Preparation method of novel recombinant humanized collagen hydrogel

Abstract

The invention provides a preparation method of a novel recombinant humanized collagen hydrogel. The method comprises the following steps: recombinant humanized collagen and a dialdehyde crosslinking agent are subjected to a reaction, the concentration of the dialdehyde crosslinking agent is 0.01-0.25%, and the recombinant humanized collagen is one or more of recombinant humanized type II collagen, recombinant humanized type III collagen and recombinant humanized type XVII collagen, and the recombinant humanized collagen has a triple helix structure. The aldehydes cross-linking agent suitable for the recombinant humanized collagen, especially the recombinant humanized collagen with a triple helix structure, and the optimal reaction conditions are determined.

Description

Technical Field

[0001] The present application relates to the field of proteins or hydrogels, and specifically to collagen hydrogels and preparation methods and uses thereof. Background Art

[0002] Collagen is a type of protein widely distributed in human connective tissues. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. Its main functions are to maintain the extracellular environment, maintain the normal physiological functions of tissues and organs, and repair body damage. Collagen is a natural biological resource with bio-tissue compatibility, cell-supporting elasticity and degradability that other polymer materials cannot match. Therefore, collagen can be widely used in industries such as medicine and cosmetics.

[0003] Natural collagen molecules can form a special superhelical structure, which is a left-handed helix with three amino acid residues as the basic repeating body. These three amino acid residues are usually Gly-XY. Gly is necessary for the formation of hydrogen bonds in collagen. It has no side chain, which makes collagen densely packed and can maintain skin tension and elasticity. Due to the shortcomings of natural collagen, such as poor stability, sensitivity to enzymes such as collagenase and matrix metalloenzymes, short retention time in the body, and lack of mechanical strength in water systems, its development and application in the biomedical field are limited. Through various chemical modifications and cross-linking reactions, the physical and chemical properties of collagen can be changed to improve its mechanical strength and anti-degradation properties, obtain new biologically active and functional collagen cross-linked derivatives, and further develop new biomaterials.

[0004] The commonly used cross-linking method at present is mainly to mix the cross-linking agent and collagen in a solution evenly and perform a cross-linking reaction. The cross-linking of recombinant humanized collagen is less, and the amount of cross-linking agent, reaction time, reaction temperature, etc. are all uncertain. Therefore, in order to overcome the problems such as poor filling support of existing recombinant humanized collagen, it is urgent to determine and improve the cross-linking reaction of recombinant humanized collagen. In order to solve the problems that the existing collagen fillers only meet the immediate filling effect and have poor support after implantation, the present invention provides a method for preparing a cross-linked recombinant collagen gel. Summary of the invention

[0005] In order to solve the problem that the existing collagen fillers only meet the immediate filling effect and need to be injected again to maintain the effect after implantation and degradation, the present invention provides a method for preparing a cross-linked recombinant collagen gel. The method prepares a cross-linked recombinant collagen gel with low toxicity and strong stability that can meet the immediate filling requirements by cross-linking the recombinant collagen gel prepared by aldehyde cross-linking agents.

[0006] The present invention is based in part on the following findings of the inventors: when cross-linking recombinant humanized collagen having a triple helical structure, the cross-linking agent and the concentration of the cross-linking agent used have an effect on the triple helical structure of the recombinant humanized collagen. Only when a specific cross-linking agent and concentration are used, the triple helical structure of the original recombinant humanized collagen is maintained by the cross-linked recombinant humanized collagen.

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

[0008] In one embodiment, the recombinant humanized collagen has a triple helical 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 in the circular dichroism spectrum.

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

[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% to 0.04% or 0.03% to 0.035%.

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

[0015] In one embodiment, the 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, 0.1M-0.4M or 0.1M-0.3M phosphate buffer solution or carbonate buffer solution.

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

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

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

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

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

[0023] In one embodiment, the recombinant humanized collagen has one or more repeating units, the repeating unit comprising any one of SEQ ID NO: 1-3 or an amino acid sequence 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.

[0024] In one embodiment, the recombinant humanized collagen has any one of SEQ ID NOs: 4-7, or 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 one of SEQ ID NOs: 4-7, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of any one of SEQ ID NOs: 4-7.

[0025] In another aspect, a method for preparing a cross-linked recombinant humanized collagen gel is provided, which comprises the method described herein and comprises removing residual dialdehyde cross-linking agent from the cross-linked recombinant collagen gel. In one embodiment, the residual dialdehyde cross-linking agent is removed by dialysis.

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

[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 sodium hydrogen phosphate.

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

[0030] In various embodiments 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 method described herein.

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

[0033] In another aspect, a composition is provided, which comprises the cross-linked recombinant humanized collagen or the cross-linked recombinant humanized collagen gel 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 an anti-wrinkle effect, skin oil control effect, skin repair effect and / or skin soothing effect cosmetic composition.

[0037] In one embodiment, the skin oil control benefit is a facial skin oil control benefit.

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

[0039] In one embodiment, the composition is one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology 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 topically administrable composition.

[0042] In one embodiment, the topical administration is topical administration to the skin.

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

[0044] In one embodiment, the collagen hydrogel further comprises saline.

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

[0046] In another aspect, a method for causing cells to adhere or adhere to a wall or promoting cell migration is provided, the method comprising contacting the cells with the cross-linked recombinant humanized collagen, the cross-linked recombinant humanized collagen gel or the composition described herein.

[0047] In another aspect, a cosmetic or beauty method is provided, which comprises applying to the skin the cross-linked recombinant humanized collagen, the cross-linked recombinant humanized collagen gel and / or the composition described herein, and optionally applying other cosmetic and / or beauty products to the skin.

