CN119390816A

CN119390816A - A novel method for preparing recombinant humanized collagen hydrogel

Info

Publication number: CN119390816A
Application number: CN202411095415.1A
Authority: CN
Inventors: 陆路; 杨霞; 姜世勃; 花晨; 王茜; 徐巍; 兰小宾; 王建; 于玉凤
Original assignee: Shanxi Jinbo Bio Pharmaceutical Co ltd; Fudan University
Current assignee: Shanxi Jinbo Bio Pharmaceutical Co ltd; Fudan University
Priority date: 2024-08-09
Filing date: 2024-08-09
Publication date: 2025-02-07

Abstract

Provides a novel preparation method of recombinant humanized collagen hydrogel. The method comprises the steps of reacting recombinant humanized collagen with a dialdehyde crosslinking agent, wherein the concentration of the dialdehyde crosslinking agent is 0.01-0.25%, 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 invention determines aldehyde crosslinking agent and optimal reaction condition suitable for recombinant humanized collagen, especially recombinant humanized collagen with triple helix structure.

Description

Novel preparation method of recombinant humanized collagen hydrogel

Technical Field

The application relates to the field of proteins or hydrogels, in particular to a collagen hydrogel, a preparation method and application thereof.

Background

Collagen is a protein widely distributed in connective tissues of human bodies, is also protein with the largest content of human bodies, and can account for 25% -35% of the total protein. Its main functions are maintaining extracellular environment, maintaining normal physiological functions of tissue and organ, repairing injury of body, etc. Collagen is a natural biological resource, has the biological tissue compatibility required by other high molecular materials, and has the supporting elasticity and the degradability for cells, so that the collagen can be widely applied to industries such as medicines, cosmetics and the like.

Natural collagen molecules can form a specific supercoiled structure, which is a left-handed helix with a basic repeat of 3 amino acid residues, typically Gly-X-Y. Gly is necessary for the formation of hydrogen bonds in collagen, and has no side chains, so that collagen is closely packed, and skin tension and elasticity can be maintained. The natural collagen has the defects of poor stability, sensitivity to collagenase, matrix metalloenzyme and other enzymes, short retention time in vivo, lack of mechanical strength in a water system and the like, so that the development and application of the natural collagen in the biomedical field are limited. The physical and chemical properties of the collagen can be changed through various chemical modification and crosslinking reactions, so that the mechanical strength and degradation resistance of the collagen are improved, novel bioactive and functional collagen crosslinked derivatives are obtained, and novel biological materials are further developed.

The crosslinking method commonly used at present mainly comprises the steps of uniformly mixing a crosslinking agent and collagen in a solution to carry out crosslinking reaction. The cross-linking of the recombinant humanized collagen is less, and the consumption of the cross-linking agent, the reaction time, the reaction temperature and the like have uncertainty. Therefore, in order to solve the problems of poor filling and supporting properties of the existing recombinant humanized collagen, the determination and improvement of the crosslinking reaction of the recombinant humanized collagen are urgent. The invention provides a preparation method of a crosslinked recombinant collagen gel, which aims to solve the problems that the existing collagen filler only meets the instant filling effect, has poor support property after implantation and the like.

Disclosure of Invention

In order to solve the problem that the existing collagen filler only meets the instant filling effect and needs to be injected again to maintain the effect after implantation degradation, the invention provides a preparation method of the crosslinked recombinant collagen gel. The method prepares the recombinant collagen gel prepared by crosslinking the aldehyde crosslinking agent, and the crosslinked recombinant collagen gel has low toxicity and strong stability and meets the requirement of instant filling.

The present invention is based, in part, on the discovery by the inventors that when cross-linking recombinant humanized collagen having a triple helix structure, the cross-linking agent and the concentration of cross-linking agent employed have an effect on the triple helix structure of the recombinant humanized collagen. The triple helix structure of the original recombinant humanized collagen is maintained by using the recombinant humanized collagen crosslinked only with a specific crosslinking agent and concentration.

In one aspect, a method of cross-linking recombinant humanized collagen is provided comprising the step of reacting the recombinant humanized collagen with a dialdehyde cross-linking agent, wherein the dialdehyde cross-linking agent is present at a concentration of 0.01-0.25%, preferably 0.02% -0.08%.

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

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

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

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

In one embodiment, the concentration of dialdehyde crosslinking agent is 0.02% to 0.07% or 0.02% to 0.06%.

In one embodiment, the concentration of dialdehyde crosslinking agent is 0.03% to 0.04% or 0.03% to 0.035%.

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

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

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

In one embodiment, 0.1M to 0.4M or 0.1M to 0.3M phosphate buffer or carbonate buffer.

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

In one embodiment, the crosslinked recombinant humanized collagen has a triple helix structure.

In one embodiment, the method further comprises reacting the recombinant humanized collagen and the dialdehyde crosslinking agent at a temperature of-20 ℃ to 50 ℃,0 to 25 ℃ (e.g., 1℃、2℃、3℃、4℃、5℃、6℃、7℃、8℃、9℃、10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃)、0-10℃、0-8℃ or 0 to 4 ℃) for a period of 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, forming a crosslinked recombinant collagen gel.

