Preparation method of a novel recombinant humanized collagen hydrogel

Abstract

A new preparation method of recombinant humanized collagen hydrogel is provided. The method comprises the following steps: reacting recombinant humanized collagen and a dialdehyde cross-linking agent, wherein the concentration of the dialdehyde cross-linking agent is 0.01-0.25%, and the recombinant humanized collagen is one or more of recombinant humanized type II, type III and type XVII collagen, and the recombinant humanized collagen has a triple helix structure. The present application determines the aldehyde cross-linking agent suitable for recombinant humanized collagen, especially the recombinant humanized collagen with a triple helix structure and the optimum reaction conditions.

Description

Technical Field

[0001] This application relates to the field of proteins or hydrogels, and more specifically to collagen hydrogels, their preparation methods, and uses. Background Technology

[0002] Collagen is a type of protein widely distributed in human connective tissue and is the most abundant protein in the human body, accounting for 25% to 35% of total protein. Its main functions include maintaining the extracellular environment, maintaining the normal physiological functions of tissues and organs, and repairing bodily damage. Collagen is a natural biological resource with biocompatibility, cell support elasticity, and biodegradability unmatched by other polymers. Therefore, collagen has wide applications in the pharmaceutical and cosmetic industries.

[0003] Natural collagen molecules can form a special superhelical structure, a left-handed helix with three amino acid residues as basic repeaters, typically Gly-XY. Gly is essential for the formation of hydrogen bonds in collagen; it lacks side chains to allow collagen to pack tightly, maintaining skin tension and elasticity. However, natural collagen suffers from poor stability, sensitivity to collagenases and matrix metalloenzymes, short in vivo retention time, and lack of mechanical strength in aqueous systems, limiting its development and application in the biomedical field. Various chemical modifications and cross-linking reactions can alter the physical and chemical properties of collagen to improve its mechanical strength and resistance to degradation, obtain new bioactive and functional collagen cross-linked derivatives, and further develop novel biomaterials.

[0004] Currently, the commonly used cross-linking method mainly involves uniformly mixing the cross-linking agent with collagen in a solution to carry out the cross-linking reaction. However, recombinant humanized collagen exhibits less cross-linking, and the amount of cross-linking agent, reaction time, and reaction temperature are all uncertain. Therefore, to overcome the problems of poor filling and support in existing recombinant humanized collagen, it is urgent to determine and improve the cross-linking reaction of recombinant humanized collagen. To address the issue that existing collagen fillers only provide immediate filling effects and have poor support after implantation, this invention provides a method for preparing a cross-linked recombinant collagen gel. Summary of the Invention

[0005] To address the issue that existing collagen fillers only provide immediate filling effects and require re-injection to maintain the effect after degradation, this invention provides a method for preparing cross-linked recombinant collagen gel. This method prepares a recombinant collagen gel by cross-linking with an aldehyde-based cross-linking agent, resulting in a cross-linked recombinant collagen gel with low toxicity, high stability, and the ability to provide immediate filling.

[0006] This invention is partly based on the inventors' discovery that when recombinant humanized collagen with a triple helix structure is crosslinked, the crosslinking agent and its concentration affect the triple helix structure of the recombinant humanized collagen. Only by using specific crosslinking agents and concentrations can the crosslinked recombinant humanized collagen maintain its initial triple helix structure.

[0007] On one hand, a method for crosslinking recombinant humanized collagen is provided, comprising the following steps: reacting the recombinant humanized collagen with a dialdehyde crosslinking agent, wherein the concentration of the dialdehyde crosslinking agent is 0.01-0.25%, preferably 0.02%-0.08%.

[0008] In one embodiment, the recombinant humanized collagen has a triple helix structure.

[0009] In one embodiment, the recombinant humanized collagen has a negative peak at 175-220 nm and a positive peak at 200-240 nm on circular dichroism spectroscopy.

[0010] In one embodiment, the recombinant humanized collagen has a negative peak at approximately 195 nm and a positive peak at approximately 221 nm on circular dichroism spectroscopy.

[0011] In one embodiment, the recombinant humanized collagen is one or more of recombinant humanized type II, type III and type XVII collagen.

[0012] In one embodiment, the concentration of the dialdehyde crosslinking agent is 0.02%-0.07% or 0.02%-0.06%.

[0013] In one embodiment, the concentration of the dialdehyde crosslinking agent is 0.03%-0.04% or 0.03%-0.035%.

[0014] In one embodiment, the dialdehyde crosslinking agent is one or more of glyoxal, malondialdehyde, succinaldehyde, and glutaraldehyde.

[0015] In one embodiment, recombinant humanized collagen is dissolved in a buffer solution, and the pH is adjusted to 5.0-11.0, preferably 5.5-8.7 or 5.7-8.

[0016] In one embodiment, the buffer solution is a phosphate buffer solution or a carbonate buffer solution.

[0017] In one embodiment, a 0.1M-0.4M or 0.1M-0.3M phosphate buffer solution or carbonate buffer solution.

[0018] In one embodiment, the concentration of recombinant humanized collagen is 10-100 mg / mL, 10-70 mg / mL, 20-40 mg / mL, or 30-40 mg / mL.

[0019] In one implementation, the cross-linked recombinant humanized collagen has a triple helix structure.

[0020] In one embodiment, the method further includes reacting recombinant humanized collagen and a dialdehyde crosslinking agent at temperatures of -20°C to 50°C, 0 to 25°C (e.g., 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C), 0 to 10°C, 0 to 8°C, or 0 to 4°C for 6 to 24 hours (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours), 12 to 24 hours, 12 to 20 hours, or 12 to 16 hours to form a crosslinked recombinant collagen gel.

[0021] In one embodiment, the cross-linked recombinant collagen gel is freeze-dried into a powder.

[0022] In one embodiment, the recombinant humanized collagen contains the sequence (Gly-XY)n, where n is 2-300.

[0023] In one embodiment, the recombinant humanized collagen has one or more repeating units, each repeating unit comprising an amino acid sequence of any one of SEQ ID NO:1-3 or having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with any one of SEQ ID NO:1-3, or an amino acid sequence in which one or more amino acid residues have been substituted, added, deleted, or inserted in the amino acid sequence of any one of SEQ ID NO:1-3. Preferably, the recombinant humanized collagen has 2-50 repeating units.

[0024] In one embodiment, the recombinant humanized collagen has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the amino acid sequences in SEQ ID NO:4-7, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted, or inserted.

[0025] In another aspect, a method for preparing cross-linked recombinant humanized collagen gel is provided, comprising the methods described herein and including the removal of residual dialdehyde cross-linking agents from the cross-linked recombinant collagen gel. In one embodiment, the residual dialdehyde cross-linking agent is removed by dialysis.

[0026] In one embodiment, cross-linked recombinant collagen powder is placed in a dialysis bag, or cross-linked recombinant collagen gel is placed directly in a dialysis bag, and stirred with buffer and / or physiological saline to remove residual dialdehyde cross-linking agents. Preferably, the collagen gel is prepared by dialysis with physiological saline for 5-48 hours, more preferably 10-24 hours. Preferably, before dialysis with physiological saline, the collagen gel is stirred and dialyzed with phosphate buffer, for example, for 1-5 hours, more preferably 2-4 hours, to obtain a cross-linked recombinant collagen gel with reduced residual dialdehyde cross-linking agent content.