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

[0049] In one embodiment, the administration is topical administration.

[0050] In one embodiment, the administration is transdermal.

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

[0052] In one embodiment, the product is a topical product.

[0053] In one embodiment, the cosmetic or beauty product is a skin anti-wrinkle, oil control, repair and / or soothing product.

[0054] In one embodiment, the product is selected from one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology 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, provided is use of the cross-linked recombinant humanized collagen or the composition described herein as a subcutaneous filling material.

[0056] In another aspect, the use of the cross-linked recombinant humanized collagen or composition described herein in the preparation of the following products is provided: dermal implants, drug carriers, medical device coatings, implant coatings, shape-forming substances, viscous surgical materials, vascular sealants; spongy substances, such as those used for three-dimensional cell culture, tissue and organ engineering substances, hemostatic agents and wound treatment agents; fibers, such as fibers used for surgical sutures; tissue implants, corneal protective membranes or matrices for cell culture; and membranous substances, such as those used for anti-adhesion membranes or artificial skin.

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

[0058] Step 1: Dissolve the recombinant humanized collagen in a phosphate buffer and adjust the pH to 7.0 to obtain a neutral solution of the recombinant collagen, wherein 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, perform cross-linking reaction at -20°C to 50°C for 6-24 hours, and freeze-dry the cross-linked recombinant humanized collagen.

[0060] Step 3: The freeze-dried cross-linked recombinant humanized collagen is washed with a buffered salt solution and / or physiological saline until mixed evenly to obtain a cross-linked recombinant collagen gel.

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

[0062] Preferably, in step 1, 0.1 mol / L-1 mol / L sodium hydroxide solution and / or 1 M HCl are used to adjust the pH to 7±0.2.

[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 (eg, anhydrous sodium dihydrogen phosphate) and disodium hydrogen phosphate.

[0065] Preferably, the volume ratio of the buffered saline solution to the gel is 10:1-50:1.

[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) The present invention determines an aldehyde cross-linking agent and optimal reaction conditions suitable for recombinant humanized collagen, especially recombinant humanized collagen with a triple helix structure. By using the aldehyde cross-linking agent and optimal reaction conditions of the present invention, cross-linked 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 a buffer solution, which can effectively reduce the residual cross-linking agent content and effectively ensure product safety. The product is more suitable for use as a subcutaneous filling material.

[0070] (3) The present invention has found 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 that does not have a triple helix structure to form a gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Circular dichroism spectra of recombinant humanized collagen T16 at different cross-linker concentrations are shown.

[0072] Figure 2 The circular dichroism spectra of cross-linked recombinant humanized collagen T16 at different buffer pH 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 recombinant humanized type III 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 recombinant humanized collagen type XVII before and after cross-linking are shown.

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

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

[0079] Fig. 9 The circular dichroism results of type I recombinant humanized collagen after cross-linking are shown, which have no characteristic peaks of collagen and do not have a triple helix structure. DETAILED DESCRIPTION

[0080] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present 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 by DNA recombinant technology, or a combination of functional fragments containing human collagen. In this article, recombinant humanized collagen is a recombinant humanized type I, type II, type III, type V, type VII and other collagen types, which are peptides or polypeptides of multiple amino acid residues connected by peptide bonds. Recombinant humanized collagen can be a recombinant humanized collagen formed by tandem repetition of multiple repeating units. Methods for preparing recombinant humanized collagen by a tandem repetition strategy are known to those skilled in the art. The selection 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 by repeating units within this range has similar functions. Prior art literature reports that recombinant collagen can be constructed by tandem repetition of repeating units. The number of repeating units may vary. For example, Chen Hua et al. Biochemical and Biophysical Research Communications 508 (2019) 1018e1023 disclosed that the T16 with 16 tandem repeats of the GERGAPGFRGPAGPNGIPGEKGPAGERGAP repeating unit and the repeating unit has cell adhesion activity, and it is believed that recombinant proteins containing multiple tandemly repeated 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., 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 microfibers for use in biopharmaceuticals and industrial fields. J. Yao et al., J. Biochem. 136 (2004) 643e649 reported that recombinant collagen-like proteins containing tandem repeats of type I collagen peptides showed high cell adhesion activity.

[0082] As used herein, hydrogel is a type of extremely hydrophilic three-dimensional network structure gel that swells rapidly in water and can hold a large volume of water without dissolving in this swollen state. In this article, when referring to a gel, the gel may be a hydrogel.

[0083] As used herein, the expression "sequence identity" refers to the degree to which two sequences (amino acids) have the same residue in the same position after alignment. Such calculations are usually 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). In addition, when determining the degree of sequence identity between two amino acid sequences, the technician may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative 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 connected by covalent bonds to form a stable structure. The crosslinking process can be achieved in a variety of ways, including but not limited to chemical crosslinking and enzymatic crosslinking. In some embodiments herein, crosslinking is performed by a chemical process. Chemical crosslinking generally uses a crosslinking agent such as a dialdehyde crosslinking agent. The crosslinking agent can trigger a crosslinking reaction to form a covalent bond, thereby stabilizing the connection between molecules.

