A mycosporin-like amino acid-protein cross-linked complex and its preparation method and application

Abstract

The present invention discloses a mycosporin-like amino acid-protein cross-linked complex and a preparation method and application thereof. The mycosporin-like amino acid-protein cross-linked complex has a structural formula as shown in the following formula (I): #imgabs0# (I); in the formula (I), R is -CH2OH or -CH(OH)CH3, and the curve connected by amide bonds represents the peptide chain of the protein. Also disclosed is a hydrogel prepared by using the mycosporin-like amino acid-protein cross-linked complex. And the mycosporin-like amino acid-protein cross-linked complex or the hydrogel is used in the preparation of ultraviolet protection, anti-aging, wound management biological scaffolds, anti-inflammatory, skin repair or hair care products, as well as in the development of biomaterials, medical dressings or cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of biology and medicine technology, and specifically relates to a mycosporin-like amino acid-protein cross-linked complex and a preparation method and application thereof. Background Art

[0002] UVA is an ultraviolet ray with a wavelength of 320 to 420 nm, also known as long-wave dark spot effect ultraviolet ray. UVA accounts for 95% of the surface ultraviolet rays and has extremely strong penetrating power. It can penetrate most transparent glass and plastics directly into the dermis and even subcutaneous layer of the skin ( JAMA Dermatol . 2014, 150, 401-406). It destroys the microstructure of the skin, such as elastic fibers and collagen fibers, basement membrane, capillaries and hair follicles, causing skin and hair follicle aging, resulting in pigmentation, skin sagging, wrinkles, hair loss, etc. This is medically known as photoaging ( J. Am. Acad. Dermatol. ,2006, 5, 1-19). For open wounds, UVA exposure can cause severe scabs and prolong healing time. UVA can produce highly active chemical intermediates, such as hydroxyl radicals and oxygen free radicals, which can damage DNA ( Free Radical Biol. Med .2009, 47, 381-388). This has a mutagenic effect on basal cell keratinocytes and can induce the immunosuppressive effect of sunlight exposure, which has a suppressive effect on the body's overall immunity. The large amount of free radicals produced by UVA can also lead to aging-related secretory phenotypes and damage-related molecular patterns, drive cell death and aggravate inflammatory responses. For open wounds, it will lead to difficulties in healing. Poor wound management may cause effects. UVA can also cause skin pigmentation and damage the durability of medical materials (such as wound dressings) or cosmetics.

[0003] Mycosporine-like amino acids (MAAs) are a class of small molecule compounds produced by marine plankton and red algae that have good photoprotective activity against ultraviolet radiation, especially the UVA band. For example, shinorine and porphyra-334 have maximum absorption at 330-334 nm, and the molar absorption coefficient is as high as 40,000-44,000 M⁻¹cm⁻¹ ( FEMS Microbiol. Lett . 2007, 269, 1-10.; Mar. Drugs2017, 15,326.), is the strongest known natural UV absorber. Currently, commercial sunscreen products mainly rely on chemically synthesized UV absorbers, such as inorganic UV absorbers such as TiO2 and ZnO, and organic UV absorbers such as fat-soluble oxybenzone. These substances have been proven to pose a serious threat to the environment, especially sensitive marine ecosystems such as coral ecosystems, and long-term use may have adverse effects on the human body ( Marine Drugs . 2017, 15). Different from commercialized UV protection agents, MAAs are a class of water-soluble small molecule compounds with important application potential in the development of medicine, cosmetics and as natural sunscreens.

[0004] However, the good water solubility of MAAs also makes them easy to be lost quickly in water, sweat, etc., or prone to excessive percutaneous absorption. Therefore, without changing their UV protection activity, cross-linking MAAs with other functional molecules is expected to effectively solve the safety and effectiveness issues of MAAs compounds in practical applications. Molecular cross-linking, especially the cross-linking of functional proteins, has a wide range of applications in biomedicine, chemical analysis, materials science and other fields. Common cross-linking agents, such as disulfide cross-linking agents, isocyanate cross-linking agents, etc., do not have specific functions other than connecting molecules. Common chemical cross-linking agents do not have other functionalities themselves. Summary of the invention

[0005] The primary purpose of the present invention is to provide a mycosporin-like amino acid-protein cross-linked complex and a preparation method thereof.

[0006] Another object of the present invention is to provide a hydrogel prepared by using the mycosporin-like amino acid-protein cross-linked complex.

[0007] Another object of the present application is to provide the use of the above-mentioned mycosporin-like amino acid-protein cross-linked complex or the hydrogel in the preparation of ultraviolet protection, anti-aging, wound management biological scaffolds, anti-inflammatory, skin repair or hair care products, as well as in the development of biomaterials, medical dressings or cosmetics.

[0008] The first object of the present invention can be achieved by the following technical solution: a mycosporin-like amino acid-protein cross-linked complex, the structural formula of which is shown in the following formula (I):

[0009] (I);

[0010] In formula (I), R is -CH2OH or -CH(OH)CH3, and the curve connected by amide bonds represents the peptide chain of the protein.

[0011] Preferably, the mycosporin-like amino acid contains at least one carboxyl group; and the protein contains a free primary amino group.

[0012] Preferably, the method of cross-linking the mycosporin-like amino acid to a protein of the present invention is achieved by forming an amide bond or a peptide bond between the carboxyl group of the mycosporin-like amino acid and the primary amino group of the protein molecule.