In one embodiment, the crosslinked recombinant collagen gel is lyophilized to a powder.

In one embodiment, the recombinant humanized collagen comprises the sequence of (Gly-X-Y) n, n being 2-300.

In one embodiment, the recombinant humanized collagen has one or more repeat units comprising an amino acid sequence of any one of SEQ ID NOS: 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 to any one of SEQ ID NOS: 1-3 or an amino acid sequence of any one of SEQ ID NOS: 1-3 with one or more amino acid residues. Preferably, the recombinant humanized collagen has 2-50 repeat units.

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

In another aspect, a method of preparing a crosslinked recombinant humanized collagen gel is provided, comprising the method described herein and comprising removing residual dialdehyde crosslinking agent from the crosslinked recombinant collagen gel. In one embodiment, the residual dialdehyde-based crosslinking agent is removed by dialysis.

In one embodiment, the crosslinked recombinant collagen powder is placed in a dialysis bag, or the crosslinked recombinant collagen gel is placed directly in a dialysis bag, and stirred with buffer and/or physiological saline to remove residual dialdehyde crosslinking agent. Preferably, the collagen gel is prepared by stirring and dialyzing with physiological saline for 5 to 48 hours, preferably 10 to 24 hours. Preferably, the collagen gel is stirred for dialysis, e.g. for 1-5 hours, preferably 2-4 hours, with phosphate buffer before stirring for dialysis with physiological saline, to obtain a crosslinked recombinant collagen gel with reduced residual dialdehyde-based cross-linking agent content.

In one embodiment, the crosslinked recombinant humanized collagen gel is a hydrogel.

In one embodiment, the buffer salt is selected from the group consisting of sodium dihydrogen phosphate and disodium hydrogen phosphate.

In one embodiment, the volume ratio of phosphate buffer or physiological saline to crosslinked recombinant collagen gel is 10:1 to 50:1.

In various embodiments herein, the recombinant humanized collagen is expressed by escherichia coli or by pichia pastoris, such as pichia pastoris.

In another aspect, there is provided a crosslinked recombinant humanized collagen prepared by the methods described herein.

In another aspect, a crosslinked recombinant humanized collagen gel is provided that comprises the crosslinked recombinant humanized collagen described herein or is prepared by the methods described herein. In one embodiment, the crosslinked recombinant humanized collagen gel is a hydrogel.

In another aspect, a composition is provided comprising a crosslinked recombinant humanized collagen or crosslinked recombinant humanized collagen gel described herein.

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

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

In one embodiment, the cosmetic composition is a cosmetic composition having anti-wrinkle efficacy, skin oil control efficacy, skin repair efficacy, and/or skin soothing efficacy.

In one embodiment, the skin oil control efficacy is facial skin oil control efficacy.

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

In one embodiment, the composition is one or more of a biological dressing, a human biomimetic material, a cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynaecological biomaterial, a nerve repair regenerating material, a liver tissue material, and a vascular repair regenerating material, a 3D printed artificial organ biomaterial, a cosmetic material, a pharmaceutical adjuvant, and a food additive.

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

In one embodiment, the composition is an orally and/or topically administered composition.

In one embodiment, the topical application is topical application to the skin.

In one embodiment, the composition is a transdermal composition.

In one embodiment, the collagen hydrogel further comprises physiological saline.

In another aspect, a method of preparing a collagen gel is provided that includes the step of dissolving the crosslinked recombinant humanized collagen described herein with a buffer and/or physiological saline.

In another aspect, a method of adhering or attaching cells or promoting cell migration is provided, the method comprising contacting cells with a crosslinked recombinant humanized collagen, crosslinked recombinant humanized collagen gel or composition described herein.

In another aspect, cosmetic or cosmesis methods are provided that include applying to skin a crosslinked recombinant humanized collagen, a crosslinked recombinant humanized collagen gel, and/or a composition according to the present disclosure, as described herein, and optionally applying to skin other cosmetic and/or cosmesis products.

In one embodiment, the cosmetic or cosmesis method is a dermatological anti-wrinkle, oil control, repair and/or soothing method.

In one embodiment, the administration is topical.

In one embodiment, the administration is transdermal.

In another aspect, there is provided the use of the crosslinked recombinant humanized collagen and/or composition described herein in the preparation of a pharmaceutical, cosmetic or cosmesis product.

In one embodiment, the product is a topical product.

In one embodiment, the cosmetic or cosmesis product is a dermatological anti-wrinkle, oil control, repair and/or soothing product.

In one embodiment, the product is selected from one or more of a biological dressing, a human biomimetic material, a cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynaecological biomaterial, a nerve repair regenerating material, a liver tissue material and a vascular repair regenerating material, a 3D printed artificial organ biomaterial.

In another aspect, there is provided the use of the crosslinked recombinant humanized collagen or composition described herein as a subcutaneous filler material.