[0085] As used herein, the term "triple helical" protein is understood to refer to a homotrimeric or heterotrimeric protein comprising at least one "triple helical domain". The term encompasses variants and fragments of triple helical proteins and functional equivalents and derivatives thereof, preferably retaining the (Gly-XY)n sequence. The term "triple helical structure" refers to a protein comprising the general formula (Gly-XY)n, wherein Gly is glycine, X and Y represent identical or different amino acids (which may vary within the range of different Gly-XY triplets), wherein n may be between 2 and 300. A triple helical domain consists of three chains, the chains being characterized by repeated (Gly-XY)n motifs that fold into a triple helical protein conformation. The triple helical character can be determined by circular dichroism. In one embodiment, the recombinant humanized collagen has a negative peak at about 195 nm (e.g., 175-220 nm, e.g., 180-210 nm, 190-200 nm) and a positive peak at about 221 nm (e.g., 200-240 nm, 210-230 nm, 215-225 nm) on the circular dichroism spectrum.

[0086] As used herein, the term "glutaraldehyde" is a five-carbon straight-chain compound with two aldehyde groups, and its molecular formula is: O=CH-CH2-CH2-CH2-CH=O, and these two symmetrical aldehyde groups can form Schiff bases or Michael-type adducts with the amino groups of basic amino acids. Glutaraldehyde can also cross-link with the sulfhydryl group of cystine, the benzene ring and the imidazole ring of tyrosine and histidine. The polymerization reaction of glutaraldehyde monomers will be accelerated by temperature increase and under alkaline conditions, but this does not affect its ability to cross-link proteins. Glutaraldehyde has now become the most widely used bifunctional cross-linking agent in protein (especially enzyme) immobilization, among which the most widely used is to chemically cross-link the adsorbed enzyme to the carrier. The glutaraldehyde concentration and pH value in the glutaraldehyde method have an impact on the cross-linking effect. In some embodiments, after forming the cross-linked recombinant collagen or cross-linked recombinant collagen gel described herein, the dialdehyde cross-linking agent, such as glutaraldehyde, can be removed. The means of 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 numerical value greater than 1. The upper limit of the numerical value is not particularly limited and can be determined by those skilled in the art. For example, one or more can be 1-50 (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 that require 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 the body and prolong its existence time in the tissue. In the fields of medicine and bioengineering, cross-linked collagen is widely used. 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. Through covalent bond connection, cross-linked collagen forms a stable structure with higher tensile strength and wear resistance. This structural stability enables 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 has improved degradation resistance. Compared with uncross-linked collagen, the degradation rate of cross-linked collagen is slowed. This is very beneficial for applications that need to exist in the body for a long time, such as tissue repair and regeneration. Uncross-linked collagen is often easily degraded by enzymes in the body, resulting in 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 that need to withstand certain mechanical loads, such as artificial bones and tissue engineering materials. In contrast, uncross-linked collagen has weaker performance in these aspects and is difficult to meet these requirements.

[0090] In addition, the long-term stability of cross-linked collagen has been increased. Due to its high structural stability and resistance to degradation, it exists in tissues for a longer time. This is very important for some applications that require long-term maintenance of repair effects, such as artificial skin and cartilage repair. The advantages of cross-linked collagen may also bring some limitations. The cross-linking process may change the structure and properties of collagen, thereby affecting its bioactivity and cellular interactions. Therefore, when applying cross-linked collagen, its performance and biocompatibility need to be fully evaluated, and it needs to be selected and optimized according to the needs of specific applications. Despite this, cross-linked collagen is still a biomaterial with great 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 recombinant humanized collagen type III, recombinant humanized collagen type II, and recombinant humanized collagen type XVII.

[0092] In some embodiments, the collagen comprises a plurality of sequences of Gly-XY, wherein X and Y can be any amino acid residues, such as any 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 embodiments, the collagen comprises 2-300 (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 , 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 therebetween).

[0093] In some embodiments, the type III recombinant humanized collagen has one or more repeating units, the repeating units comprising SEQ ID NO: 1 or an amino acid sequence 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 to SEQ ID NO: 1, or 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, the repeating units comprising SEQ ID NO: 1 or an amino acid sequence 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 to SEQ ID NO: 1, or 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, the repeating units comprising SEQ ID NO: 2 or 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 SEQ ID NO: 2, or 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, the repeating units comprising SEQ ID NO: 3 or 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 SEQ ID NO: 3, or one or more amino acid substitutions, insertions, deletions or additions compared to SEQ ID NO: 2. 43, 44, 45, 46, 47, 48, 49, or 50 or any range therebetween).

[0094] In one embodiment, the recombinant humanized collagen has one or more repeating units, which comprise any one of SEQ ID NOs: 1-3, or 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 one of SEQ ID NOs: 1-3, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of any one of SEQ ID NOs: 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 any one of SEQ ID NOs: 4-7, or 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 one of SEQ ID NOs: 4-7, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of any one of SEQ ID NOs: 4-7.

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

[0097] Preparation of recombinant collagen

[0098] The exemplary recombinant humanized collagens in this article can all be conventionally prepared according to the methods of the prior art, 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. According to the amino acid sequence of the recombinant collagen, a commercial synthesis company is commissioned to synthesize the gene fragment and the constructed expression plasmid is transformed into Escherichia coli competent cells BL21 (DE3) (Merck). The specific process is:

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

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

[0102] 3: Add 600 μl of LB without resistance to the mixture and culture at 37°C and 220 rpm for 1 h.

[0103] 4: Take 200 μl of the bacterial solution and spread it evenly on an LB 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 antibiotic).

[0104] 5: Place the plate upside down in a 37°C incubator for about 20 hours until clearly visible colonies appear.