[0013] Further preferably, the mycosporin-like amino acid-protein complex is mainly formed by cross-linking the carboxyl group of the mycosporin-like amino acid shown in the following formula (II) and the primary amino group of the protein shown in the following formula (III):

[0014] (II) (III);

[0015] In formula (II), R is -CH2OH or -CH(OH)CH3.

[0016] That is, preferably, the mycosporin-like amino acid is porphyra-334 (P334) or shinorine (SH).

[0017] The mycosporin-like compounds described in the present invention are natural small molecule ultraviolet protective agents, have multiple good biological activities such as good water properties and good safety, can be efficiently prepared through a synthetic biology system, and have methods and good application scenarios in the fields of biomedicine and cosmetics.

[0018] Preferably, the mass ratio of the mycosporin-like amino acid to the protein molecule is 2-4:50.

[0019] Preferably, the protein may be any natural or engineered protein, and may also be an enzyme or a polypeptide.

[0020] Preferably, the protein is recombinant human type XVII collagen, which has one or more sequences as shown in SEQ ID NO:1.

[0021] More preferably, the protein is recombinant human type XVII collagen, the sequence of which is shown in SEQ ID NO:2.

[0022] Preferably, the mycosporin-like amino acid-protein cross-linked complex of the present invention is rhCOL17-P334 or rhCOL17-SH.

[0023] As an improvement of the present invention, the method of cross-linking mycosporin-like amino acids to proteins is achieved by a carboxyl activator or a cross-linking agent.

[0024] Preferably, the carboxyl activation reagent, or cross-linking reagent, includes but is not limited to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0025] Therefore, the method of cross-linking mycosporin-like amino acids to proteins is achieved by carboxyl activating agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC / N-hydroxysuccinimide NHS or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride DMTMM.

[0026] Preferably, the mycosporin-like amino acid is first activated by an activator before cross-linking, and the activator is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride DMTMM. When a mixture of EDC and NHS is used as the activator, the molar ratio of the mycosporin-like amino acid to the activators EDC and NHS is 1:2:2; when the activator is DMTMM, the molar ratio of the mycosporin-like amino acid to the activator DMTMM is 1:2.

[0027] Preferably, the bio-crosslinking process of the present invention is to first activate the carboxyl group of mycosporin-like amino acids with a crosslinking reagent such as EDC / NHS or DMTMM to generate activated mycosporin-like amino acids (amine-reactive MAAs) that can react with primary amino groups, referred to as ar-MAAs; react the ar-MAAs with primary amino donor proteins or polypeptides to generate biomacromolecule-MAA crosslinked complexes; preferably, the MAAs contain two carboxyl groups, such as porphyra-334 (P334) or shinorine (SH), and MAAs act as linker molecules to crosslink different protein molecules to form crosslinked complexes of protein biomacromolecules crosslinked by MAA.

[0028] In the present invention, a carboxyl activator or a molecular cross-linking agent, such as EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide) or DMTMM (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride) cross-linking technology is used to activate the carboxyl group of the mycosporin-like amino acid, so that it is cross-linked to a protein molecule rich in free primary amino groups to form a new hydrogel and other composite materials; not only can the properties of the macromolecular material itself be improved, but also the problems of easy loss and easy absorption of MAAs due to their good water solubility are effectively solved.

[0029] The present invention also provides a method for preparing the above-mentioned mycosporin-like amino acid-protein cross-linked complex, comprising the following steps:

[0030] (S1) protein preparation;

[0031] (S2) activating the mycosporin-like amino acid with an activating agent;

[0032] (S3) Cross-linking the activated mycosporin-like amino acid with the protein in step (S1) to obtain a mycosporin-like amino acid-protein cross-linked complex.

[0033] The protein-MAA cross-linked complex of the present invention, namely protein-P334 or protein-SH itself can be used as a functional material for application development; it can also be prepared together with other materials into a macromolecular composite system such as a hydrogel.

[0034] The above second object of the present invention can be achieved by the following technical scheme: a hydrogel is prepared by adding the mycosporin-like amino acid-protein cross-linked complex into a matrix material methacrylated gelatin GleMA containing a photoinitiator.

[0035] Preferably, the mass percentage of protein in the mycosporin-like amino acid-protein cross-linked complex in the hydrogel is 0.2-1%.

[0036] More preferably, the mass percentage of protein in the mycosporin-like amino acid-protein cross-linked complex in the hydrogel is 0.5%.

[0037] Preferably, the photoinitiator is phenyl-2,4,6-trimethylbenzoylphosphine lithium LAP, and the mass percentage of the photoinitiator in the hydrogel is 0.2-0.3%.

[0038] More preferably, the photoinitiator is phenyl-2,4,6-trimethylbenzoylphosphine lithium LAP, and the mass percentage of the photoinitiator in the hydrogel is 0.25%.

[0039] Preferably, the methacrylated gelatin GleMA is methacrylated gelatin in which 30-90% of the amino groups are substituted by methacryloyl groups, and the mass percentage of the methacrylated gelatin GelMA in the hydrogel is 8-15%.

[0040] More preferably, the methacrylated gelatin GleMA is methacrylated gelatin in which 30% of the amino groups are substituted by methacryloyl groups, and the mass percentage of the methacrylated gelatin GelMA in the hydrogel is 10%.