[0027] In one embodiment, the cross-linked recombinant humanized collagen gel is a hydrogel.

[0028] In one embodiment, the buffer salt is selected from sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0029] In one embodiment, the volume ratio of phosphate buffer or saline to cross-linked recombinant collagen gel is 10:1 to 50:1.

[0030] In the various embodiments described herein, the recombinant humanized collagen is expressed by *Escherichia coli* or by *Pichia pastoris*, such as *Pichia pastoris*.

[0031] In another aspect, cross-linked recombinant humanized collagen is provided, which is prepared by the methods described herein.

[0032] In another aspect, a cross-linked recombinant humanized collagen gel is provided, comprising the cross-linked recombinant humanized collagen described herein or prepared by the methods described herein. In one embodiment, the cross-linked recombinant humanized collagen gel is a hydrogel.

[0033] In another aspect, compositions are provided comprising the cross-linked recombinant humanized collagen or cross-linked recombinant humanized collagen gel as described herein.

[0034] In one embodiment, the composition further comprises a pharmaceutically or cosmetically acceptable carrier, diluent, or excipient.

[0035] In one embodiment, the composition is a pharmaceutical composition, a food composition, or a cosmetic composition.

[0036] In one embodiment, the cosmetic composition is a cosmetic composition with anti-wrinkle effects, oil control effects, skin repair effects, and / or skin soothing effects.

[0037] In one implementation, the skin oil control effect is the facial skin oil control effect.

[0038] In one embodiment, the composition is a kit, preferably containing physiological saline.

[0039] In one embodiment, the composition is one or more of the following: bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0040] In one embodiment, the composition is a solid, liquid, or gel composition.

[0041] In one embodiment, the composition is an oral and / or topical composition.

[0042] In one implementation, topical application is application to the skin.

[0043] In one embodiment, the composition is a transdermal composition.

[0044] In one embodiment, the collagen hydrogel also contains physiological saline.

[0045] In another aspect, a method for preparing collagen gels is provided, comprising the step of dissolving the cross-linked recombinant humanized collagen described herein in a buffer solution and / or physiological saline.

[0046] In another aspect, methods are provided for enabling cells to adhere or adhere to a wall or promote cell migration, said methods comprising contacting cells with cross-linked recombinant human collagen, cross-linked recombinant human collagen gel or composition described herein.

[0047] In another aspect, cosmetic or beauty methods are provided, which include applying to the skin cross-linked recombinant humanized collagen, cross-linked recombinant humanized collagen gel and / or compositions according to the present invention, and optionally applying other beauty and / or cosmetic products to the skin.

[0048] In one implementation, the cosmetic or beauty method is a method for anti-wrinkle, oil control, repair, and / or soothing the skin.

[0049] In one implementation, the application is a local application.

[0050] In one implementation, the application is transdermal.

[0051] In another aspect, the use of the cross-linked recombinant humanized collagen and / or compositions described herein in the preparation of pharmaceutical, cosmetic or beauty products is provided.

[0052] In one implementation, the product is an external use product.

[0053] In one implementation, the cosmetic or beauty product is a skin anti-wrinkle, oil-controlling, repairing, and / or soothing product.

[0054] In one implementation scheme, the product is selected from one or more of the following: bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, and 3D printed artificial organ biomaterials.

[0055] In another aspect, the use of the cross-linked recombinant humanized collagen or compositions described herein as subcutaneous filler materials is provided.

[0056] In another aspect, the use of the cross-linked recombinant humanized collagen or compositions described herein in the preparation of products such as: dermal implants, drug carriers, medical device coatings, implant coatings, shape-forming substances, adhesive surgical materials, and vascular occlusives; sponge-like substances, such as those used for three-dimensional cell cultures, tissue and organ engineering materials, hemostatic agents, and wound treatment agents; fibers, such as those used for surgical sutures; tissue implants, corneal protective films, or matrices for cell culture; and membrane-like substances, such as those used for anti-adhesion films or artificial skin.

[0057] The preparation method of cross-linked recombinant collagen gel may include one or more of the following steps:

[0058] Step 1: Dissolve recombinant humanized collagen in phosphate buffer and adjust the pH to 7.0 to obtain a neutral solution of recombinant collagen. The recombinant humanized collagen is prepared by fermentation of Escherichia coli or Pichia pastoris.

[0059] Step 2: Add aldehyde cross-linking agents (glyoxal, glutaraldehyde, etc.) to the solution of recombinant humanized collagen, and perform cross-linking reaction at -20℃~50℃ for 6-24 hours. Then freeze-dry the cross-linked recombinant humanized collagen.

[0060] Step 3: Wash the freeze-dried cross-linked recombinant humanized collagen with buffer salt solution and / or physiological saline until it is mixed evenly to obtain cross-linked recombinant collagen gel.

[0061] Preferably, in step 1, the amount of recombinant humanized collagen protein is 10mg-60mg.

[0062] Preferably, in step 1, the pH is adjusted to 7 ± 0.2 using a 0.1 mol / L to 1 mol / L sodium hydroxide solution and / or 1 M HCl.

[0063] Preferably, in step 2, the crosslinking agent is glutaraldehyde. Preferably, in step 2, the crosslinking reaction time is 12 hours. Preferably, the concentration of glutaraldehyde is 0.01-0.25%.

[0064] Preferably, in step 3, the buffer salt is selected from sodium dihydrogen phosphate (e.g., anhydrous sodium dihydrogen phosphate) and disodium hydrogen phosphate.

[0065] Preferably, the volume ratio of the buffer salt solution to the gel is 10:1 to 50:1.

[0066] Preferably, in step 3, the total cleaning time is 10-24 hours.

[0067] Compared with the prior art, the present invention has the following advantages:

[0068] (1) This invention identifies aldehyde crosslinking agents and optimal reaction conditions suitable for recombinant humanized collagen, particularly recombinant humanized collagen with a triple helix structure. Using the aldehyde crosslinking agents and optimal reaction conditions of this invention, crosslinked recombinant humanized collagen can be prepared while retaining the triple helix structure of the recombinant humanized collagen.

[0069] (2) After the cross-linking reaction of recombinant humanized collagen, the gel is washed with buffer solution, which can effectively promote the reduction of residual cross-linking agent content, effectively ensure product safety, and make the product more suitable for use as a subcutaneous filling material.

[0070] (3) The present invention finds that the method of the present invention is suitable for cross-linking recombinant collagen with a triple helix structure to form a gel, while the method of the present invention cannot cross-link type I recombinant collagen without a triple helix structure to form a gel. Attached Figure Description

[0071] Figure 1 The circular dichroism chromatograms of recombinant humanized collagen T16 at different cross-linking agent concentrations are shown.

[0072] Figure 2 The circular dichroism chromatograms of cross-linked recombinant humanized collagen T16 at different buffer pH values ​​are shown.

[0073] Figure 3 The morphological changes of type III recombinant humanized collagen T8 before and after cross-linking are shown.

[0074] Figure 4 The morphological changes of type III recombinant humanized collagen T16 before and after cross-linking are shown.