[0105] 6. Pick a single clone from the transformed LB plate and culture it in 10ml LB (containing 100μg / ml ampicillin) medium for 12h-16h, then transfer it to 2×YT medium (16g / L peptone, 10g / L yeast extract, 5g / L sodium chloride) at a ratio of 1:100 for expansion culture, culture at 37℃, 220rpm until the bacterial solution OD600 is 0.4-0.6, add 0.5mM IPTG (Sigma, catalog number: I5502-1G) at a final concentration to induce expression, and the induction conditions are 18℃, 180rpm and culture for 20h. Finally, collect the bacteria by centrifugation and store at -20℃ or immediately proceed to the next step of purification.

[0106] 7. Resuspend (1 L) the bacterial pellet with about 50 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride), break the bacteria using a high-pressure lysis instrument (Xinzhi Biotechnology), and centrifuge at 13,000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0107] 8. Use 5 column volumes of binding buffer (40mM NaH2PO3, 500mM NaCl, pH7.8) to balance the Ni-NTA (Qiagen, Cat. No. 30210) affinity column. Then add the protein supernatant and incubate at 4°C for 0.5-1h to allow the target recombinant protein to fully bind to the column material. Then rinse the impurities with 200ml of washing buffer (10mM imidazole, 40mM NaH2PO3, 500mM NaCl, pH 7.8) containing 10mM imidazole (Sigma). Finally, add an appropriate amount of Prescission Protease (PPase for short) (Sigma, SAE0045) protease with a His tag, incubate at 4°C for 16h, and collect the flow-through, which is the target collagen with the carrier protein removed. The obtained product is dialyzed overnight and lyophilized into dry powder for standby use.

[0108] 9. The purity of the obtained protein was detected by SDS-PAGE. The specific process is: take 40μl of the purified protein solution, add 10μl 5x protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), place it in 100℃ boiling water for 10min, then add 10μl to each well in SDS-PAGE protein gel, run it at 80V for 2h, use Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. The molecular weight of the purified protein is consistent with the expected molecular weight, indicating that the above recombinant collagen is correctly expressed.

[0109] For type III collagen T8, commercial synthesis was commissioned based on its amino acid sequence. The encoding nucleotide sequence was inserted into the pPiczalαA expression vector and the pPiczalαA expression vector was introduced into yeast competent cells (X33). The transformed yeast cells were induced to express to obtain type III collagen T8. Electrophoresis detection was performed. Confirm that type III collagen T8 was correctly expressed (for more detailed process, see the embodiment of CN 202311272338.8). 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 may 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] Recombinant humanized collagen described herein is suitable for use as biomaterials, materials for manufacturing, cosmetics or food additives. Methods for expressing recombinant triple helical proteins include standard expression methods generally known in the art, such as those described in Molecular Cloning (Sambrook and Russell (2001)). Expression systems for producing triple helical proteins are described in, for example, US20120116053. Transformation, positive transformant selection and culture methods of Pichia pastoris are disclosed in, for example, U.S. Patent Nos. 4,837,148; 4,855,231; 4882,279; 4929,555; 5,122,465; 5,324,639; 5,593,859 and 6,472,171. Methods for producing triple helical proteins are known in the art and are described in, for example, US20120282817, EP1809751 and WO2012 / 117406. Expression systems for producing triple helical proteins are described in, for example, US20120116053.

[0113] Composition

[0114] The cross-linked recombinant humanized collagen of the present invention can be prepared into a composition. The composition may include the cross-linked recombinant humanized collagen hydrogel described herein. The composition may also include a pharmaceutically and / or cosmetically acceptable carrier or solvent. The composition may be a pharmaceutical composition or a cosmetic composition for the purpose of medicine and / or cosmetics. For example, the composition is one or more of a biological dressing, a human bionic material, a plastic surgery 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 vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

[0115] The cosmetic composition may be a cosmetic composition having anti-wrinkle effect, skin oil control effect, skin repair effect and / or skin soothing effect. The part to which the cosmetic composition is applied is not particularly limited, and may be the face, hands, legs, trunk, etc. For example, the skin oil control effect is the facial skin oil control effect.

[0116] The form of the composition is not particularly limited as long as the intended function can be achieved. For example, the composition is a solid, liquid or gel composition.

[0117] The composition can be applied in any suitable manner, for example, for oral and / or topical compositions. The composition can also be prepared as a test kit. The test kit can include additional ingredients, such as auxiliary ingredients, such as buffers, and include instructions for use. In particular, the composition can be formulated into a suitable formulation, such as a liquid formulation. The formulation can include a buffer, such as a D-PBS buffer or a PBS buffer. If purified triple helical protein is used as a biomedical material, they must be able to be made into an appropriate form. To help reach these forms, purified triple helical protein can be stabilized (such as the case of animal collagen) before being used in medical applications to improve its long-term stability and mechanical strength when needed. A variety of suitable stabilization strategies are possible. Glutaraldehyde is a suitable reagent 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] Provided herein is 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, such as 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 numerical range therebetween. Recombinant humanized collagen can be any type of recombinant humanized collagen with triple helical structure, preferably but not limited to one or more of recombinant humanized type II, type III and type XVII collagen. The concentration of recombinant humanized collagen can be 10-100mg / mL, 10-60mg / mL, 20-40mg / mL or 30-40mg / mL, such as 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL, 60mg / mL, 70mg / mL, 80mg / mL, 90mg / mL, 100mg / mL or any range therebetween. The concentration of recombinant humanized collagen is the final concentration of recombinant humanized collagen in reaction volume or system. The exemplary recombinant humanized collagen herein can be conventionally prepared according to the methods of 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, succinaldehyde and glutaraldehyde.