[0041] The last object of the present invention can be achieved by the following technical solution: application of the above-mentioned mycosporin-like amino acid-protein cross-linked complex or the above-mentioned hydrogel in the preparation of UV protection, anti-aging, wound management biological scaffold, anti-inflammatory, skin repair or hair care products.

[0042] Furthermore, the present invention also discloses the application of the mycosporin-like amino acid-protein cross-linked complex or the hydrogel in the development of biomaterials, medical dressings or cosmetics.

[0043] The application in biomaterials refers to the use of the above method to modify or cross-link proteins so that they have different material properties, biological functions, medical treatment functions or skin protection and repair functions, and to develop corresponding products as components.

[0044] The material properties include, but are not limited to, UV protection, mechanical properties, hydrophilicity, adhesion, ease of use, transparency or wearability.

[0045] The biological functions include but are not limited to antioxidant activity, cell aging protection activity, mitochondrial dysfunction or cell proliferation promotion.

[0046] The medical treatment functions include but are not limited to wound repair, skin disease treatment and management.

[0047] The cosmetic applications include but are not limited to skin protection, basement membrane repair, hair care, whitening and spot removal functions.

[0048] The present invention has the following advantages:

[0049] (1) The present invention utilizes the carboxyl group contained in the side chain of the mycosporin-like amino acid itself as a reactive group to undergo a coupling reaction with the primary amino group of the protein to form a mycosporin-like amino acid-protein covalent complex, wherein the mycosporin-like amino acid containing a double carboxyl group can cross-link the protein or polypeptide containing the primary amino group to form a novel polymer network structure. The cross-linking chemistry does not affect the mycosporin-like amino acid's own protective activity against ultraviolet radiation, especially UVA, and also avoids the problem that the mycosporin-like amino acid with good water solubility is easily lost in water, sweat, etc. and excessively absorbed through the skin in actual applications;

[0050] (2) The present invention activates the carboxyl group of MAAs through carboxyl activator DMTMM or EDC / NHS, and cross-links with proteins or polypeptides containing free primary amino groups, thereby improving the material properties while maintaining the UV protection activity of MAAs themselves, effectively improving the problem that MAAs are easily lost through water or overabsorbed, and providing a new method for developing new functional materials with UVA protection activity and applying them to biomaterials, polymers, drug delivery systems, medicine, and beauty and cosmetics development;

[0051] (3) The mycosporin-like amino acid-protein covalent complex of the present invention is modified by chemical coupling of mycosporin-like amino acids, and the protein is endowed with ultraviolet protection activity and good mechanical properties, adhesion properties, and good biological, medical and skin protection and repair activities, and can be applied to the development of new biomaterials, dressings, medical devices, pharmaceutical products or cosmetics and other industry products;

[0052] (4) The method for preparing the protein-MAAs complex or further preparing the hydrogel using the complex provided by the present invention is simple and efficient, and can quickly prepare functional biomaterials or related preparations;

[0053] (5) The protein-MAAs-based hydrogel provided by the present invention has good UV protection and protective effects on skin, wounds, hair follicles, etc., and has excellent practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described below with reference to the accompanying drawings in conjunction with embodiments;

[0055] In all quantitative comparison graphs, all data are expressed as mean ± SD (n ≥ 3); statistical significance was determined by one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test;

[0056] Figure 1 is the expression plasmid map of the recombinant human collagen rhCOL17 with the collagen sequence Q4R3T in Example 1;

[0057] Figure 2 This is the SDS-PAGE gel electrophoresis analysis of the recombinant human collagen rhCOL17 in Example 1. In each lane, P: precipitate, S: supernatant, FT: flow-through, W: wash, E: elution, AC: TEV enzyme cleavage product at 4°C;

[0058] Figure 3 is a chemical schematic diagram of preparing a protein-porphyra-334 cross-linked complex using EDC / NHS in Example 1;

[0059] Figure 4 is a schematic diagram of preparing a double-layer hydrogel GCP using rhCOL17-P334 and GelMA in Example 1;

[0060] Figure 5 The pore size diagram and scanning electron microscope diagram of the GCP hydrogel in Example 1, wherein Figure A is the pore size diagram of the GCP hydrogel, Figure B is the scanning electron microscope diagram, l-GCP, m-GCP and h-GCP represent GCP with rhCOL17 contents of 0.2%, 0.5% and 1.0% (w / w), respectively, the same below;

[0061] Figure 6 This is the 1H NMR analysis in Example 1. The colored shaded resonances correspond to the protons indicated in the structure in the upper right portion of the figure. G: GelMA; C: rhCOL17; GC: GelMA containing rhCOL17;

[0062] Figure 7 The FTIR spectrum of the biomaterial functional groups analyzed in Example 1, G: GelMA; C: rhCOL17; GC: GelMA containing rhCOL17, GP: GelMA containing porphyra-334;

[0063] Figure 8 It is the mechanical property analysis of the GCP hydrogel in Example 2, Figure A: average storage modulus (G') and loss modulus (G"), Figure B: cyclic compression curve; Figure C: stress-strain curve;

[0064] Fig. 9 is the adhesion performance of the GCP hydrogel in Example 2, Figure A: Adhesion of GCP to different organs and finger joints, Figure B: Adhesion strength test of GCP hydrogel on pig skin, second harmonic generation (SHG) imaging shows that the hydrogel adheres to the pig skin, and the interface is highlighted by a yellow dotted line, Figure C: Adhesion strength data of the adhesion test experiment on the bar graph (n = 3), l-GCP, m-GCP and h-GCP represent GCP with rhCOL17 contents of 0.2%, 0.5% and 1.0% (w / w), respectively;