[0075] Figure 5 The morphological changes of type II recombinant humanized collagen before and after cross-linking are shown.

[0076] Figure 6 The morphological changes of type XVII recombinant humanized collagen before and after cross-linking are shown.

[0077] Figure 7 The cell adhesion activity of the cross-linked recombinant humanized collagen was demonstrated.

[0078] Figure 8 The morphology of cross-linked type I recombinant humanized collagen is shown.

[0079] Figure 9 The results of circular dichroism spectroscopy of type I recombinant humanized collagen after cross-linking are shown. No collagen characteristic peaks are observed, and it does not possess a triple helix structure. Detailed Implementation

[0080] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0081] As used herein, recombinant humanized collagen is a full-length or partial amino acid sequence fragment encoded by a sex-specific gene of human collagen, prepared using DNA recombination technology, or a combination of functional fragments containing human collagen. In this document, recombinant humanized collagen refers to recombinant humanized type I, II, III, V, and VII collagen types, which are peptides or polypeptides consisting of multiple amino acid residues linked by peptide bonds. Recombinant humanized collagen can be formed by the tandem repetition of multiple repeating units. Methods for preparing recombinant humanized collagen using a tandem repetition strategy are known to those skilled in the art. The choice of the number of repeating units is conventional for those skilled in the art. For example, recombinant humanized collagen can have 2-50 repeating units, such as 2-10. It should be understood that recombinant humanized collagen formed from repeating units within this range possesses similar functions. Prior art literature reports that recombinant collagen can be constructed by the tandem repetition of repeating units. The number of repeating units can vary. For example, Chen Hua et al. in Biochemical and Biophysical Research Communications 508(2019)1018e1023 disclosed that the T16, which contains the GERGAPGFRGPAGPNGIPGEKGPAGERGAP repeat unit and 16 tandem repeats of this repeat unit, possesses cell adhesion activity. They also suggested that recombinant proteins containing multiple tandem repeats of triple helical fragments can have a more stable helical conformation or a more favorable ligand-binding configuration to promote cell membrane attachment and adhesion. R. Strawn et al. in Biopolymers 109(2018),e23226 demonstrated that recombinant collagen-like proteins with multiple identical triple helical peptide units arranged in a tandem repeat manner can self-assemble into microfibrils for use in biopharmaceuticals and industrial applications. J. Yao et al. in J. Biochem. 136(2004)643e649 reported that recombinant collagen-like proteins containing tandem repeats of type I collagen peptides exhibit high cell adhesion activity.

[0082] As used in this article, a hydrogel is a type of highly hydrophilic three-dimensional network gel that swells rapidly in water and can retain a large volume of water without dissolving in this swollen state. In this article, when referring to a gel, the term "gel" can refer to a hydrogel.

[0083] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. Such calculations are typically performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984). Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

[0084] As used herein, the term "crosslinking" refers to the chemical process by which collagen molecules are linked together by covalent bonds to form a stable structure. Crosslinking can be achieved in various ways, including but not limited to chemical crosslinking and enzymatic crosslinking. In some embodiments described herein, crosslinking is performed through a chemical process. Chemical crosslinking typically uses crosslinking agents such as dialdehyde crosslinking agents. These agents initiate the crosslinking reaction to form covalent bonds, thus strengthening the intermolecular connections.

[0085] As used herein, the term "triple-helix" protein is understood to refer to a homotrimeric or heterotrimeric protein containing at least one "triple-helix domain". This term encompasses variants and fragments of triple-helix proteins and their functional equivalents and derivatives, preferably retaining the (Gly-XY)n sequence. The term "triple-helix structure" refers to a protein containing the general formula (Gly-XY)n, where Gly is glycine, X and Y represent the same or different amino acids (the specifics can vary within the range of different Gly-XY triplets), and n can be between 2 and 300. The triple-helix domain consists of three chains characterized by repeating (Gly-XY)n motifs folded into a triple-helix protein conformation. Triple-helix characteristics can be determined by circular dichroism spectroscopy. In one embodiment, the recombinant humanized collagen has a negative peak at approximately 195 nm (e.g., 175-220 nm, 180-210 nm, 190-200 nm) and a positive peak at approximately 221 nm (e.g., 200-240 nm, 210-230 nm, 215-225 nm) on circular dichroism spectroscopy.

[0086] As used herein, the term "glutaraldehyde" is a five-carbon straight-chain compound with two aldehyde groups, having the molecular formula O=CH-CH2-CH2-CH2-CH=O. These two symmetrical aldehyde groups can form Schiff bases or Michael adducts with the amino groups of basic amino acids. Glutaraldehyde can also crosslink with the thiol group of cysteine, and the benzene and imidazole rings of tyrosine and histidine. Increased temperature and alkaline conditions accelerate the polymerization of glutaraldehyde monomers, but this does not affect its ability to crosslink proteins. Glutaraldehyde is now the most widely used bifunctional crosslinking agent in protein (especially enzyme) immobilization, with its most common application being the chemical crosslinking of adsorbent enzymes onto a carrier. In the glutaraldehyde process, the concentration of glutaraldehyde and pH value affect the crosslinking effect. In some embodiments, the dialdehyde crosslinking agent, such as glutaraldehyde, can be removed after the formation of the crosslinked recombinant collagen or crosslinked recombinant collagen gel described herein. Methods for removing glutaraldehyde from the gel are known to those skilled in the art. In some embodiments, glutaraldehyde is removed by dialysis.

[0087] As used herein, the term "one or more" refers to any value greater than 1. There is no particular upper limit to the value, and it can be determined by those skilled in the art. For example, one or more can be 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

[0088] Cross-linked collagen

[0089] The structural stability and mechanical strength of cross-linked collagen are enhanced. The cross-linked structure can not only improve the tensile strength of collagen, but also enhance its wear resistance, making it widely used in applications requiring high mechanical properties, such as artificial bones and tissue engineering materials in the medical field. In addition, the cross-linked structure can also adjust the degradation rate of collagen in vivo and prolong its existence time in tissues. In the fields of medicine and bioengineering, cross-linked collagen has a wide range of applications. It can be made into artificial skin, used for cartilage repair and bone tissue engineering, and is also an ideal carrier material for drug delivery and cell culture. In the treatment of tissue defects such as trauma and burns, cross-linked collagen has also shown its unique advantages and played an important role in plastic surgery. The unique properties of cross-linked collagen have greatly enhanced its application potential in the fields of medicine and bioengineering. Its increased structural stability and mechanical strength provide an important material basis for tissue repair and regeneration. Compared with uncross-linked collagen, cross-linked collagen has the following significant advantages: (1) The structural stability of cross-linked collagen is significantly enhanced. Cross-linked collagen forms a stable structure through covalent bonding, resulting in higher tensile strength and abrasion resistance. This structural stability allows cross-linked collagen to better maintain its shape and mechanical properties during application, reducing the risk of structural loss or degradation. (2) Cross-linked collagen exhibits improved resistance to degradation. Compared to uncross-linked collagen, the degradation rate of cross-linked collagen is slowed. This is highly advantageous for applications requiring prolonged presence in the body, such as tissue repair and regeneration. Uncross-linked collagen is often easily degraded by enzymes in the body, leading to loss of its function and structure. (3) The mechanical properties of cross-linked collagen are enhanced. Its tensile strength, rigidity, and stability are improved, making it more suitable for applications requiring certain mechanical loads, such as artificial bones and tissue engineering materials. In contrast, uncross-linked collagen is weaker in these aspects and cannot meet these requirements.