[0121] The method can also include dissolving the 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. The pH can be adjusted using sodium hydroxide solution or hydrochloric acid, such as 0.5 mol / L sodium hydroxide solution or 1 M hydrochloric acid. The buffer solution can be a phosphate buffer solution or a carbonate buffer solution, such as a buffer salt selected from sodium dihydrogen phosphate and sodium dihydrogen phosphate. The cross-linked recombinant humanized collagen can still retain a triple helical structure.

[0122] The method can also include reacting the recombinant humanized collagen and the dialdehyde crosslinking agent at a temperature of -20°C-50°C, 0-25°C, 0-10°C, 0-8°C or 0-4°C for 6-24 hours, 12-24 hours, 12-20 hours or 12-16 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 therebetween. 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 therebetween.

[0123] In one embodiment, the method includes removing residual dialdehyde cross-linking agents from the cross-linked recombinant collagen gel.

[0124] In one embodiment, residual dialdehyde cross-linking agent is removed by dialysis.

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

[0126] In one embodiment, the collagen gel is prepared by stirring and dialysis with normal saline for 5-48 hours, preferably stirring and dialysis for 10-24 hours. The selection of dialysis time is conventional for those skilled in the art, for example, it can be 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 therebetween.

[0127] In one embodiment, the collagen gel is stirred and dialyzed with phosphate buffer before stirring with normal saline, for example, stirred and dialyzed for 1-5 hours, preferably 2-4 hours, for example 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 normal saline to 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 phosphate buffer is conventional for those skilled in the art. For example, the phosphate buffer can be a 0.2M sodium dihydrogen phosphate aqueous solution (liquid A, NaH2PO4·H2O 27.6g, dissolved in distilled water, diluted to 1000mL) and a 0.2M sodium dihydrogen phosphate aqueous solution (liquid B, Na2HPO4·7H2O 53.6g or Na2HPO4·12H2O 71.6g or Na2HPO4·2H2O 35.6g dissolved in distilled water, diluted to 1000mL). pH 7.0 phosphate buffer can be prepared by adding 39mL of liquid A to 61mL of liquid B. pH 7.5 phosphate buffer can be prepared by adding 16mL of liquid A to 84mL of liquid B.

[0128] Methods of using cross-linked recombinant collagen

[0129] Also provided herein is a method for causing cell adhesion or attachment, the method comprising contacting the cell with a cross-linked recombinant humanized collagen or a cross-linked recombinant humanized collagen composition as described herein. The composition is preferably a cross-linked recombinant humanized collagen hydrogel as described herein. The method of the present invention can be performed in vitro to increase cell adhesion to a culture container. Alternatively, the method of the present invention can also be performed in vivo.

[0130] Also provided herein is a method for promoting cell migration, the method comprising contacting cells with cross-linked recombinant humanized collagen or a cross-linked recombinant humanized collagen composition. The method of the present invention can be performed in vitro or in vivo.

[0131] Also provided herein is a makeup or beauty method, which includes applying the cross-linked recombinant humanized collagen or the cross-linked recombinant humanized collagen composition described herein to the skin. Application to the skin can be topical application, for example, application to a skin part that needs anti-wrinkle, oil control, repair and / or soothing. The method can also include applying other beauty and / or makeup products to the skin. The types of other beauty and / or makeup products are not particularly limited, and can be other cosmetics with skin anti-wrinkle, oil control, repair and / or soothing effects, such as collagen. These makeup or beauty methods can be skin anti-wrinkle, oil control, repair and / or soothing methods.

[0132] Also provided herein is a method for administering the cross-linked recombinant humanized collagen or the cross-linked recombinant humanized collagen composition described herein to a subject in need thereof. The method can be used to treat or prevent conditions associated with collagen deficiency, or can be used for anti-wrinkle, oil control, repair and / or soothing of the skin in a subject. The administration can be oral administration.

[0133] use

[0134] The present invention also provides the use of cross-linked recombinant humanized collagen or cross-linked recombinant humanized collagen composition in medicine, makeup or beauty products. These products can be external products, such as external cosmetic products or beauty products. These cosmetics or beauty products can be skin anti-wrinkle, oil control, repair and / or soothing products. For example, the product can be selected from biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair regeneration materials, liver tissue materials and vascular repair regeneration materials, 3D printing artificial organ biomaterials. The cross-linked recombinant humanized collagen of the present invention can be used for various applications and procedures, including recovery, regeneration and beauty procedures, vascular procedures, osteogenesis and chondrogenesis procedures, cartilage reconstruction, transplant bone substitutes, hemostasis, wound treatment and management, tissue reinforcement and support, incontinence, etc. Non-limiting examples of biomedical products that can be produced from the cross-linked recombinant humanized collagen of the present invention and their possible applications include, but are not limited to: dermal implants, drug carriers, medical device coatings, implant coatings (bone and blood vessels), shape-forming substances, viscosurgery, vascular sealants, cosmetics; sponge-like substances, such as for three-dimensional cell culture, tissue and organ engineering, hemostatic agents and wound treatment (artificial skin and wound dressings); fibers, such as for surgical sutures and hemostatic agents; gel-like substances, such as for tissue implants, corneal protective membranes, contact lenses and matrices for cell culture; and membrane-like substances, such as for anti-adhesion membranes, drug delivery systems, artificial skin and the like.

[0135] Also provided herein is a method for preparing a medicine, a cosmetic or a beauty product, the method comprising the steps of using the cross-linked recombinant humanized collagen, the cross-linked recombinant humanized collagen gel or the composition 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 control, repair and / or soothing product. In some embodiments, the product is selected from medical devices, biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials One or more.