[0065] Fig.10 It is the UV protection performance test in Example 2. Figure A: Schematic diagram of the UV protection experimental device, evaluating the protective effect of the sample on human dermal fibroblasts cultured in phenol red-free medium under UVA (40 μW / cm²), Figure B: UVA shielding rate of GC, GCP and ANESSA (Shiseido) measured over time, GC: GelMA containing rhCOL17;

[0066] Fig.11 is the evaluation of the protective effect of GCP hydrogel on cells in Example 3, Figure A: Immunofluorescence images showing mitochondrial aggregates (red) and monomers (green) in fibroblasts under different treatments, and the cell nuclei were stained with DAPI (blue), (scale bar = 20 μm), Figure B: Stimulated emission depletion (STED) imaging depicts the changes in mitochondrial structure in different treatment groups, and the yellow dotted outline indicates the cell boundary (scale bar = 5 μm), Figure C: Quantitative analysis of mitochondrial structure based on STED images, Figure D: NADP+ / NADPH ratio and SOD activity measurements in fibroblasts;

[0067] Fig.12 The GCP gel in Example 4 promotes wound healing. Figure A: Photos and heat maps showing the progress of wound healing over time under UVA irradiation. Figure B: Quantitative analysis of wound healing effect: The upper and lower figures show the percentage of wound contraction and healing over time at each time point, respectively;

[0068] Fig.13 This is the reaction process of preparing a protein-shinorine cross-linked complex by activating shinorine with DMTMM in Example 5;

[0069] Fig.14 is a schematic diagram of preparing a double-layer cross-linked GCH hydrogel using rhCOL17, shinorine and GelMA in Example 5;

[0070] Fig.15 5 is an evaluation of the anti-aging effect of GCH hydrogel on fibroblasts in Example 5, Figure A: Schematic diagram of fibroblast culture setting, Figure B: After the cells were irradiated with UVA for 48 hours, the supernatant was collected for ELISA analysis, and the bar graph shows the expression level of IL-8 after treatment, Figure C: After the cells were irradiated with UVA for 48 hours, the supernatant was collected for ELISA analysis, and the bar graph shows the expression level of TNF-α after treatment, Figure D: After the cells were irradiated with UVA for 48 hours, the supernatant was collected for ELISA analysis, and the bar graph shows the expression level of IL-6 after treatment, Figure E: NETs (neutrophil extracellular traps) induced by IL-8 (50 ng / mL) were displayed by staining with Sytox Green and DAPI, Figure F: Average fluorescence intensity of NETs, ​​Figure G: Immunofluorescence staining with CitH3 (green), MPO (red) and DAPI (blue) showed NETs formation induced by IL-8;

[0071] Fig.16 Figure A is a diagram showing the repair effect of GCH hydrogel on mouse skin wounds exposed to UVA by H&E in Example 5, Figure B is a diagram showing the collagen deposition of GCH hydrogel in the wound repair area by Masson staining in Example 5, Figure C is a diagram showing the effect of GCH hydrogel on the volume fraction of skin collagen in Example 5, and Figure D is a quantitative comparison diagram of the number of hair follicles observed in the wound sections stained by H&E in Example 5;

[0072] Fig.17The GCH hydrogel in Example 5 promotes collagen expression and accelerates skin repair, wherein Figure A and Figure B are immunofluorescence staining of collagen IV (COL4) and collagen XVII (COL17) in wound tissue on the 5th and 10th days of skin trauma, respectively, and the yellow dotted line highlights the repaired skin surface (scale bar = 500 μm). DETAILED DESCRIPTION

[0073] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0074] The reagents and materials used in the following examples, unless otherwise specified, are from commercial sources. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0075] Example 1 A GelMA-human recombinant collagen XVII-porphyra-334 cross-linked hydrogel and its construction method

[0076] 1. Human recombinant collagen XVII (rhCOL17)

[0077] 1.1 Artificial sequence design: The human collagen 17 (rhCOL17) sequence was selected from GenBank NP_000485, in which a partial fragment Q4R3T was selected, and its amino acid sequence is shown in SEQ ID NO: 1;

[0078] GPVGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAK (as shown in SEQ IDNO: 1).

[0079] The sequence shown in SEQ ID NO: 1 is repeated three times in series to form the final artificial collagen sequence having human collagen rhCOL17 collagen domain 15, namely human recombinant collagen XVII, whose amino acid sequence is shown in SEQ ID NO: 2;

[0080] GPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGPVGLQGLRGEVGLPGVGEPGAKGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAK (as SEQ ID NO:2 shown).

[0081] 1.2 Molecular cloning: A DNA sequence encoding a 6×His affinity chromatography tag at the N-terminus as shown in SEQ ID NO: 2 and optimized by codons was synthesized and inserted into the KpnⅠ and XhoⅠ restriction sites of the pET-32a(+) vector by conventional molecular cloning techniques to obtain the rhCOL17 recombinant expression plasmid Q4R3T_pET32. The specific recombinant expression plasmid map is shown in Figure 1 shown.