In another aspect, there is provided the use of the crosslinked recombinant humanized collagen or compositions described herein in the preparation of dermal implants, drug carriers, medical device coatings, implant coatings, shape-forming substances, adhesive surgical materials, vascular sealants, spongy substances, such as for three-dimensional cell cultures, tissue and organ engineering substances, hemostatic agents and wound healing agents, fibers, such as for surgical sutures, tissue implants, cornea protective films or matrices for cell culture, and membranous substances, such as for anti-adhesive films or artificial skin.

The method of preparing the crosslinked recombinant collagen gel may include one or more of the following steps:

Step 1, dissolving recombinant humanized collagen in phosphate buffer solution, and regulating pH to 7.0 to obtain neutral solution of the recombinant collagen, wherein the recombinant humanized collagen is prepared by fermenting escherichia coli or pichia pastoris.

And 2, adding aldehyde cross-linking agents (glyoxal, glutaraldehyde and the like) into the solution of the recombinant humanized collagen, performing cross-linking reaction for 6-24 hours at the temperature of 20-50 ℃, and freeze-drying the cross-linked recombinant humanized collagen.

And 3, washing the freeze-dried crosslinked recombinant humanized collagen with a buffer salt solution and/or physiological saline until the mixture is uniformly mixed to obtain crosslinked recombinant collagen gel.

Preferably, in step 1, the recombinant humanized collagen has a mass of 10mg to 60mg.

Preferably, in step 1, the pH is adjusted to 7.+ -. 0.2 using 0.1mol/L to 1mol/L sodium hydroxide solution and/or 1M HCl.

Preferably, in step 2, the cross-linking agent is glutaraldehyde. Preferably, in step 2, the crosslinking reaction time is 12 hours. Preferably, the glutaraldehyde concentration is 0.01-0.25%.

Preferably, in step 3, the buffer salt is selected from the group consisting of sodium dihydrogen phosphate (e.g., anhydrous sodium dihydrogen phosphate) and disodium hydrogen phosphate.

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

Preferably, in step 3, the total time of the washing is 10 to 24 hours.

Compared with the prior art, the invention has the following advantages:

(1) The invention determines aldehyde crosslinking agent and optimal reaction condition suitable for recombinant humanized collagen, especially recombinant humanized collagen with triple helix structure. By adopting the aldehyde cross-linking agent and the optimal reaction conditions, the cross-linked recombinant humanized collagen can be prepared under the condition of retaining the triple helix structure of the recombinant humanized collagen.

(2) After the crosslinking reaction of the recombinant humanized collagen, the gel is cleaned by adopting a buffer solution, so that the content of the residual crosslinking agent can be effectively promoted and reduced, the safety of the product is effectively ensured, and the product is more suitable for being used as a subcutaneous filling material.

(3) The present invention finds that the method of the present invention is suitable for cross-linking recombinant collagen having a triple helix structure to form a gel, whereas for type I recombinant collagen not having a triple helix structure, the method of the present invention is not capable of cross-linking it to form a gel.

Drawings

FIG. 1 shows circular dichroism spectrum of recombinant humanized collagen T16 at different cross-linker concentrations.

FIG. 2 shows circular dichroism spectrum of cross-linked recombinant humanized collagen T16 at different buffer pH.

Figure 3 shows the morphological changes of the recombinant humanized collagen type III T8 before and after cross-linking.

Figure 4 shows the morphological changes of the recombinant humanized collagen type III T16 before and after cross-linking.

Figure 5 shows the morphological changes of the recombinant humanized collagen type II before and after cross-linking.

Figure 6 shows the morphological changes of the recombinant humanized collagen of type XVII before and after cross-linking.

FIG. 7 shows cell adhesion activity of the recombinant humanized collagen after crosslinking.

FIG. 8 shows the morphology of the type I recombinant humanized collagen after the crosslinking treatment.

FIG. 9 shows the circular dichromatic results of the type I recombinant humanized collagen after crosslinking, without collagen characteristic peaks and without triple helix structure.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

As used herein, recombinant humanized collagen is a full-length or partial amino acid sequence fragment encoded by a human collagen specific sex gene prepared by DNA recombinant techniques, or a combination of functional fragments comprising human collagen. In this context, recombinant humanized collagen is a recombinant humanized type I, II, III, V, VII or the like collagen, which is a peptide or polypeptide of multiple amino acid residues linked by peptide bonds. The recombinant humanized collagen may be a recombinant humanized collagen formed by tandem repeat of a plurality of repeating units. Methods for preparing recombinant humanized collagen by tandem repeat strategies are known to those skilled in the art. The choice of the number of repeating units is conventional to the person skilled in the art. For example, the recombinant humanized collagen may have 2-50 repeat units, such as 2-10. It will be appreciated that recombinant humanized collagen formed from repeat units within this range has similar functionality. The prior art document reports that recombinant collagen can be obtained by tandem repeat construction of repeat units. There may be some variation in the number of repeating units. For example Chen huan et al Biochemical and Biophysical Research Communications (2019) 1018e1023 discloses that both the GERGAPGFRGPAGPNGIPGEKGPAGERGAP repeat unit and T16 of the 16 tandem repeats of the repeat unit may possess cell adhesion activity, and that recombinant proteins containing triple helix fragments of multiple tandem repeats may be considered to 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 demonstrate that recombinant collagen-like proteins having multiple identical triple helical peptide units arranged in tandem repeats can self-assemble into microfibers for use in biopharmaceutical 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.