[0090] Furthermore, cross-linked collagen exhibits increased long-term stability. Due to its higher structural stability and resistance to degradation, it persists in tissues for a longer period. This is crucial for applications requiring long-term maintenance of repair effects, such as artificial skin and cartilage repair. However, the advantages of cross-linked collagen may also present some limitations. The cross-linking process can alter the structure and properties of collagen, thereby affecting its bioactivity and cell-cell interactions. Therefore, when applying cross-linked collagen, a comprehensive evaluation of its performance and biocompatibility is necessary, and selection and optimization should be tailored to the specific application requirements. Nevertheless, cross-linked collagen remains a biomaterial with immense potential, providing strong support for tissue repair and regeneration.

[0091] In some embodiments, the collagen is recombinant humanized collagen. In some embodiments, the recombinant humanized collagen is type III, type II, or type XVII recombinant humanized collagen.

[0092] In some embodiments, the collagen contains multiple Gly-XY sequences, where X and Y can be any amino acid residue, such as any one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. In some implementations, collagen contains 2-300 units (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64). The number of Gly-XY values ​​in the ranges 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290 and any range thereof.

[0093] In some embodiments, the type III recombinant humanized collagen has one or more repeating units, each repeating unit comprising an amino acid sequence of SEQ ID NO:1 or having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:1, or having undergone one or more amino acid substitutions, insertions, deletions, or additions compared to SEQ ID NO:1. In some embodiments, the type III recombinant humanized collagen has one or more repeating units, each repeating unit comprising an amino acid sequence of SEQ ID NO:1 or having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:1, or having undergone one or more amino acid substitutions, insertions, deletions, or additions compared to SEQ ID NO:1. In some embodiments, the type II recombinant humanized collagen has one or more repeating units, each repeating unit comprising an amino acid sequence of SEQ ID NO:2 or having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:2, or having undergone one or more amino acid substitutions, insertions, deletions, or additions compared to SEQ ID NO:2. In some embodiments, the type XVII recombinant humanized collagen has one or more repeating units, each repeating unit comprising an amino acid sequence of SEQ ID NO:3 or having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:3, or having undergone one or more amino acid substitutions, insertions, deletions, or additions compared to SEQ ID NO:2. In this article, multiples are 2 to 50 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or any range therein).

[0094] In one embodiment, the recombinant humanized collagen has one or more repeating units, each repeating unit comprising an amino acid sequence of any one of SEQ ID NO:1-3 or having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity with any one of SEQ ID NO:1-3, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted, or inserted in the amino acid sequence of any one of SEQ ID NO:1-3. Preferably, the recombinant humanized collagen has 2-50 repeating units (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50).

[0095] In one embodiment, the recombinant humanized collagen has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the amino acid sequences in SEQ ID NO:4-7, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted, or inserted.

[0096] In one implementation, the recombinant humanized collagen is expressed by *Escherichia coli* or by Pichia pastoris such as *Pichia pastoris*.

[0097] Preparation of recombinant collagen

[0098] The exemplary recombinant humanized collagens described herein can all be prepared conventionally using existing methods, such as CN201811254050.7; CN201811254050.7; CN201811438582.6 and CN202311272338.8; CN201811254050.7 and CN201811438582.6; CN201911051106.3.

[0099] An exemplary method for preparing recombinant collagen is as follows: Based on the amino acid sequence of the recombinant collagen, a commercial synthesis company was commissioned to synthesize the gene fragment. The successfully constructed expression plasmid was then transformed into *E. coli* competent cells BL21(DE3) (Merck). The specific process is as follows:

[0100] 1: Take 1 μl of plasmid and put it into 100 μl of E. coli competent cells BL21(DE3), and incubate on ice for 30 min.

[0101] 2: Heat shock the mixture in a 42°C water bath for 90 seconds, then quickly place it on ice and let it stand for 2 minutes.

[0102] 3: Add 600 μl of non-resistant LB to the mixture and incubate at 37°C and 220 rpm for 1 h.

[0103] 4: Take 200 μl of the bacterial suspension and spread it evenly on an LB agar plate containing ampicillin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 μg / ml ampicillin).

[0104] 5: Invert the plate and incubate it in a 37°C incubator for about 20 hours until clearly visible colonies grow.

[0105] 6. Pick single colonies from the transformed LB agar plates and incubate them in 10 ml LB medium (containing 100 μg / ml ampicillin) for 12-16 h. Then, transfer them at a 1:100 ratio to 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) for scale-up culture. Incubate at 37℃ and 220 rpm until the OD600 reaches 0.4-0.6. Add 0.5 mM IPTG (Sigma, catalog number: I5502-1G) to induce expression. Induction conditions are 18℃ and 180 rpm for 20 h. Finally, centrifuge to collect the cells and store at -20℃ or proceed immediately to the next purification step.

[0106] 7. Resuspend the bacterial pellet (1L) in about 50ml of phosphate buffer (pH 7.8) (40mM sodium dihydrogen phosphate, 500mM sodium chloride), break the bacteria using an autoclave (Xinzhi Biotechnology), and centrifuge at 13000rpm for 30min to fully separate the soluble protein from the inclusion bodies.

[0107] 8. Equilibrate the Ni-NTA (Qiagen, catalog number 30210) affinity column with 5 column volumes of binding buffer (40 mM NaH2PO3, 500 mM NaCl, pH 7.8). Then add the protein supernatant and incubate at 4°C for 0.5–1 h to allow the target recombinant protein to fully bind to the column. Wash away any contaminating proteins with 200 ml of wash buffer containing 10 mM imidazole (Sigma, 10 mM imidazole, 40 mM NaH2PO3, 500 mM NaCl, pH 7.8). Finally, add an appropriate amount of His-tagged Prescission Protease (PPase) (Sigma, SAE0045) and incubate at 4°C for 16 h. Collect the flow-through, which contains the target collagen with the carrier protein removed. Dialyze the resulting product overnight and lyophilize it into a powder for later use.

[0108] 9. The purity of the obtained protein was determined using SDS-PAGE. The specific procedure was as follows: 40 μl of the purified protein solution was added to 10 μl of 5x protein loading buffer (250 mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), and the mixture was boiled in water at 100℃ for 10 min. Then, 10 μl of the buffer was added to each well of an SDS-PAGE protein gel, and the gel was run at 80V for 2 h. The protein was then stained with Coomassie Brilliant Blue (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20 min, followed by destaining with protein destaining solution (10% acetic acid, 5% ethanol). The molecular weight of the purified protein matched the expected molecular weight, indicating that the recombinant collagen was correctly expressed.