[0082] 1.3 Induced expression of collagen: Transform competent Escherichia coli with the above plasmid Q4R3T_pET32 E. coli BL21(DE3) (Invitrogen) was used to obtain the production engineering strain of rhCOL17 E. coli rhCOL17a; induced by 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) 600 Position 0.6-0.8 E. coli rhCOL17c, incubate at 16°C with shaking for 16-24 h.

[0083] 1.4 Protein separation and purification: Recover the bacteria in 1.3, suspend them in a 20 mM phosphate lysis buffer (20 mM) containing 20 mM imidazole and 500 mM sodium chloride, and centrifuge for 20 min after ultrasonic disruption of the cells. Collect the supernatant and separate and purify the target protein using an NTA-Ni affinity chromatography column; first use 10 column volumes of balanced 20 mM phosphate buffer (containing 20 mM imidazole, 500 mM sodium chloride, pH 7.4) to elute the impurities; then use 20 mM phosphate buffer (containing 400 mM imidazole, 500 mM NaCl, pH 7.4) to elute, and the target protein rhCOL17a is obtained. The specific protein expression and purification conditions are as follows. Figure 2 shown.

[0084] 2. Preparation of P334 Refer to application number 202411738807.5 for the construction, preparation, separation and purification of porphyra-334 production engineering strain.

[0085] 3. Preparation of GelMA-rhCOL17-Porphyra-334 cross-linked complex

[0086] 3.1 Activation of P334

[0087] First, P334 (molecular weight 346 g / mol) was dissolved in phosphate buffered saline (PBS) to prepare a 2 mg / mL stock solution. 1.5 mL of the p334 solution was slowly added with 6.3 mg of EDC and 3.8 mg of NHS, respectively, so that the molar ratio of P334, EDC and NHS in the solution was 1:2:2. If DMTMM was used to activate the carboxyl group, 9.0 mg of DMTMM was added to make the molar ratio of P334 to the activator DMTMM 1:2. The solution was stirred at 40°C for 30-60 minutes to activate the two carboxyl groups of P334 in the molecule, generating ar-P334 ( Figure 3 ).

[0088] 3.2 Preparation of GelMA-rhCOL17-P334 (GCP) hydrogel

[0089] GCP Preparation:

[0090] Prepare a 100 mg / mL rhCOL17 solution with PBS, take 0.5 mL (50 mg) and add it to the above 1.5 mL of ar-P334 to react with P334 to form 2 mL of rhCOL17-P334 cross-linked complex solution ( Figure 3 );

[0091] Prepare a GelMA solution containing the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP): take 0.25 g of LAP and add it to 8 mL of PBS for ultrasonic dissolution, then add 1 g of GelMA and heat it in a 70 °C water bath in the dark until it is completely dissolved;

[0092] Then, the above 2 mL of rhCOL17-P334 cross-linked complex solution was slowly added to the 8 mL of LAP-containing GelMA solution while stirring to prepare a semi-solid, injectable single-layer cross-linked GCP hydrogel, which was stored in the dark for later use; in the GelMA molecules used, 30% of the amino groups had been replaced by methacryloyl groups; the GCP hydrogel was m-GCP with a rhCOL17 content of 0.5% (w / w); for l-GCP and h-GCP, the initial rhCOL17 concentrations were adjusted to 40 mg / mL and 200 mg / mL, respectively, so that the final contents of rhCOL17 were 0.2% and 1%, respectively, and the other preparation conditions remained unchanged.

[0093] When the above-mentioned single-layer cross-linked GCP hydrogel is injected into the wound or skin surface, it can be irradiated with 405 nm visible light (at a distance of 20 cm) to induce the cross-linking reaction of the photoGelMA molecules to generate the final gelled double-layer cross-linked molecular structure of the GCP hydrogel ( Figure 4 ); The cross-linking reaction can also be easily initiated by exposure to sunlight, with an average gelation time of ~8 s ( Figure 5 Figure A in the middle); The microstructure of the freeze-dried GCP hydrogel was analyzed by Sigma 300 scanning electron microscope (SEM, Zeiss, Germany); The hydrogel samples that were frozen in liquid nitrogen and freeze-dried for 48 hours were gold-sprayed on the SEM slicer and their morphology was analyzed in detail; The results showed that all hydrogel groups showed an interconnected porous structure, and the pore size decreased with the increase of rhCOL17 concentration; The average pore size was 2.17±0.09 μm (containing 0.5% rhCOL17), which was significantly smaller than that of GelMA (3.17±0.03 μm) ( Figure 5 These experimental results show that GCP hydrogel has the expected chemical structure and exhibits good microscopic morphology and nanoscale structural properties.

[0094] GCP structure characterization: In order to confirm the success of the cross-linking reaction and characterize the microstructure of GCP, the hydrogel liquid sample was analyzed using a Bruker AvanceII hydrogen nuclear magnetic resonance spectrometer (D2O solvent). 1 H NMR (Bruker AVANCE III 500, Germany) was used to confirm the success of the cross-linking reaction; Nicolet iS50 Fourier transform infrared spectrometer (ThermoFisher Scientific, USA) was used to analyze the fluorescence intensity at 400–4000 cm −1 FTIR spectra of the hydrogel samples were recorded in the range.