As used herein, hydrogels (Hydrogel) are a class of extremely hydrophilic three-dimensional network structure gels that swell rapidly in water and in this swollen state can hold a large volume of water without dissolution. Herein, when referring to a gel, the gel may be a hydrogel.

As used herein, the expression "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at identical positions after alignment. Such calculations are typically performed using a computer program. Exemplary procedures for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman,1988; pearson, 1990), 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, the skilled artisan may consider so-called "conservative" amino acid substitutions, which may 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 have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

As used herein, the term "cross-linking" refers to the chemical process of building a stable structure by covalent linkage between collagen molecules. The crosslinking process may be accomplished 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 employs a crosslinking agent such as a dialdehyde-type crosslinking agent. The crosslinking agent can initiate a crosslinking reaction to form a covalent bond, and the intermolecular association is stabilized.

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 helix proteins and functional equivalents and derivatives thereof, preferably retaining the (Gly-X-Y) n sequence. The term "triple helix structure" refers to a protein comprising the general formula (Gly-X-Y) n, wherein Gly is glycine, X and Y represent the same or different amino acids (which in particular may vary within the scope of different Gly-X-Y triplets), wherein n may be between 2 and 300. The triple-helical domain consists of three chains characterized by a repeat (Gly-X-Y) n motif folded into the triple-helical protein conformation. Triple helix characteristics can be determined by circular dichroism. In one embodiment, the recombinant humanized collagen has a negative peak at about 195nm (e.g., 175-220nm, e.g., 180-210nm,190-200 nm) and a positive peak at about 221nm (e.g., 200-240nm,210-230nm,215-225 nm) on a circular dichroism spectrum.

As used herein, the term "glutaraldehyde" is a five-carbon straight-chain compound having two aldehyde groups of the formula o=ch-CH ₂-CH₂-CH₂ -ch=o, which can form Schiff bases or Michael adducts with the amino groups of basic amino acids. Glutaraldehyde also can crosslink with the benzene and imidazole rings of the sulfhydryl, tyrosine and histidine groups of cystine. The temperature rise and under alkaline conditions accelerate the polymerization of glutaraldehyde monomers, but this does not affect their ability to crosslink proteins. Glutaraldehyde has now become the most widely used bifunctional cross-linking agent in protein (especially enzyme) immobilization, with the most widely used being the chemical cross-linking of the sorbed enzyme to the support. Glutaraldehyde concentration, pH value, etc. in the glutaraldehyde method have an influence on the crosslinking effect. In some embodiments, after forming the crosslinked recombinant collagen or crosslinked recombinant collagen gel described herein, a dialdehyde-based crosslinking agent, such as glutaraldehyde, may be removed. Means for removing glutaraldehyde from the gel are known to those skilled in the art. In some embodiments, glutaraldehyde is removed by dialysis means.

The term "one or more" as used herein refers to any number above 1. The upper limit of the numerical value is not particularly limited and may be determined by one skilled in the art. For example, one or more may 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).

Crosslinked collagen

The structural stability and mechanical strength of the crosslinked collagen are enhanced. The cross-linked structure not only can improve the tensile strength of the collagen, but also can enhance the wear resistance of the collagen, so that the collagen can be widely applied to application fields requiring high mechanical properties, such as artificial bones and tissue engineering materials in the medical field. In addition, the crosslinked structure can also adjust the degradation rate of collagen in vivo and prolong the existence time of the collagen in tissues. In the fields of medicine and bioengineering, crosslinked collagen has wide application. It can be made into artificial skin, cartilage repair and bone tissue engineering, and is also an ideal carrier material for drug delivery and cell culture. Crosslinked collagen also exhibits its unique advantages in the treatment of tissue defects such as wounds, burns, and the like, and plays an important role in orthopedic surgery. The unique property of the crosslinked collagen greatly improves the application potential of the crosslinked collagen in the fields of medicine and bioengineering. The increased structural stability and mechanical strength thereof provide an important material basis for tissue repair and regeneration. Crosslinked collagen has the significant advantage over uncrosslinked collagen that (1) the structural stability of crosslinked collagen is significantly enhanced. Through covalent bond connection, the crosslinked collagen forms a stable structure, and has higher tensile strength and wear resistance. The structural stability enables the cross-linked collagen to better maintain the shape and mechanical properties of the cross-linked collagen in application, and reduces the risk of structural loss or degradation. (2) crosslinked collagen has improved resistance to degradation. The degradation rate of crosslinked collagen is retarded compared to uncrosslinked collagen. This is very advantageous for applications requiring a long time to be present in the body, such as tissue repair and regeneration. Uncrosslinked collagen tends to be easily degraded by enzymes in the body, resulting in loss of its function and structure. (3) the mechanical properties of the crosslinked collagen are enhanced. The performance in the aspects of tensile strength, rigidity, stability and the like is improved, so that the composite material is more suitable for applications which need to bear a certain mechanical load, such as artificial bones and tissue engineering materials. In contrast, uncrosslinked collagen is weak in these respects, and it is difficult to meet these requirements.