[0109] For type III collagen T8, its amino acid sequence was commercially synthesized. Its encoding nucleotide sequence was inserted into the pPiczalαA expression vector, and the pPiczalαA expression vector was introduced into competent yeast cells (X33). The transformed yeast cells were induced to express type III collagen T8. Electrophoresis was performed to confirm that type III collagen T8 was correctly expressed (see the example in CN 202311272338.8 for more detailed procedures). In other words, collagen (e.g., type III collagen) can be expressed by yeast (e.g., Pichia pastoris, such as Pichia pastoris) containing the pPiczalαA expression vector. The pPiczalαA expression vector can contain a nucleic acid molecule encoding collagen.

[0110] The above preparation method is applicable to the preparation and purification of recombinant collagen in the examples.

[0111] Triple helix sequence

[0112] The recombinant humanized collagen described herein is suitable for use as a biomaterial, a material for manufacture, a cosmetic, or a food additive. Methods for expressing the recombinant triple-helix protein include standard expression methods generally known in the art, such as those described in Molecular Cloning (Sambrook and Russell (2001)). Expression systems for generating the triple-helix protein are described, for example, in US20120116053. Methods for transforming Pichia pastoris, selecting positive transformants, and culturing it are disclosed, for example, in US Patent Nos. 4,837,148; 4,855,231; 4,882,279; 4,929,555; 5,122,465; 5,324,639; 5,593,859, and 6,472,171. Methods for generating triple-helical proteins are known in the art and described, for example, in US20120282817, EP1809751, and WO2012 / 117406. Expression systems for generating triple-helical proteins are described, for example, in US20120116053.

[0113] Composition

[0114] The cross-linked recombinant humanized collagen of the present invention can be prepared into compositions. The compositions may include the cross-linked recombinant humanized collagen hydrogel described herein. The compositions may also contain pharmaceutically and / or cosmetically acceptable carriers or solvents. The compositions may be pharmaceutical or cosmetic compositions for pharmaceutical and / or cosmetic purposes. For example, the compositions are one or more of the following: bio-dressings, biomimetic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D-printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, and food additives.

[0115] Cosmetic compositions can be those with anti-wrinkle, oil-controlling, skin-repairing, and / or skin-soothing effects. There are no particular restrictions on the application area of ​​the cosmetic composition; it can be the face, hands, legs, torso, etc. For example, oil-controlling effects refer to oil-controlling effects on the face.

[0116] There are no particular limitations on the form of the composition, as long as it can achieve the intended function. For example, the composition can be a solid, liquid, or gel composition.

[0117] The composition can be applied in any suitable manner, such as for oral and / or topical application. The composition can also be prepared as a kit. The kit may contain additional components, such as excipients like buffers, and includes instructions for use. In particular, the composition can be formulated into suitable formulations, such as liquid formulations. Formulations may contain buffers, such as D-PBS buffer or PBS buffer. If purified triple-helix proteins are used as biomedical materials, they must be able to be formulated into appropriate forms. To help achieve these forms, purified triple-helix proteins can be stabilized before use in medical applications (as in the case of animal collagen) to improve their long-term stability and mechanical strength when needed. A variety of suitable stabilization strategies are possible. Glutaraldehyde is a suitable agent for cross-linking and is widely used to improve the in vivo stability of collagen substances.

[0118] Method for preparing cross-linked recombinant humanized collagen

[0119] This article provides a method for preparing cross-linked recombinant humanized collagen, the method comprising reacting a cross-linking agent with the cross-linked recombinant humanized collagen described herein.

[0120] The method may include cross-linking recombinant humanized collagen having a triple helix structure. In some embodiments, the recombinant humanized collagen is reacted with a dialdehyde cross-linking agent, wherein the concentration of the dialdehyde cross-linking agent is 0.01-0.25%. The concentration of the dialdehyde cross-linking agent is the final concentration of the dialdehyde cross-linking agent in the reaction system, for example, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, or any range thereof. Recombinant humanized collagen can be any type of recombinant humanized collagen with a triple helix structure, preferably, but not limited to, one or more of recombinant humanized type II, III, and XVII collagen. The concentration of recombinant humanized collagen can be 10-100 mg / mL, 10-60 mg / mL, 20-40 mg / mL, or 30-40 mg / mL, for example, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or any range therebetween. The concentration of recombinant humanized collagen is the final concentration of recombinant humanized collagen in the reaction volume or system. The exemplary recombinant humanized collagens described herein can all be prepared conventionally using methods available in the prior art, such as CN201811254050.7; CN201811254050.7; CN201811438582.6 and CN202311272338.8; CN201811254050.7 and CN201811438582.6; CN201911051106.3. In some embodiments, the dialdehyde crosslinking agent is one or more of glyoxal, malondialdehyde, succinate, and glutaraldehyde.

[0121] The method may also include dissolving recombinant humanized collagen in a buffer solution and adjusting the pH to 5.0–11.0, such as 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 8.7, 9, 9.5, 10, 10.5, or 11, or any range therebetween. Sodium hydroxide solution or hydrochloric acid can be used to adjust the pH; for example, 0.5 mol / L sodium hydroxide solution or 1 M hydrochloric acid can be used. The buffer solution can be a phosphate buffer solution or a carbonate buffer solution, for example, the buffer salt is selected from sodium dihydrogen phosphate and sodium hydrogen phosphate. The cross-linked recombinant humanized collagen can still retain its triple helix structure.

[0122] The method may also include reacting recombinant humanized collagen with a dialdehyde crosslinking agent at temperatures of -20°C to 50°C, 0 to 25°C, 0 to 10°C, 0 to 8°C, or 0 to 4°C for 6 to 24 hours, 12 to 24 hours, 12 to 20 hours, or 12 to 16 hours to form a crosslinked recombinant collagen gel. The crosslinking temperature can be, for example, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or any range thereof. The crosslinking time can be, for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or any range thereof.

[0123] In one embodiment, the method includes removing residual dialdehyde crosslinking agents from the crosslinked recombinant collagen gel.

[0124] In one implementation, residual dialdehyde crosslinking agents are removed by dialysis.

[0125] In one embodiment, cross-linked recombinant collagen is freeze-dried into cross-linked recombinant collagen powder; the cross-linked recombinant collagen powder is then placed in a dialysis bag. In one embodiment, cross-linked recombinant collagen gel is placed directly in a dialysis bag. In one embodiment, the mixture is stirred with buffer and / or physiological saline to remove residual dialdehyde cross-linking agents.

[0126] In one embodiment, collagen gel is prepared by dialysis with physiological saline with stirring for 5-48 hours, preferably 10-24 hours. The selection of dialysis time is conventional for those skilled in the art and can be, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours or any range thereof.

[0127] In one embodiment, the collagen gel is dialyzed with phosphate buffer before stirring with physiological saline, for example, for 1-5 hours, preferably 2-4 hours, such as 1, 2, 3, 4, or 5 hours. In one embodiment, the buffer salt is selected from sodium dihydrogen phosphate and disodium hydrogen phosphate. In one embodiment, the volume ratio of phosphate buffer or physiological saline to the cross-linked recombinant collagen gel is 10:1-50:1, for example, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, or any range therebetween. The composition of the phosphate buffer is conventional to those skilled in the art. For example, phosphate buffer can be prepared as a 0.2M sodium dihydrogen phosphate aqueous solution (solution A: 27.6g NaH2PO4·H2O dissolved in distilled water and diluted to 1000mL) and a 0.2M disodium hydrogen phosphate aqueous solution (solution B: 53.6g Na2HPO4·7H2O or 71.6g Na2HPO4·12H2O or 35.6g Na2HPO4·2H2O dissolved in distilled water and diluted to 1000mL). A pH 7.0 phosphate buffer can be prepared by adding 61mL of solution B to 39mL of solution A. A pH 7.5 phosphate buffer can be prepared by adding 84mL of solution B to 16mL of solution A.