[0095] The results showed that after GCP was introduced into P334 1 H-NMR gives the weakest resonance signal at δ2.91 (methylene adjacent to the primary amino group) Figure 6 ), indicating that the activated carboxyl group of P334 effectively captures the free primary amino group of rhCOL17 and couples it with amide bonds; at the same time, in the infrared spectrum of GCP, the C=O stretching vibration of amide I band, the NH bending and CN stretching vibration of II band are all enhanced, indicating that a cross-linking structure dominated by amide bonds is formed between P334 and the main material ( Figure 7 ).

[0096] Example 2 Mechanical properties and UVA protection function of GCP hydrogel

[0097] Mechanical properties: Compression tests were performed using a universal testing instrument (INSTRON 5943; MA, US). The samples were prepared as standard cylinders (10 mm in diameter and 5 mm in height) and a compressive load was applied at a strain rate of 2 mm / min to obtain a strain-stress curve. The compression modulus was calculated based on the curve. The hydrogels were subjected to a cyclic compression test, in which 60% compression was performed at a strain rate of 5 mm / min for 20 consecutive cycles. Frequency sweep tests were performed at room temperature (25°C) at a strain amplitude of 1.0% (axial force was controlled at 0.6 N ± 10%) over a frequency range of 10 -2 ~10 1 Hz.

[0098] The results show that the storage modulus (G′) of GCP is in the range of 300–500 Pa (Young’s modulus: 0.9–1.5 kPa, Figure 8 Figure A in the middle), which is lower than or close to the elastic limit of human skin (4.5–8.0 kPa), and exhibits the rheological characteristics of irreversible gel (G′>G″); after the GCP hydrogel was subjected to 20 cycles of compression testing in the strain range of 0-60%, no obvious plastic deformation was observed, the hysteresis loop was small, and it exhibited good fatigue resistance ( Figure 8 Middle B); The elongation at break of GCP increased by about 1 times compared with GelMA (from ~100% to >200%) ( Figure 8 (Figure C).

[0099] These results indicate that the introduction of rhCOL17-P334 can significantly enhance the tensile properties of the hydrogel, enabling it to maintain higher strength and ductility within a higher strain range; as expected, the double-network hydrogel GCP has robust mechanical properties, ensuring that it will not break or fail during daily activities.

[0100] Adhesion performance: Adhesion is the basic performance of GCP in practical applications, durability, functional effectiveness and wearability.

[0101] To test adhesion: GCP was tested for adhesion to mouse organs and human finger joint skin surfaces, and a lap shear test was applied to determine the adhesion strength of GCP hydrogel on tissues. GCP hydrogel was applied to two pieces of pig skin with a bonding area of ​​20 × 20 mm. After the samples were connected, they were stretched to failure (0.1 mm / min) using a universal testing instrument at a crosshead speed of 2 mm / min. Fig. 9); Before the GCP is subjected to light-induced CC cross-linking, the hydrogel exhibits good fluidity and can be injected into the wound site and adaptively diffuse into the irregular wound interior, which is essential for forming a tight connection between the base surface and the substrate; under natural light, the dressing can gel quickly and can still adhere to the skin surface under relatively severe vibration and shaking, and can form a firm adhesion with organs and skin when covering organs and joints ( Fig. 9 Using the characteristics of skin tissue autofluorescence under second harmonic generation, it was observed that the GCP (indicated by the yellow dashed line and arrow) can completely and tightly adhere to the surface of pig skin (auto-green fluorescence), showing a strong bonding strength ( Fig. 9 The adhesion strength of GCP is 8.22±0.53 KPa, which is about twice that of GelMA and exceeds the adhesion strength of standard commercial dressings (usually about 5 kPa). Fig. 9 (C in the middle); both exhibited excellent injectability, wound conformability, adhesion, and wearability.

[0102] UV shielding performance: Comparison of GCP hydrogel and commercial sunscreen shielding agent ANESSA ® UV Milk (containing ethylhexyl methoxycinnamate, bis-ethylhexyloxyphenol methoxyphenyl triazine, and TiO2 / ZnO) was deposited on clean quartz plates, and these quartz plates were immersed in artificial sweat (pH 6.6); the quartz plates were illuminated under 330 nm UV-LED (Longpro LTD., Guangzhou, China) light, and the light power was measured at preset time points using a UV light power meter (Lianhuicheng LH-125, Guangzhou, China). Fig.10 The UV shielding rate is calculated as follows: UV shielding rate = ((J0-J) / J)*100%, where J0 and J are the light powers under the clean quartz plate and the quartz plate coated with different samples.

[0103] The results showed that under the experimental conditions, the shielding effect of GCP against ultraviolet rays remained above 55%, significantly reducing the ultraviolet power; the ANESSA group showed better UVA shielding performance at the beginning of the experiment, but because it was difficult to maintain the coating state for a long time and aggregated into spots, the UVA shielding performance gradually weakened to 16.68%±5.26%; in comparison, GCP showed an extremely stable ultraviolet protection effect during the test process of up to 500 minutes, indicating that the addition of P334 provides a continuous and stable UVA protection function for the transparent GCP hydrogel system ( Fig.10 (Figure B).

[0104] Example 3 Protective effect of GCP hydrogel on cells in vitro

[0105] Cell photoaging experiment and UV shielding measurement: The UV protection experimental device in reference example 2 was used to treat fibroblasts ( Fig.10 (Figure A in the middle).