[0136] Also provided herein are methods for preparing the following products: dermal implants, drug carriers, medical device coatings, implant coatings, shape-forming substances, viscous surgical materials, vascular sealants; spongy substances, such as those used for three-dimensional cell culture, tissue and organ engineering substances, hemostats and wound treatment agents; fibers, such as those used for surgical sutures; tissue implants, corneal protective membranes or matrices for cell culture; and membranous substances, such as those used for anti-adhesion membranes or artificial skin, the method comprising the steps of using the cross-linked recombinant humanized collagen, cross-linked recombinant humanized collagen gel or composition described herein.

[0137] Example

[0138] The following examples are provided to illustrate the present invention. It should be understood by those skilled in the art that the examples are merely illustrative and not limiting. The present invention is limited only by the scope of the appended claims.

[0139] General methods of PCR, cloning, ligation, etc. of nucleotides are well known to those skilled in the art and can be found, for example, in the following documents: "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, New York; 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); "APractical Guide To Molecular Cloning [A Practical Guide]", B. Perbal (1984).

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

[0141] Collagen Type I

[0142] GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS, SEQ ID NO: 8 (preparation method and purification method, see 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 method and purification method, see CN201811254050.7, CN201811438582.6 and CN202311272338.8; expressed in Pichia pastoris);

[0144] Morphological characteristics of type III recombinant humanized collagen T16 before and after cross-linking (motif GERGAPGFRGPAGPNGIPGEKGPAGERGAP, protein sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP, SEQ ID NO: 5) (preparation method and purification method, see CN201811254050.7 and CN201811438582.6; recombinant expression in Escherichia coli);

[0145] Type II recombinant humanized collagen (repeating unit, GEPGLQGPAGPPGEKGEPGDDGPSGAEGPP (SEQ ID NO: 2), sequence GEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPP, SEQ ID NO: 6) (preparation method and purification method, see CN201910111553.7; recombinant expression in Escherichia coli);

[0146] Type XVII recombinant humanized collagen (basic repeating unit GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLP GAVGEPGAKGAMGPA (SEQ ID NO: 3), GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA, SEQ ID NO: 7) (preparation method and purification method, see CN201911051106.3; recombinant expression in Escherichia coli).

[0147] The above recombinant humanized collagens are all prepared in-house by Shanxi Jinbo Biopharmaceutical Co., Ltd. They can also be prepared according to the steps in the "Preparation of recombinant collagen" 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 humanized collagen (type I collagen, type III collagen T8, type III recombinant humanized collagen T16, type II recombinant humanized collagen or type XVII recombinant humanized collagen) was fully dissolved in 1 mL of 0.2 M sodium phosphate buffer solution, and pH was adjusted to 7.0 and 7.5 using 0.5 mol / L sodium hydroxide solution and 1 M hydrochloric acid to obtain a solution of recombinant humanized collagen. Glutaraldehyde crosslinking agent was added to the recombinant humanized collagen solution at a final concentration of 0.01%, 0.03%, 0.08%, 0.1%, 0.15% and 0.20%, and placed in a 4°C constant temperature water bath for 12 hours of crosslinking reaction to obtain a crosslinked recombinant humanized collagen gel (the morphology before and after crosslinking is as shown in FIG. 1 ). Figure 3-Figure 6 and Figure 8 show).

[0150] In order to reduce the residual glutaraldehyde cross-linking agent in the recombinant humanized collagen gel, the cross-linked 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 normal saline for 8 hours to form a glutaraldehyde cross-linked recombinant humanized collagen gel.

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

[0152] 30 mg of recombinant humanized collagen (type III recombinant humanized collagen T16) was fully dissolved in 1 mL of phosphate and carbonate buffer solution, and 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 a solution of recombinant humanized collagen. 0.03% of glutaraldehyde crosslinking agent was added to the recombinant humanized collagen solution, placed in a 4°C constant temperature water bath, and crosslinked for 12 hours.

[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-cross-linked recombinant humanized collagen gel.

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

[0155] 1) Sample preparation

[0156] Prepare phosphate buffer solution (PBS) 1X: dissolve 8g NaCl, 0.2g KCl, 3.62g Na2HPO4.12H2O and 0.24g KH2PO4 in 800ml distilled water, adjust the pH value of the solution to 7.4 with HCl, add water to 1L, sterilize by autoclave, and store at room temperature. If the storage time is more than 1 week, filter through a 0.45 filter membrane if needed.

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

[0158] Sample testing:

[0159] Serial dilution sample configuration:

[0160] 1) Use an ice box to make ice, dilute the overnight sample at 4°C by two times (note to change the pipette tip), take 300 μl of the overnight sample, and test it on the machine.

[0161] 2) Instrument (JASCO circular dichroism spectrometer, JASCO J-815) parameter setting

[0162] Band width: 1.0nm

[0163] Step:1.0nm

[0164] Measurement range: 190-260nm

[0165] Time-per-point: 1s

[0166] Scanning speed: 50nm / min

[0167] Repeats: 3 times

[0168] Measurement temperature: 4°C.

[0169] 3) Scanning of standard product CD

[0170] The scanning wavelength was set to 190-260 nm for blank buffer baseline test, and the circular dichroism absorption of the sample solution PBS in the range of 190-260 nm was collected.

[0171] 4) Scanning and graph processing: Use the machine's own software to process the data, export it to Excel, and use Graphpad to draw the graph.