In addition, the long-term stability of the crosslinked collagen is increased. It has higher structural stability and degradation resistance, and its existence time in tissue is longer. This is important for applications where long-term maintenance of repair effects is required, such as artificial skin and cartilage repair. The advantages of cross-linked collagen may also bring about some limitations. The cross-linking process may alter the structure and properties of collagen, thereby affecting its bioactivity and cellular interactions. Therefore, when cross-linked collagen is used, it is necessary to comprehensively evaluate its properties and biocompatibility, and to select and optimize it according to the needs of a specific application. Nevertheless, cross-linked collagen is still a biological material with great potential, providing powerful support for tissue repair and regeneration.

In some embodiments, the collagen is recombinant humanized collagen. In some embodiments, the recombinant humanized collagen is a type III recombinant humanized collagen, a type II recombinant humanized collagen, and a type XVII recombinant humanized collagen.

In some embodiments, the collagen comprises a sequence of multiple Gly-X-Y, where X and Y may be any amino acid residue, 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 a number of between 2 and 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) Gly-X-Y.

In some embodiments, the recombinant humanized collagen type III has one or more repeat units comprising SEQ ID NO. 1 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. 1 or is one or more amino acid substitutions, insertions, deletions or additions compared to SEQ ID NO. 1. In some embodiments, the recombinant humanized collagen type III has one or more repeat units comprising SEQ ID NO. 1 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. 1 or is one or more amino acid substitutions, insertions, deletions or additions compared to SEQ ID NO. 1. In some embodiments, the recombinant humanized collagen type II has one or more repeat 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 is one or more amino acid substitutions, insertions, deletions or additions compared to SEQ ID NO. 2. In some embodiments, the recombinant humanized collagen type XVII has one or more repeat 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 is one or more amino acid substitutions, insertions, deletions or additions compared to SEQ ID NO. 2. Herein, a plurality is 2-50 (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 therebetween).

In one embodiment, the recombinant humanized collagen has one or more repeat units comprising an amino acid sequence of any one of SEQ ID NOS: 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 to any one of SEQ ID NOS: 1-3 or an amino acid sequence of any one of SEQ ID NOS: 1-3 with one or more amino acid residues. Preferably, the recombinant humanized collagen has 2-50 repeat 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).

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

Preparation of recombinant collagen

The exemplary recombinant humanized collagens herein can be prepared conventionally according to prior art methods, such as CN201811254050.7, CN201811254050.7, CN201811438582.6 and CN202311272338.8, CN201811254050.7 and CN201811438582.6, CN201911051106.3.

An exemplary method for the preparation of recombinant collagen is as follows. The successfully constructed expression plasmid was transformed into E.coli competent cell BL21 (DE 3) (Merck) by commercial synthesis company based on the amino acid sequence of recombinant collagen. The specific process is as follows:

1. Mu.l of plasmid were taken in 100. Mu.l of E.coli competent cells BL21 (DE 3) and allowed to stand on ice for 30min.

And 2, heating the mixture in a water bath at 42 ℃ for 90 seconds, and then rapidly standing on ice for 2 minutes.

To this mixture, 600. Mu.l of non-resistant LB was added and incubated at 37℃for 1h at 220 rpm.

4 200. Mu.l of the bacterial liquid was uniformly spread on LB plates containing ampicillin resistance (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100. Mu.g/ml ampicillin).

5, Culturing the plate upside down in a 37 ℃ incubator for about 20 hours until clearly visible colonies are grown.

6. After picking up a monoclonal colony from the transformed LB plate and culturing in 10ml LB (100. Mu.g/ml ampicillin) medium for 12h-16h, transferring the colony into 2 XYT medium (16G/L peptone, 10G/L yeast extract, 5G/L sodium chloride) according to the ratio of 1:100 for expansion culture, culturing at 37 ℃ and 220rpm until the bacterial liquid OD600 is 0.4-0.6, adding 0.5mM IPTG (Sigma Co., product number: I5502-1G) for induction expression under the conditions of 18 ℃ and 180rpm for 20h. Finally, the cells were collected by centrifugation and stored at-20 ℃ or immediately subjected to further purification.

7. About 50ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500mM sodium chloride) was used to re-suspend (1L) the bacterial pellet, and after disruption by a high-pressure disruption instrument (Xinzhi organism), the pellet was centrifuged at 13000rpm for 30 minutes to separate the soluble protein from the inclusion body.