[0106] JC-1 staining results showed that UVA exposure caused a significant decrease in mitochondrial membrane potential of fibroblasts and severe damage to mitochondrial function; GCP treatment significantly alleviated this damage and, like the ANESSA group, almost completely protected the mitochondrial membrane potential ( Fig.11 Figure A); STED super-resolution imaging technology was used to image mitochondria in fibroblasts under UVA irradiation ( Fig.11 The results showed that the number of mitochondrial fragmentation in the GCP group was not significantly different from that in the NC group and the ANESSA group, indicating an excellent UVA protection effect. Compared with the NC group, the number of fragmentation in the UVA group without P334 cross-linking and the GelMA / rhCOL group increased by 157.65% and 143.53% (P < 0.01) and (P < 0.01), respectively. Fig.11 Figure C); Due to their good UVA shielding performance, the GCP group and the ANESSA group reduced the consumption of NADPH and maintained the SOD content in the cells at the same level as the NC group and the ANESSA group (P>0.05) ( Fig.11 In summary, UVA caused a series of mitochondrial dysfunction and oxidative stress responses; GelMA / rhCOL without P334 showed consistent cell and mitochondrial damage with the UVA direct exposure group; in contrast, P334-crosslinked GCP hydrogel almost completely eliminated the effect of UVA on fibroblast mitochondria; these results demonstrated the necessity of UVA shielding for wound management and the effectiveness of introducing P334 functional components.

[0107] Example 4 Effect of GCP hydrogel on promoting wound healing

[0108] Wound healing promotion: The effect of GCP gel on wound healing was evaluated by full-thickness skin excision wound without splinting (d>6 mm); the repair effect of GCP hydrogel was tested under UVA exposure conditions; first, mice were anesthetized with 2% isoflurane, and after removing the back hair, the skin was disinfected alternately with 70% alcohol and iodine; a full-thickness skin excision wound with a diameter of 1 cm was formed in the middle of the mouse's back using a sterile round punch to ensure that the excision depth reached the subcutaneous tissue; then, the GCP hydrogel was injected into the wound area using a sterile syringe (without the needle) to ensure uniform coverage of the wound; after the injection, the wound area was irradiated with a 405 nm light source at a distance of about 20 cm to perform a photocuring reaction to ensure cross-linking of the hydrogel and form a stable protective layer; dressings were changed every two days, and GCP hydrogel was re-injected and the photocuring process was repeated; after wound treatment, the wound was irradiated with a UVA lamp at an intensity of 10 J / cm² every day to simulate the wound healing environment under UV exposure; wound samples were obtained on the 5th and 10th days after surgery by excising the wound and its surrounding 5 mm marginal tissue was collected and subsequently processed histologically according to standard procedures;

[0109] Calculation of relative wound size: relative wound size (%) = (wound area on day n / wound area on day 0) × 100%;

[0110] The results showed that GCP gel could effectively adhere to wound tissue and maintain adhesion for at least 24 hours. On the fifth day of the inflammatory stage, the healing rate of the GCP group was 36.62%±7.76%, an increase of 8.06% compared with the NC group (P<0.01). On the tenth day of the hyperplasia stage, the healing rate of the GCP group reached 87.71%±4.91%, an increase of 28.65% compared with the NC group (P<0.01), and there was no adverse reaction to important organs. Interestingly, on the fifth day, there was no statistical difference between the rhCOL17 / GelMA group and the NC group, but entering the hyperplasia stage significantly promoted the healing efficiency of the wound, reflecting the potential of GCP hydrogel in tissue regeneration and repair. In contrast, GelMA, which functions as a basic scaffold, did not show the same effect ( Fig.12 Figures A and B in the figure).

[0111] Example 5 Preparation and application of GleMA-rhCOL17-shinorine (GCH) hydrogel

[0112] 1) Preparation of human recombinant collagen XVII (rhCOL17): see Example 1.

[0113] 2) Preparation of Shinorine (SH): Refer to the construction, preparation, separation and purification methods of shinorine production engineering strains in application number 202411738807.5.

[0114] 3) Preparation of cross-linked complexes

[0115] Preparation of ar-SH by SH activation Fig.13 ) and preparation of GCH hydrogel ( Fig.14 ) are all referred to Reference Example 1.

[0116] Preparation of GCH hydrogel: GCH hydrogel was prepared according to Example 1.

[0117] 4) Anti-cell aging effect of GCH hydrogel: Neutrophils are key immune effector cells in the early stage of wound healing. They mediate defense responses by releasing active molecules and NETs. The aging of fibroblasts exacerbates the complexity of the inflammatory microenvironment. After irradiation with UVA ( Fig.15 Figure A), the expression level of neutrophil chemokine IL8 was significantly increased, and the aging-related secretory phenotypes MMP1 and MMP2 were significantly increased ( Fig.15 In contrast, the expression level of IL-8 in the GCH group decreased by 83.65%±3.71 (P<0.001), the expression level of MMP-1 was 76.64%±13.49 (P<0.001), and the expression level of MMP-2 decreased by 92.53%±6.48 (P<0.001) compared with the UVA exposure group; IL8 (50 ng / mL) treatment of neutrophils induced NETs formation, and there was no significant difference with the classic inducer PMA ( Fig.15 E-F in the middle); This indicates that after UVA irradiation, the increased IL-8 in fibroblasts plays a key role in promoting the formation of NETs; this is also confirmed by immunofluorescence staining of MPO and Cit H3 (Cit H3, MPO and DAPI) of the filamentous structure ( Fig.15 GCH can significantly reduce the level of IL8, which is helpful for preventing excessive production of NETs and aging of fibroblasts.