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

[0173] The method for detecting the adhesion activity of collagen can refer to 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 sample to be tested is detected by ultraviolet absorption method, including bovine type I collagen (China Food and Drug Inspection Institute, No.: 380002), the cross-linked collagen provided by the present invention (cross-linked type XVII collagen, cross-linked type II collagen and cross-linked type III collagen T16). Specifically, the ultraviolet absorption of the sample at 215nm and 225nm is measured respectively, and the protein concentration is calculated using the empirical formula C (μg / mL) = 144x (A215-A225). Note that the detection needs to be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far ultraviolet light, it is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that are not colorable with Coomassie Brilliant Blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After the protein concentration is detected, the concentration of all proteins to be tested is 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 5 3T3 cells in good culture state were incubated at 37℃ for 60min.

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

[0178] (5) Use LDH detection kit (Roche, 04744926001) to detect the absorbance at OD492nm according to the manufacturer's instructions. The cell adhesion rate can be calculated based on the value of the blank control.

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

[0180]

[0181] The cell adhesion rate can reflect the adhesion activity of each protein. The higher the activity of the protein, the better the external environment it can provide to the cells in a short time, helping the cells to adhere to the wall.

[0182] Experimental Results

[0183] Figure 1 The circular dichroism spectra of recombinant humanized collagen T16 cross-linked at different glutaraldehyde concentrations are shown. Unexpectedly, most glutaraldehyde cross-linked recombinant collagen cannot maintain its structural characteristics and will damage its protein structure. The results of this example prove that 0.03% glutaraldehyde concentration can maintain the triple helical structure of recombinant humanized collagen while playing a cross-linking role. Figure 1 It can be seen that the circular dichroism spectrum has typical positive and negative peaks of collagen, that is, a negative peak near 195nm and a positive peak near 221nm, with a triple helical structure. At a glutaraldehyde concentration of 0.03%, recombinant humanized collagen T8, recombinant humanized collagen type II and recombinant humanized collagen type XVII also have similar triple helical structures.

[0184] Figure 2 The circular dichroism spectra of recombinant humanized collagen glutaraldehyde cross-linked in buffers with different pH values ​​are shown. To determine the optimal pH cross-linking conditions for buffers (phosphate and carbonate), cross-linking experiments were performed at different pH values ​​in the buffer system using the same glutaraldehyde concentration (0.03%), and the cross-linked samples were analyzed using circular dichroism. Figure 2 The results show that recombinant humanized collagen T16 can maintain a triple helix structure in the range of pH 5.7-8.0, with a negative peak near 195nm and a positive peak near 221nm, and has a triple helix structure. The triple helix structure cannot be maintained in the range of pH 9.5-10.5. In the range of pH 5.7-8.0, recombinant humanized collagen T8, type II recombinant humanized collagen, and type XVII recombinant humanized collagen also have similar triple helix structures.

[0185] Figure 3 The morphological changes of recombinant humanized collagen T8 expressed in yeast system before and after cross-linking (PH7.0). The cross-linked recombinant humanized collagen has a typical hydrogel morphology. Compared with the recombinant humanized collagen before cross-linking, the cross-linked recombinant humanized collagen can have a certain morphological support.

[0186] Figure 4 The morphological characteristics of recombinant humanized type III collagen T16 expressed and purified in the E. coli system before and after cross-linking are shown. Figure 4 The morphological changes at pH 7.0 and 7.5 are shown. The cross-linked recombinant collagen formed at pH 7.0 and 7.5 can maintain a good mechanical support morphology.

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

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

[0189] Figure 7 It is shown that, compared with bovine type I collagen (PC group, 0.5 mg / ml), the cross-linked recombinant humanized collagen hydrogel of the present invention can still maintain its bioadhesion activity. It is unexpected that the cross-linked recombinant humanized collagen can maintain cell adhesion activity.

[0190] Figure 8 The morphology of the type I recombinant humanized collagen after cross-linking treatment is shown. The type I recombinant humanized collagen does not have a triple helix structure. After being treated by the method of Example 1, the type I recombinant humanized collagen is still in a fluid state and fails to form a gel.

[0191] Fig. 9 The circular dichroism results of type I recombinant humanized collagen after cross-linking are shown, which have no characteristic peaks of collagen and do not have a triple helix structure.

[0192] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for cross-linking recombinant humanized collagen, comprising the following steps: 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%, preferably 0.02%-0.08%, Preferably, the recombinant humanized collagen has a triple helical structure. Preferably, the recombinant humanized collagen has a negative peak at 175-220 nm and a positive peak at 200-240 nm on the circular dichroism spectrum. Preferably, the recombinant humanized collagen has a negative peak at about 195 nm and a positive peak at about 221 nm on the circular dichroism spectrum; Preferably, the recombinant humanized collagen is one or more of recombinant humanized type II, type III and type XVII collagen; Preferably, the concentration of the dialdehyde crosslinking agent is 0.02%-0.07%; Preferably, the concentration of the dialdehyde crosslinking agent is 0.03%-0.04%, preferably 0.03%-0.035%; Preferably, the dialdehyde cross-linking agent is one or more of glyoxal, malondialdehyde, succinaldehyde and glutaraldehyde; Preferably, the 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; Preferably, the buffer solution is a phosphate buffer solution or a carbonate buffer solution, preferably a 0.1M-0.4M or 0.1M-0.3M phosphate buffer solution or a carbonate buffer solution; Preferably, the buffer salt is selected from sodium dihydrogen phosphate and disodium hydrogen phosphate; Preferably, the concentration of the recombinant humanized collagen is 10-100 mg / mL, 10-70 mg / mL, 20-40 mg / mL or 30-40 mg / mL; Preferably, the recombinant humanized collagen retains a triple helical structure after cross-linking; Preferably, the recombinant humanized collagen comprises a sequence of (Gly-XY)n, where n is 2-300; Preferably, the recombinant humanized collagen has one or more repeating units, the repeating unit comprising any one of SEQ ID NOs: 1-3, or an amino acid sequence 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 NOs: 1-3, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of any one of SEQ ID NOs: 1-3, preferably, the recombinant humanized collagen has 2-50 repeating units; Preferably, the recombinant humanized collagen has any one of SEQ ID NOs: 4-7, or an amino acid sequence 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 to any one of SEQ ID NOs: 4-7, or an amino acid sequence in which one or more amino acid residues are substituted, added, deleted or inserted into the amino acid sequence of any one of SEQ ID NOs: 4-7; Preferably, the recombinant humanized collagen is expressed by Escherichia coli or by yeast, such as Pichia pastoris; Preferably, the method comprises reacting recombinant humanized collagen and a dialdehyde crosslinking agent at a temperature of -20°C-50°C, 0-25°C, 0-10°C, 0-8°C or 0-4°C for 6-24 hours, 12-24 hours, 12-20 hours or 12-16 hours to form a cross-linked recombinant collagen gel; Preferably, the cross-linked recombinant collagen gel is freeze-dried into a powder.