[0128] Method using cross-linked recombinant collagen

[0129] This document also provides methods for cell adhesion or fixation, the methods comprising contacting cells with the cross-linked recombinant human collagen or a cross-linked recombinant human collagen composition described herein. The composition is preferably a cross-linked recombinant human collagen hydrogel described herein. The methods of the present invention can be performed in vitro to increase cell adhesion to a culture vessel. Alternatively, the methods of the present invention can also be performed in vivo.

[0130] This article also provides a method for promoting cell migration, the method comprising contacting cells with cross-linked recombinant human collagen or a cross-linked recombinant human collagen composition. The method of the present invention can be performed in vitro or in vivo.

[0131] This document also provides cosmetic or beauty methods that include applying the cross-linked recombinant humanized collagen or cross-linked recombinant humanized collagen compositions described herein to the skin. Skin application can be topical, such as application to areas of skin requiring anti-wrinkle, oil-control, repair, and / or soothing effects. The method may also include applying other beauty and / or cosmetic products to the skin. There are no particular limitations on the types of other beauty and / or cosmetic products; they can be other cosmetics with anti-wrinkle, oil-control, repair, and / or soothing effects, such as collagen. These cosmetic or beauty methods can be methods for anti-wrinkle, oil-control, repair, and / or soothing the skin.

[0132] This article also provides a method for administering the cross-linked recombinant humanized collagen or cross-linked recombinant humanized collagen compositions described herein to subjects in need. This method can be used to treat or prevent conditions related to collagen deficiency, or for skin rejuvenation, oil control, repair, and / or soothing in subjects. The administration may be oral.

[0133] use

[0134] This article also provides the use of cross-linked recombinant humanized collagen or cross-linked recombinant humanized collagen compositions in pharmaceutical, cosmetic, or beauty products. These products can be topical, such as topical cosmetics or beauty products. These cosmetics or beauty products can be anti-wrinkle, oil-controlling, repairing, and / or soothing skin products. For example, the products can be selected from bio-dressings, biomimetic materials, plastic surgery materials, organoid culture materials, cardiovascular scaffold materials, coating materials, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, and 3D-printed artificial organ biomaterials. The cross-linked recombinant humanized collagen of this invention can be used in various applications and procedures, including recovery, regeneration, and cosmetic procedures, vascular procedures, osteogenic and cartilage formation procedures, cartilage remodeling, bone graft substitutes, hemostasis, wound treatment and management, tissue strengthening and support, incontinence, etc. Non-limiting examples and possible applications of biomedical products derived from the cross-linked recombinant humanized collagen of the present invention include, but are not limited to: dermal implants, drug carriers, medical device coatings, implant coatings (bone and blood vessels), shape-forming substances, viscosurgery, vascular occlusives, cosmetics; sponge-like substances, such as those used in three-dimensional cell cultures, tissue and organ engineering, hemostatic agents, and wound treatment (artificial skin and wound dressings); fibers, such as those used in surgical sutures and hemostatic agents; gel-like substances, such as those used in tissue implants, corneal protective films, contact lenses, and matrices for cell culture; and membrane-like substances, such as those used in anti-adhesion membranes, drug delivery systems, artificial skin, and the like.

[0135] This document also provides methods for preparing pharmaceutical, cosmetic, or beauty products, including the steps of using cross-linked recombinant humanized collagen, cross-linked recombinant humanized collagen gel, or compositions described herein. In some embodiments, the product is a topical product. In some embodiments, the cosmetic or beauty product is a skin anti-wrinkle, oil-controlling, repairing, and / or soothing product. In some embodiments, the product is selected from one or more of the following: medical devices, bio-dressings, biomimetic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, and 3D-printed artificial organ biomaterials.

[0136] This document also provides methods for preparing products in the following categories: dermal implants, drug carriers, medical device coatings, implant coatings, shape-forming substances, adhesive surgical materials, and vascular occlusive agents; sponge-like substances, such as those for three-dimensional cell cultures, tissue and organ engineering materials, hemostatic agents, and wound healing agents; fibers, such as those for surgical sutures; tissue implants, corneal protective films, or matrices for cell cultures; and membrane-like substances, such as those for anti-adhesion films or artificial skin, the methods comprising the steps of using cross-linked recombinant human collagen, cross-linked recombinant human collagen gel, or compositions described herein.

[0137] Example

[0138] The following embodiments are provided to illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative and not restrictive. The invention is limited only by the scope of the appended claims.

[0139] The general methods of PCR, cloning, and ligation of nucleotides are well known to those skilled in the art and can be found, for example, in the following literature: "Molecular cloning: A laboratory manual," Sambrook et al. (1989), Cold Spring Harbor lab, Cold Spring Harbor, NY; Ausubel, FM et al. (eds.); "Current protocols in Molecular Biology," John Wiley and Sons (1995); Harwood, CR and Cutting, SM (eds.); "DNA Cloning: A Practical Approach, Volumes I and II," DN Glover (ed.) (1985); "Oligonucleotide Synthesis," MJ Gait (ed.) (1984); "Nucleic Acid Hybridization," BD Hames & S.J. Higgins (eds.) (1985); "A Practical Guide to..." "Molecular Cloning: A Practical Guide" by B. Perbal (1984).

[0140] The specific types of recombinant humanized collagen are as follows:

[0141] Type I collagen

[0142] GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS, SEQ ID NO:8 (Preparation and purification methods are described in CN201811254050.7; Recombinant expression in Escherichia coli);

[0143] Type III collagen T8 (motif GERGAPGFRGPAGPNGIPGEKGPAGERGAP, SEQ ID NO:1, protein sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGE KGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP, SEQ ID NO:4) (preparation and purification methods are described in CN201811254050.7, CN201811438582.6 and CN202311272338.8; expression in Pichia pastoris);

[0144] Morphological characteristics of type III recombinant humanized collagen T16 before and after crosslinking (motif: GERGAPGFRGPAGPNGIPGEKGPAGERGAP, protein sequence: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP) GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP, SEQ ID NO:5 (Preparation and purification methods are described in CN201811254050.7 and CN201811438582.6; recombinant expression in Escherichia coli);

[0145] Type II recombinant humanized collagen (repeat unit, GEPGLQGPAGPPGEKGEPGDDGPSGAEGPP (SEQ ID NO:2), sequence GEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPP, SEQ ID NO:6) (Preparation and purification methods are described in CN201910111553.7; recombinant expression in Escherichia coli);

[0146] Type XVII recombinant humanized collagen (basic repeating units GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLP GAVGEPGAKGAMGPA (SEQ ID NO:3), GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA, SEQ ID NO:7) (preparation and purification methods are described in CN201911051106.3; recombinant expression in E. coli).