[0118] 5) Skin repair effect of GCH hydrogel:

[0119] UVA exposure, trauma, and a variety of environmental and physiological factors can accelerate skin aging, collagen loss, and hair follicle aging. These events are usually accompanied by inflammation. Trauma to the skin and hair follicles is caused by trauma, and the repair effect of GCH on the skin and hair follicles is evaluated.

[0120] Inflammation inhibition: H&E staining of wound tissue showed that compared with the UVA-irradiated model group, the GCH group showed less inflammatory cell infiltration on the 5th day, indicating that the early inflammatory response was inhibited; on the 10th day, the GCH group significantly promoted epithelial regeneration, the epidermal thickness was close to normal, and the tissue structure tended to be complete ( Fig.16 (Figure A in the middle).

[0121] Collagen deposition and remodeling: During the skin injury reconstruction phase, collagen deposition is a crucial process and serves as a benchmark for evaluating wound healing; MASSON staining ( Fig.16 Figure B) shows that the GCH group had the most collagen deposition in the wound repair area on the 10th day, with a CV value of 75.37%±5.05%, which was about 19.3% higher than that of NC ( Fig.16 (Figure C).

[0122] Hair follicle repair effect: Hair follicle repair is one of the important signs of the skin remodeling stage of skin damage or wound healing. It relies on COL17 on the basement membrane to maintain the adhesion and positioning of hair follicle stem cells. The number of hair follicles in the wound healing area of ​​the GCH group increased significantly on the tenth day, showing a good hair follicle repair effect ( Fig.16 (D in the middle).

[0123] Basement membrane repair effect: Immunofluorescence results showed that the expression of COL4 in the GCH group was significantly higher than that in other groups on the 5th and 10th days, indicating that it effectively promoted the repair of the basement membrane in the early stage of wound healing, thereby accelerating the epithelial regeneration process ( Fig.17 At the same time, COL17 expression also increased significantly, further indicating that it plays a key role in the repair of dermal-epidermal junction and collagen deposition, promoting the overall remodeling and repair of the skin ( Fig.17 (Figure B).

[0124] It can be seen that GCH has good application prospects in skin protection, repair, anti-aging and wound recovery.

[0125] The above embodiments are merely examples for demonstrating the technical solutions of the present invention and are not intended to be limitative descriptions of the invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A mycosporin-like amino acid-protein cross-linked complex, characterized in that: Its structural formula is shown in the following formula (I): （I）； In formula (I), R is -CH2OH or -CH(OH)CH3, and the curves connected by amide bonds represent the peptide chains of the protein; The mycosporin-like amino acid-protein complex is mainly formed by cross-linking the carboxyl group of the mycosporin-like amino acid shown in the following formula (II) and the primary amino group of the protein molecule shown in the following formula (III): （Ⅱ）， （Ⅲ）； In formula (II), R is -CH2OH or -CH(OH)CH3; The protein is recombinant human type XVII collagen, and its sequence is shown in SEQ ID NO:

2.

2. The mycosporin-like amino acid-protein cross-linked complex according to claim 1, characterized in that: The mass ratio of the mycosporin-like amino acid to the protein is 2-4:

50.

3. The mycosporin-like amino acid-protein cross-linked complex according to claim 1, characterized in that: The mycosporin-like amino acid is firstly activated by an activator before cross-linking. The activator is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EDC and N -hydroxysuccinimide NHS or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride DMTMM, when a mixture of EDC and NHS is used as the activator, the molar ratio of the mycosporin-like amino acid to the activator EDC and NHS is 1:2:2; when the activator is DMTMM, the molar ratio of the mycosporin-like amino acid to the activator DMTMM is 1:

2.

4. The method for preparing the mycosporin-like amino acid-protein cross-linked complex according to any one of claims 1 to 3, characterized in that: The following steps are involved: (S1) protein preparation; (S2) activating the mycosporin-like amino acid with an activating agent; (S3) Cross-linking the activated mycosporin-like amino acid with the protein in step (S1) to obtain a mycosporin-like amino acid-protein cross-linked complex.

5. A hydrogel, characterized in that: The mycosporin-like amino acid-protein cross-linked complex according to any one of claims 1 to 3 is added to a matrix material methacrylated gelatin containing a photoinitiator to obtain the complex.

6. The hydrogel according to claim 5, characterized in that The mass percentage of protein in the mycosporin-like amino acid-protein cross-linked complex in the hydrogel is 0.2-1%; the photoinitiator is phenyl-2,4,6-trimethylbenzoylphosphine lithium LAP, and the mass percentage of the photoinitiator in the hydrogel is 0.2-0.3%; the methacrylated gelatin is methacrylated gelatin in which 30-90% of the amino groups are replaced by methacryloyl groups, and the mass percentage of the methacrylated gelatin in the hydrogel is 8-15%.

7. Use of the mycosporin-like amino acid-protein cross-linked complex according to any one of claims 1 to 3 or the hydrogel according to claim 5 or 6 in the preparation of UV protection, anti-aging, wound management bioscaffold, anti-inflammatory, skin repair or hair care products.

8. Use of the mycosporin-like amino acid-protein cross-linked complex according to any one of claims 1 to 3 or the hydrogel according to claim 5 or 6 in the preparation of biomaterials, medical dressings or cosmetics development.

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