiment, ten male C57BLKS-LeprdbDock7misty (i.e., BKS-db or db / db transgenic diabetic mice) mice aged 12-13 weeks (Guangzhou Yaokang) were used. After shaving the hair on the back of the mice, four full-thickness skin puncture biopsy wounds with a diameter of 6 mm were made on the back of each mouse using a skin biopsy device. Subsequently, one of the aforementioned LN111+IL-10 hydrogel, LN111+IL-10 / VEGF-A165 hydrogel dressing, and LN111 covalently doped hydrogel without any immune or growth factors were placed on three of the wounds, respectively. At the same time, a 6 mm diameter circular 3M cellulose acetate dressing (Adaptic®) was used to cover the fourth wound as a control. The wound size was measured and photographed daily.

[0054] Fourteen days later, the mice were euthanized, and the skin around the wound and the underlying muscle layer were immediately excised along the wound edge to assess the full-thickness tissue repair. The obtained tissue samples were fixed and preserved in 4% paraformaldehyde. Immunosectioning and scanning sectioning were performed on each sample in a paraffin-embedded cassette: Each sample was placed in a labeled embedding cassette and dehydrated using a gradient ethanol program: 75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, and anhydrous ethanol in chambers I and II for 30 min each; followed by clearing with ethanol-xylene for 10 min and xylene for 10 min. After clearing, the samples were immersed in molten paraffin at 65°C three times consecutively (paraffin chambers I–III), each time for 1 h. After paraffin infiltration, the molten paraffin was embedded using a Leica embedding machine. The molten paraffin was injected into a pre-marked mold and then cooled at –20°C to allow the paraffin block to solidify fully and reach a suitable hardness for sectioning. Before histological staining, the paraffin block was cut into sections of 3–5 μm thickness using a Leica microtome. Twenty sections were cut from each sample. The sections were spread in 37°C warm water and then attached to adhesive slides. They were then dried in a 60°C oven to remove residual paraffin and enhance the adhesion of the sections.

[0055] One slice from each sample was used for HE and Masson trichrome staining, respectively. The results are as follows: Figure 9 As shown. Figure 9 These are photographs of the mouse experimental endpoint and HE and Masson staining results of wound tissue sections in this embodiment, scale bar = 1000 μm. It can be seen that LN111 hydrogel loaded with IL-10 (regardless of whether VEGF-A165 is added) provides the best healing effect. In HE staining, LN111 hydrogel loaded with IL-10 (regardless of whether VEGF-A165 is added) provides the best healing effect, with the wound surface filled with relatively uniform granulation tissue. In contrast, the granulation tissue in the center of the wound treated with LN111 hydrogel without IL-10 loading was significantly thinner. In Masson staining, the wound area covered by LN111 hydrogel loaded with IL-10 (regardless of whether VEGF-A165 is added) showed relatively more blue collagen deposition. Both HE and Masson staining showed that the Adaptic dressing (i.e., the control group) showed the worst wound healing.

[0056] Figure 10The figure shows the statistical results of wound healing in mice under various conditions from day 8 to day 14 of this embodiment. a) is the curve showing the percentage of wound healing relative to the initial area as a function of daily changes. In all four conditions, regardless of whether VEGF-A165 was added, LN111 hydrogel loaded with IL-10 significantly improved wound healing, which was superior to unloaded LN111 hydrogel dressing or 3MAdaptic dressing. Statistical analysis was performed using two-way ANOVA. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. b) to e) represent the ranking of healing rates on the backs of each mouse under the fourteenth day of the experiment, and the results were then summarized. The best and second-best healing rates in most mice were mainly produced by the IL-10-loaded LN111 hydrogel (regardless of whether VEGF-A165 was added), indicating that the IL-10-loaded LN111 hydrogel constructed in this embodiment has the effect of promoting wound healing in diabetic patients. In most mice, the wound healing rate of the LN111 hydrogel without IL-10 ranked third, while the healing rate of the Adaptic dressing ranked fourth.

[0057] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A polypeptide with non-covalent adsorption capacity for immune factors, characterized in that, The amino acid sequence of the polypeptide is selected from one or more of SEQ ID NO.1 to SEQ ID NO.

38.

2. The application of the polypeptide as described in claim 1 in the preparation of biomaterial composites.

3. A biomimetic non-covalently adsorbed and sustained-release immune factor biomaterial composite, characterized in that, This includes two-dimensional or three-dimensional biomaterial substrates, carrier proteins and immune factors that are non-covalently adsorbed as immune factors.

4. The biomaterial composite as described in claim 3, characterized in that, The carrier protein for the non-covalently adsorbed immune factors is one or more of the following: complete recombinant protein of each subtype of laminin LN containing the polypeptide with non-covalent adsorption capacity for immune factors as described in claim 1, and complete recombinant fibronectin FN.

5. The biomaterial composite as described in claim 3, characterized in that, The immune factor is either an interleukin-type immune factor or a non-interleukin-type immune factor.

6. A method for preparing a biomaterial composite as described in any one of claims 3 to 5, characterized in that, Includes the following steps: S1. The carrier protein that non-covalently adsorbs immune factors is covalently or non-covalently bound to a two-dimensional or three-dimensional biomaterial substrate to obtain a substrate modified with the carrier protein. S2. Immerse or infiltrate the substrate modified with carrier protein obtained in step S1 into immune factors and incubate at room temperature to obtain the final product.

7. The use of the polypeptide as described in claim 1 in the preparation of an anti-inflammatory hydrogel dressing for diabetic wounds.

8. A hydrogel dressing for anti-inflammatory wounds in diabetic patients, characterized in that, It is formed by cross-linking a hydrogel precursor solution through a copolymerization reaction. The hydrogel precursor solution includes a hydrogel material, a carrier protein that can be copolymerized into the hydrogel material after functionalization, an immune factor that is non-covalently adsorbed with the carrier protein, a cross-linking agent, and an aqueous medium.

9. The anti-inflammatory hydrogel dressing for diabetic wounds as described in claim 8, characterized in that, The hydrogel material is one or more of acryloyl polyethylene glycol, maleyl polyethylene glycol, and diazo-based polyethylene glycol; the carrier protein that can be copolymerized into the hydrogel material after functionalization is generated by reacting one or more of the intact recombinant protein of each subtype of laminin LN containing the polypeptide with non-covalent adsorption capacity for immune factors as described in claim 1, and intact recombinant fibronectin FN with acryloyl-succinimide valerate, maleyl-succinimide valerate, or diazo-succinimide valerate; the immune factor that is non-covalently adsorbed with the carrier protein is an interleukin-type immune factor or a non-interleukin-type immune factor; the crosslinking agent is p-dithioglycol polyethylene glycol, and the dispersion concentration in the hydrogel precursor solution is 0.1~100 mg / mL; the aqueous medium is deionized water, physiological saline, or phosphate buffer solution.

10. The use of complete recombinant proteins or complete recombinant fibronectins of various subtypes of laminin comprising the polypeptides of claim 1 that have non-covalent adsorption capacity for immune factors in the preparation of biomaterials for the adsorption and sustained release of immune factors.

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