[0147] All the recombinant humanized collagen proteins mentioned above were prepared internally by Shanxi Jinbo Biopharmaceutical Co., Ltd. They can also be prepared using the steps outlined in the "Preparation of Recombinant Collagen Proteins" section above.

[0148] Example 1: Preparation of cross-linked recombinant humanized collagen hydrogels at different glutaraldehyde cross-linking agent concentrations

[0149] 30 mg of recombinant human collagen (type I collagen, type III collagen T8, type III recombinant human collagen T16, type II recombinant human collagen, or type XVII recombinant human collagen) was fully dissolved in 1 mL of 0.2 M sodium phosphate buffer solution. The pH was adjusted to 7.0 and 7.5 using 0.5 mol / L sodium hydroxide solution and 1 M hydrochloric acid, respectively, to obtain a solution of recombinant human collagen. Glutaraldehyde crosslinking agent was added to the recombinant human collagen solution at final concentrations of 0.01%, 0.03%, 0.08%, 0.1%, 0.15%, and 0.20%. The solution was placed in a 4°C constant temperature water bath and the crosslinking reaction was carried out for 12 hours to obtain the crosslinked recombinant human collagen gel (the morphology before and after crosslinking is shown in the figure). Figures 3-6 and Figure 8 show).

[0150] To reduce the residual glutaraldehyde crosslinking agent in recombinant humanized collagen gel, the crosslinked recombinant humanized collagen can be freeze-dried, washed with 500 mL of 0.2 M phosphate buffer for 2 hours, and then washed with 500 mL of physiological saline for 8 hours to form glutaraldehyde-crosslinked recombinant humanized collagen gel.

[0151] Example 2: Preparation of cross-linked recombinant humanized collagen hydrogels under different pH conditions

[0152] 30 mg of recombinant human collagen (type III recombinant human collagen T16) was fully dissolved in 1 mL of phosphate and carbonate buffer solution. The pH was adjusted to 5.7, 6, 6.5, 7, 7.5, 8.0, 9.5, 10.0, and 10.5 using 0.5 mol / L sodium hydroxide solution and 1 M hydrochloric acid to obtain solutions of recombinant human collagen. 0.03% glutaraldehyde crosslinking agent was added to the recombinant human collagen solution, and the mixture was placed in a 4°C water bath for 12 hours for the crosslinking reaction.

[0153] The cross-linked recombinant humanized collagen was freeze-dried, washed with 500 mL of 0.2 M phosphate buffer for 2 hours, and then washed with 500 mL of physiological saline for 8 hours to form a glutaraldehyde-crosslinked recombinant humanized collagen gel.

[0154] Example 3: Circular dichroism spectroscopy of recombinant humanized collagen hydrogel

[0155] 1) Sample preparation

[0156] To prepare 1X phosphate-buffered saline (PBS): Dissolve 8g NaCl, 0.2g KCl, 3.62g Na₂HPO₄·12H₂O, and 0.24g KH₂PO₄ in 800ml distilled water. Adjust the pH of the solution to 7.4 with HCl, and add water to a final volume of 1L. Autoclave and store at room temperature. If storage time exceeds one week, filter through a 0.45mm filter membrane before use.

[0157] Sample dilution: Dilute the cross-linked hydrogel sample with PBS to a final concentration of 0.8 mg / mL.

[0158] Sample testing:

[0159] Gradual dilution sample preparation:

[0160] 1) Use an ice box ice maker to obtain ice, and perform a two-fold serial dilution of the overnight sample at 4 degrees Celsius (note to change the pipette tip). Take 300 μl of the overnight sample and test it on the instrument.

[0161] 2) Instrument (JASCO Circular Dichroism Spectrometer, JASCO J-815) Parameter Settings

[0162] Band width: 1.0nm

[0163] Step: 1.0nm

[0164] Measurement range: 190-260nm

[0165] Time per point: 1 second

[0166] Scanning speed: 50 nm / min

[0167] Repeats: 3 times

[0168] Measurement temperature: 4℃.

[0169] 3) Standard CD scanning

[0170] Set the scanning wavelength to 190-260 nm for baseline testing of blank buffer, and collect the circular dichroism absorption of the sample solution PBS in the range of 190-260 nm.

[0171] 4) Scanned graph processing. Process the data using the machine's built-in software, export it to Excel, and then use GraphPad to create graphs.

[0172] Example 4: Bioactivity assay of cross-linked recombinant humanized collagen hydrogel

[0173] The method for detecting the adhesion activity of collagen can be found in the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0174] (1) The concentration of the protein samples to be tested was detected using ultraviolet absorption, including bovine type I collagen (China National Institutes for Food and Drug Control, No.: 380002) and cross-linked collagen (cross-linked type XVII collagen, cross-linked type II collagen, and cross-linked type III collagen T16) provided in this invention. Specifically, the ultraviolet absorption of the samples at 215 nm and 225 nm was measured respectively, and the protein concentration was calculated using the empirical formula C(μg / mL) = 144 x (A215 - A225). Note that the detection must be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far-ultraviolet light, which is not affected by the content of chromophores, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues that are not colorimetric by Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After the protein concentration was measured, the concentration of all the proteins to be tested was adjusted to 0.5 mg / mL with PBS.

[0175] (2) Add 100 μL of various protein solutions (bovine type I collagen or cross-linked collagen) to a 96-well plate.

[0176] (3) Add 10 to each hole 5 A well-cultured 3T3 cell was incubated at 37°C for 60 minutes.

[0177] (4) Wash each well with PBS 4 times.

[0178] (5) The absorbance at OD492nm was measured using an LDH detection kit (Roche, 04744926001) according to the manufacturer's instructions. The cell adhesion rate could be calculated based on the values ​​of the blank control.

[0179] The formula for calculating the wall adhesion rate is as follows:

[0180]

[0181] Cell adhesion rate reflects the adhesion activity of various proteins. The higher the activity of a protein, the better it can provide a high-quality external environment for the cell in a short time, helping the cell to adhere.

[0182] Experimental results

[0183] Figure 1 Circular dichroism spectroscopy is shown for the cross-linking of recombinant humanized collagen T16 at different glutaraldehyde concentrations. Surprisingly, most of the glutaraldehyde-crosslinked recombinant collagen failed to maintain its structural characteristics, exhibiting some degree of structural damage. The results of this example demonstrate that a 0.03% glutaraldehyde concentration can maintain the triple helix structure of recombinant humanized collagen while achieving cross-linking. Figure 1 As can be seen in its circular dichroism chromatogram, there are typical positive and negative peaks of collagen, namely a negative peak near 195 nm and a positive peak near 221 nm, exhibiting a triple helix structure. At a concentration of 0.03% glutaraldehyde, recombinant humanized collagen T8, type II recombinant humanized collagen, and type XVII recombinant humanized collagen also have similar triple helix structures.

[0184] Figure 2 Circular dichroism chromatograms of recombinant humanized collagen glutaraldehyde crosslinking in buffer solutions at different pH values ​​are shown. To determine the optimal pH crosslinking conditions for the buffer solutions (phosphate and carbonate), crosslinking experiments were performed at different pH conditions in the buffer systems using the same glutaraldehyde concentration (0.03%), and the crosslinked samples were analyzed using circular dichroism chromatograms. Figure 2 The results showed that recombinant humanized collagen T16 maintained its triple helix structure within the pH range of 5.7-8.0, exhibiting a negative peak near 195 nm and a positive peak near 221 nm, thus maintaining its triple helix structure. However, it could not maintain its triple helix structure within the pH range of 9.5-10.5. Recombinant humanized collagen T8, type II, and type XVII also exhibited similar triple helix structures within the pH range of 5.7-8.0.