8. The Ni-NTA (Qiagen, cat# 30210) affinity column was equilibrated with 5 column volumes of Binding buffer (40mM NaH2PO3,500mM NaCl,pH 7.8). Then adding the protein supernatant and incubating for 0.5-1h at 4 ℃ to enable the target recombinant protein to be fully bound to the column. The hybrid protein was rinsed with 200ml of wash buffer (10 mM imidazole, 40mM NaH2PO3,500mM NaCl,pH 7.8) containing 10mM imidazole (Sigma). Finally adding a proper amount of Prescission Protease (PPase for short) protease with His tag (Sigma, SAE 0045), incubating for 16h at 4 ℃, and collecting the flow-through to obtain the target collagen with carrier protein removed. The resulting product was dialyzed overnight and lyophilized to a dry powder for use.

9. The purity of the obtained protein was checked by SDS-PAGE. The specific process comprises taking 40 μl of purified protein solution, adding 10 μl of 5x protein loading buffer (250 mM Tris-HCl (pH: 6.8), 10% SDS,0.5% bromophenol blue, 50% glycerol, 5% beta-mercaptoethanol), decocting in 100deg.C boiling water for 10min, adding 10 μl of each well into SDS-PAGE protein gel, running at 80V for 2 hr, staining with Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250,25% isopropanol, 10% glacial acetic acid) for 20min, and decolorizing with protein decolorizing solution (10% acetic acid, 5% ethanol). The molecular weight of the purified protein was consistent with the expected molecular weight, indicating that the recombinant collagen was expressed correctly.

For type III collagen T8, commercial synthesis was commissioned based on its amino acid sequence. Its coding nucleotide sequence was inserted into pPiczal a expression vector and pPiczal a expression vector was introduced into yeast competent cells (X33). The transformed yeast cells were induced to express type III collagen T8. And (5) performing electrophoresis detection. It was confirmed that collagen type III T8 was expressed correctly (see the examples of CN 202311272338.8 for more details). That is, collagen (e.g., type III collagen) may be expressed by a yeast (e.g., pichia pastoris, such as pichia pastoris) that includes pPiczal a expression vectors. pPiczal a expression vectors may comprise a nucleic acid molecule encoding collagen.

The preparation method is suitable for preparing and purifying the recombinant collagen in the examples.

Triple helix sequence

The recombinant humanized collagen described herein is suitable for use as a biomaterial, a material for manufacturing, a cosmetic or a food additive. Methods of expressing recombinant triple helix 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 helix proteins are described, for example, in US 20120116053. Methods for transformation, selection of positive transformants and cultivation of pichia are disclosed, for example, in U.S. Pat. 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 of producing triple helical proteins are known in the art and are described, for example, in US20120282817, EP1809751 and WO 2012/117406. Expression systems for producing triple helix proteins are described, for example, in US 20120116053.

Composition and method for producing the same

The crosslinked recombinant humanized collagen of the present invention can be prepared as a composition. The composition may comprise a crosslinked recombinant humanized collagen hydrogel as described herein. The composition may also comprise a pharmaceutically and/or cosmetically acceptable carrier or solvent. The composition may be a pharmaceutical or cosmetic composition for pharmaceutical and/or cosmetic purposes. For example, the composition is one or more of a biological dressing, a human biomimetic material, a cosmetic material for plastic, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynaecological biomaterial, a nerve repair regenerating material, a liver tissue material, a blood vessel repair regenerating material, a 3D printing artificial organ biomaterial, a cosmetic material, a pharmaceutical adjuvant, and a food additive.

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 efficacy is facial skin oil control efficacy.

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.

The composition may be applied in any suitable manner, for example as an orally and/or topically applied composition. The composition may also be prepared as a kit. The kit may comprise additional components, for example auxiliary components, such as buffers, and instructions for use. In particular, the compositions may be formulated as suitable formulations, such as liquid formulations. The formulation may comprise a buffer, such as a D-PBS buffer or a PBS buffer. If purified triple helical proteins are used as biomedical materials, they must be able to be made into the appropriate form. To help achieve these forms, the purified triple helix protein may be stabilized prior to use in medical applications (as is the case with animal collagen) to improve its long term stability and mechanical strength when needed. A wide variety of suitable stabilization strategies are possible. Glutaraldehyde is a suitable agent for crosslinking and is widely used to improve the in vivo stability of collagen materials.

Method for preparing cross-linked recombinant humanized collagen

Provided herein are methods of making cross-linked recombinant humanized collagen comprising reacting a cross-linking agent with cross-linked recombinant humanized collagen described herein.

The method may include cross-linking the recombinant humanized collagen having a triple helix structure. In some embodiments, the recombinant humanized collagen is reacted with a dialdehyde crosslinking agent, wherein the dialdehyde crosslinking agent is present at a concentration of 0.01-0.25%. The concentration of the dialdehyde crosslinking agent is the final concentration of the dialdehyde crosslinking agent in the reaction system, e.g., 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 of values therebetween. The recombinant humanized collagen may be any type of recombinant humanized collagen having a triple helix 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 may be 10-100mg/mL,10-60mg/mL,20-40mg/mL, or 30-40mg/mL, such as10 mg/mL,20 mg/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 the reaction volume or system. The exemplary recombinant humanized collagens herein can be prepared conventionally according to prior art methods, 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.