2. A method for preparing a cross-linked recombinant humanized collagen gel, comprising the method of claim 1 and comprising removing residual dialdehyde cross-linking agents from the cross-linked recombinant collagen gel; preferably, removing the residual dialdehyde cross-linking agents by dialysis; The cross-linked recombinant collagen powder is placed in a dialysis bag, or the cross-linked recombinant collagen gel is directly placed in a dialysis bag, and stirred with a buffer solution and / or physiological saline to remove the residual dialdehyde cross-linking agent; preferably, the collagen gel is prepared by stirring and dialyzing with physiological saline for 5-48 hours, preferably 10-24 hours; preferably, before stirring and dialyzing with physiological saline, the collagen gel is stirred and dialyzed with a phosphate buffer, for example, stirred and dialyzed for 1-5 hours, preferably 2-4 hours, to obtain a cross-linked recombinant collagen gel with a reduced content of residual dialdehyde cross-linking agents; Preferably, the cross-linked recombinant humanized collagen gel is a hydrogel; Preferably, the volume ratio of phosphate buffer or physiological saline to the cross-linked recombinant collagen gel is 10:1-50:

1.

3. Cross-linked recombinant humanized collagen prepared by the method according to claim 1.

4. A cross-linked recombinant humanized collagen gel, comprising the cross-linked recombinant humanized collagen of claim 3 or prepared by the method of claim 2, preferably, the cross-linked recombinant humanized collagen gel is a hydrogel.

5. A composition comprising the cross-linked recombinant humanized collagen of claim 3 or the cross-linked recombinant humanized collagen gel of claim 4; Preferably, the composition further comprises a pharmaceutically or cosmetically acceptable carrier, diluent or excipient; Preferably, the composition is a pharmaceutical composition, a food composition or a cosmetic composition; Preferably, the cosmetic composition is a cosmetic composition with anti-wrinkle effect, skin oil control effect, skin repair effect and / or skin soothing effect; preferably, the skin oil control effect is facial skin oil control effect; Preferably, the composition is a kit, preferably comprising physiological saline; Preferably, the composition is one or more of medical devices, biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives; Preferably, the composition is a solid, liquid or gel composition; Preferably, the composition is an oral and / or topical composition; Preferably, the topical administration is topical administration to the skin; preferably, the composition is a transdermal composition; Preferably, the collagen hydrogel further comprises physiological saline.

6. A method for preparing a collagen gel, comprising the step of dissolving the cross-linked recombinant humanized collagen of claim 3 with a buffer and / or physiological saline, preferably, the gel is a hydrogel.

7. A method for causing cells to adhere or adhere to a wall or promoting cell migration, the method comprising contacting the cells with the cross-linked recombinant humanized collagen according to claim 3, the cross-linked recombinant humanized collagen gel according to claim 4, or the composition according to claim 5.

8. A cosmetic or beauty method, comprising applying to the skin the cross-linked recombinant humanized collagen according to claim 3, the cross-linked recombinant humanized collagen gel according to claim 4, or the composition according to claim 5, and optionally applying other cosmetic and / or make-up products to the skin; Preferably, the cosmetic or beauty method is a skin anti-wrinkle, oil control, repair and / or soothing method; Preferably, the administration is topical; Preferably, administration is transdermal.

9. Use of the cross-linked recombinant humanized collagen according to claim 3, the cross-linked recombinant humanized collagen gel according to claim 4 or the composition according to claim 5 in the preparation of medicines, cosmetics or beauty products, Preferably, wherein the product is a product for external use, Preferably, the cosmetic or beauty product is a skin anti-wrinkle, oil control, repair and / or soothing product; Preferably, the product is selected from one or more of medical devices, biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, and 3D printed artificial organ biomaterials.

10. Use of the cross-linked recombinant humanized collagen according to claim 3, the cross-linked recombinant humanized collagen gel according to claim 4 or the composition according to claim 5 in the preparation of the following products: dermal implants, drug carriers, medical device coatings, implant coatings, shape-forming substances, viscous surgical materials, vascular sealants; spongy substances, such as those used for three-dimensional cell culture, tissue and organ engineering substances, hemostatic agents and wound treatment agents; fibers, such as fibers used for surgical sutures; tissue implants, corneal protective membranes or matrices for cell culture; and membranous substances, such as those used for anti-adhesion membranes or artificial skin.

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