[0185] Figure 3 The morphological changes of recombinant humanized collagen (T8) expressed in a yeast system before and after cross-linking are shown (pH 7.0). The cross-linked recombinant humanized collagen exhibits a typical hydrogel morphology. Compared to the uncross-linked recombinant humanized collagen, the cross-linked recombinant humanized collagen provides a certain degree of morphological support.

[0186] Figure 4 The morphological characteristics of type III recombinant humanized collagen T16, expressed and purified in Escherichia coli system, are shown before and after cross-linking.Figure 4 The study showed morphological changes at pH 7.0 and 7.5, and that the cross-linked recombinant collagen formed at pH 7.0 and 7.5 maintained good mechanical support.

[0187] Figure 5 The morphological changes of recombinant humanized type II collagen before and after cross-linking are shown. Recombinant humanized type II collagen completes the transition from liquid to solid state under cross-linking in glutaraldehyde buffer at pH 7.0, maintaining its mechanically supportive morphological characteristics.

[0188] Figure 6 The morphological changes of recombinant humanized collagen type XVII before and after cross-linking are shown. Recombinant humanized collagen type XVII can complete the liquid-to-solid transition under cross-linking conditions in glutaraldehyde buffer at pH 7.0.

[0189] Figure 7 The results show that, compared to bovine type I collagen (PC group, 0.5 mg / ml), the cross-linked recombinant humanized collagen hydrogel of the present invention still maintains its bioadhesive activity. The ability of the cross-linked recombinant humanized collagen to maintain cell adhesion activity is unexpected.

[0190] Figure 8 The morphology of cross-linked type I recombinant humanized collagen is shown. Type I recombinant humanized collagen does not possess a triple helix structure. After treatment using the method of Example 1, this type I recombinant humanized collagen remained in a fluid state and failed to form a gel.

[0191] Figure 9 The results of circular dichroism spectroscopy of type I recombinant humanized collagen after cross-linking are shown. No collagen characteristic peaks are observed, and it does not possess a triple helix structure.

[0192] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of crosslinking recombinant humanized collagen comprising the steps of: reacting the recombinant humanized collagen with a dialdehyde cross-linking agent, wherein the dialdehyde cross-linking agent is glutaraldehyde at a concentration of 0.01-0.08%, the recombinant humanized collagen consists of an amino acid sequence of any one of SEQ ID NOs: 4-7, and the recombinant humanized collagen is dissolved in a buffer solution, and the pH is adjusted to 5.7-8.

2. The method of claim 1, wherein, The buffer solution is a phosphate buffer solution or a carbonate buffer solution.

3. The method of claim 2, wherein the concentration of the phosphate buffer solution or the carbonate buffer solution is 0.1-0.4 M.

4. The method of claim 1, wherein the concentration of the recombinant humanized collagen is 10-100 mg / mL.

5. The method of claim 1, wherein the cross-linked recombinant humanized collagen retains a triple helix structure.

6. The method of claim 1, wherein the recombinant humanized collagen is expressed by E. coli or expressed by yeast.

7. The method of claim 1, comprising reacting the recombinant humanized collagen with the dialdehyde cross-linking agent at a temperature of 0-25 °C for a period of 6-24 hours to form a cross-linked recombinant collagen gel.

8. The method of claim 7, comprising lyophilizing the cross-linked recombinant collagen gel into a powder.

9. A method of preparing a cross-linked recombinant humanized collagen gel, comprising the method of any one of claims 1-8 and comprising removing residual dialdehyde cross-linking agent from the cross-linked recombinant collagen gel.

10. The method of claim 9, wherein the residual dialdehyde cross-linking agent is removed by dialysis.

11. A cross-linked recombinant humanized collagen prepared by the method of any one of claims 1-8.

12. A cross-linked recombinant humanized collagen gel prepared by the method of any one of claims 1-10.

13. A composition comprising the cross-linked recombinant humanized collagen of claim 11 or the cross-linked recombinant humanized collagen gel of claim 12.

14. The composition of claim 13, which is a pharmaceutical composition, a food composition, or a cosmetic composition.

15. The composition of claim 14, further comprising a pharmaceutically or cosmetically acceptable carrier, diluent, or excipient.

16. The composition of claim 13, which is a kit.

17. The composition of claim 13, which is one or more of a medical device, a biological dressing, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material, and a blood vessel repair and regeneration material, a 3D-printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

18. The composition of claim 13, which is a solid, a liquid, or a gel composition.

19. The composition of claim 13, which is an orally and / or topically administrable composition.

20. The composition of claim 19, wherein the topical administration is to the skin.

21. A method of preparing a collagen gel comprising the step of dissolving the crosslinked recombinant humanized collagen of claim 11 with a buffer and / or physiological saline.

22. A method of cell adhesion or attachment or promoting cell migration, said method comprising contacting a cell with the crosslinked recombinant humanized collagen of claim 11, the crosslinked recombinant humanized collagen gel of claim 12 or the composition of any one of claims 13-20, for a non-disease prevention or treatment purpose.

23. A cosmetic or dermatological method comprising applying to the skin the crosslinked recombinant humanized collagen of claim 11, the crosslinked recombinant humanized collagen gel of claim 12 or the composition of any one of claims 13-20, for a non-disease prevention or treatment purpose.

24. The method of claim 23, comprising applying to the skin other cosmetic and / or dermatological products.

25. The method of claim 23, which is a skin anti-wrinkle, oil control, repair and / or soothing method.

26. The method of claim 23, wherein said applying is topical application.

27. The method of claim 23, wherein said applying is transdermal application.

28. Use of the crosslinked recombinant humanized collagen of claim 11, the crosslinked recombinant humanized collagen gel of claim 12 or the composition of any one of claims 13-20 for the preparation of a cosmetic or dermatological product.

29. The use of claim 28, wherein the cosmetic or dermatological product is a skin anti-wrinkle, oil control, repair and / or soothing product.

30. The use of claim 28, said product being selected from one or more of the group consisting of medical devices, biological dressings, human biomimetic materials, orthopedic and aesthetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials.

31. Use of the crosslinked recombinant humanized collagen of claim 11, the crosslinked recombinant humanized collagen gel of claim 12 or the composition of any one of claims 13-20 for the preparation of dermal implants, pharmaceutical carriers, medical device coatings, implant coatings, shape forming substances, adhesive surgical materials, vascular sealants; sponge-like substances; fibers; tissue implants, corneal protective films or substrates for cell culture; and film-like substances.

32. The use of claim 31, wherein the sponge-like substances are selected from the group consisting of substances for three-dimensional cell culture, tissue and organ engineering, hemostatic agents and wound treatment agents.

33. The use of claim 31, wherein the fibers are fibers for surgical suturing.

34. The use of claim 31, wherein the film-like substances are for anti-adhesion films or artificial skin.

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