The method may further comprise dissolving the recombinant humanized collagen in a buffer solution and adjusting the pH to a pH of 5.0-11.0, e.g., 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 with sodium hydroxide solution or hydrochloric acid, for example 0.5mol/L sodium hydroxide solution or 1M hydrochloric acid can be used. The buffer solution may be a phosphate buffer solution or a carbonate buffer solution, for example the buffer salt is selected from sodium dihydrogen phosphate and sodium dihydrogen phosphate. The crosslinked recombinant humanized collagen may still retain the triple helix structure.

The method may further comprise reacting the recombinant humanized collagen and the dialdehyde crosslinking agent at a temperature of-20 ℃ to 50 ℃, 0 to 25 ℃, 0 to 10 ℃, 0 to 8 ℃, or 0 to 4 ℃ for a period of 6 to 24 hours, 12 to 20 hours, or 12 to 16 hours to form a crosslinked recombinant collagen gel. The crosslinking temperature may be, for example, 1 ℃,2 ℃,3 ℃,4 ℃,5 ℃,6 ℃,7 ℃,8 ℃,9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃,16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃ or any range therebetween. The crosslinking time may 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.

In one embodiment, the method includes removing residual dialdehyde crosslinking agent from the crosslinked recombinant collagen gel.

In one embodiment, the residual dialdehyde-based crosslinking agent is removed by dialysis.

In one embodiment, the crosslinked recombinant collagen is freeze-dried to crosslinked recombinant collagen powder and the crosslinked recombinant collagen powder is placed in a dialysis bag. In one embodiment, the crosslinked recombinant collagen gel is placed directly in a dialysis bag. In one embodiment, the residual dialdehyde crosslinking agent is removed by stirring with buffer and/or physiological saline.

In one embodiment, the collagen gel is prepared by stirring dialysis with physiological saline for 5-48 hours, preferably stirring dialysis for 10-24 hours. The choice of dialysis time is conventional to the person skilled in the art and may 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 in between, for example.

In one embodiment, the collagen gel is stirred for dialysis, e.g., for 1-5 hours, preferably 2-4 hours, e.g., 1,2, 3, 4 or 5 hours, with phosphate buffer prior to stirring with physiological saline. In one embodiment, the buffer salt is selected from the group consisting of sodium dihydrogen phosphate and disodium hydrogen phosphate. In one embodiment, the volume ratio of phosphate buffer or physiological saline to crosslinked recombinant collagen gel is 10:1 to 50:1, such as 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, the phosphate buffer may be 0.2M aqueous sodium dihydrogen phosphate (solution A, naH2PO4.H2O 27.6g, dissolved in distilled water, diluted to 1000 mL) and 0.2M aqueous disodium hydrogen phosphate (solution B, na2HPO4.7H2O 53.6g, na2HPO4.12H2O 71.6g, or Na2HPO4.2H2O 35.6g, dissolved in distilled water, diluted to 1000 mL). The pH7.0 phosphate buffer can be prepared by adding 61mL of solution B to 39mL of solution A. phosphate buffer pH7.5 can be prepared by adding 84mL of solution B to 16mL of solution A.

Method of using cross-linked recombinant collagen

Also provided herein are methods of adhering or attaching cells comprising contacting the cells with a crosslinked recombinant humanized collagen or crosslinked recombinant humanized collagen composition described herein. The composition is preferably a crosslinked recombinant humanized collagen hydrogel as described herein. The methods of the invention can be performed in vitro to increase the adhesion of cells to culture vessels. Alternatively, the method of the invention may be performed in vivo.

Also provided herein are methods of promoting cell migration comprising contacting a cell with a crosslinked recombinant humanized collagen or a crosslinked recombinant humanized collagen composition. The method of the invention may be performed in vitro or in vivo.

Also provided herein are cosmetic or cosmesis methods comprising applying to the skin the crosslinked recombinant humanized collagen or crosslinked recombinant humanized collagen composition described herein. The application to the skin may be topical, such as to a skin site in need of anti-wrinkling, oil control, repair, and/or soothing. The method may further comprise applying other cosmetic and/or makeup products to the skin. The kind of other cosmetic and/or makeup products is not particularly limited, and may be other cosmetics having anti-wrinkle, oil control, restoration and/or soothing effects on skin, such as collagen. These cosmetic or cosmesis methods may be anti-wrinkle, oil control, repair and/or soothing methods.

Also provided herein are methods of administering the crosslinked recombinant humanized collagen or crosslinked recombinant humanized collagen compositions described herein to a subject in need thereof. The method may be used to treat or prevent conditions associated with collagen deficiency, or may be used to anti-wrinkle, oil control, repair and/or soothe skin in a subject. The administration may be oral administration.

Use of the same

Also provided herein is the use of the crosslinked recombinant humanized collagen or crosslinked recombinant humanized collagen composition in a pharmaceutical, cosmetic or cosmesis product. These products may be topical products, such as cosmetic or cosmeceutical products for external use. These cosmetic or cosmesis products may be anti-wrinkle, oil control, repair and/or soothing products. For example, the product may be selected from the group consisting of biological dressings, biomimetic materials for the human body, cosmetic materials for plastic, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrical and gynecological biomaterials, nerve repair regeneration materials, liver tissue materials and vascular repair regeneration materials, 3D printing artificial organ biomaterials. The crosslinked recombinant humanized collagen of the present invention can be used in a variety of applications and procedures, including recovery, regeneration and cosmetic procedures, vascular procedures, osteogenic and chondrogenic procedures, cartilage remodeling, bone graft substitutes, hemostasis, wound treatment and management, tissue augmentation and support, incontinence, and the like. Non-limiting examples of biomedical products that can be produced from the crosslinked 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, adhesive surgery (viscosurgery), vascular sealants, cosmetics, spongy substances such as for three-dimensional cell cultures, tissue and organ engineering, hemostatic agents and wound treatments (artificial skin and wound coatings), fibers such as for surgical sutures and hemostatic agents, gelatinous substances such as for tissue implants, cornea protective films, contact lenses and matrices for cell cultures, and membranous substances such as for anti-adhesive films, drug delivery systems, artificial skin and the like.

Also provided herein are methods of making a pharmaceutical, cosmetic or cosmesis product, the method comprising the step of using the crosslinked recombinant humanized collagen, crosslinked recombinant humanized collagen gel or composition described herein. In some embodiments, the product is a topical product. In some embodiments, the cosmetic or cosmesis product is a dermatological anti-wrinkle, oil control, repair, and/or soothing product. In some embodiments, the product is selected from one or more of medical devices, biological dressings, biomimetic materials for humans, cosmetic materials for plastic surgery, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrical and gynecological biomaterials, nerve repair regeneration materials, liver tissue materials and vascular repair regeneration materials, 3D printed artificial organ biomaterials.

Also provided herein are methods of making dermal implants, drug carriers, medical device coatings, implant coatings, shape-forming substances, adhesive surgical materials, vascular sealants, spongy substances, such as those used in three-dimensional cell cultures, tissue and organ engineering substances, hemostatic agents, and wound healing agents, fibers, such as those used in surgical sutures, tissue implants, cornea protective films, or matrices used in cell culture, and membranous substances, such as those used in anti-adhesive films or artificial skin, the methods comprising the step of using the crosslinked recombinant humanized collagen, crosslinked recombinant humanized collagen gels, or compositions described herein.

Examples

The following examples are provided to illustrate the invention. Those skilled in the art will appreciate that the embodiments are merely illustrative and not limiting. The invention is limited only by the scope of the appended claims.

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 "Molecular cloning: alaboratory manual [ molecular cloning: laboratory Manual ]", sambrook et al (1989), cold Spring Harbor lab [ Cold spring harbor laboratory ], cold spring harbor, N.Y., ausubel, F.M. et al (code); "Current protocols in Molecular Biology [ guidelines for molecular biology ]", john Wiley and Sons (1995); harwood, C.R. and Cutting, S.M. (code); "DNACloning: APRACTICAL APPROACH, volumes I and II [ DNA cloning: practical methods, volume I and II ]", D.N.Glover code (1985); "Oligonucleotide Synthesis [ oligonucleotide synthesis ]", M.J.Gait code (1984); "Nucleic Acid Hybridization [ nucleic acid hybridization ]", B.D.Hames & S.J.Hiins code (351985); molecular cloning (1984).

The specific types of recombinant humanized collagen are as follows:

Type I collagen

GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS,SEQ ID NO:8( The preparation method and the purification method are shown in CN201811254050.7;

type III collagen T8 (motif GERGAPGFRGPAGPNGIPGEKGPAGERGAP, SEQ ID NO:1, methods for preparation and purification of protein sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGE KGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP,SEQ ID NO:4)(, see CN201811254050.7, CN201811438582.6 and CN202311272338.8; pichia pastoris expression);

Morphological characteristics of the III type recombinant humanized collagen T16 before and after crosslinking (motif GERGAPGFRGPAGPNGIPGEKGPAGERGAP, a preparation method and a purification method of a protein sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP,SEQ ID NO:5)( are shown in CN201811254050.7 and CN201811438582.6; recombinant expression of escherichia coli);

Type II recombinant humanized collagen (repeat unit, GEPGLQGPAGPPGEKGEPGDDGPSGAEGPP (SEQ ID NO: 2), sequence GEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGEPGLQGPAGPPGEKGEPGDDGPSGAEGPP,SEQ ID NO:6)( preparation method and purification method see CN201910111553.7; E.coli recombinant expression);

XVII type recombinant humanized collagen (basic repeat unit GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLP GAVGEPGAKGAMGPA(SEQ ID NO:3),GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA,SEQ ID NO:7)( preparation method and purification method see CN201911051106.3; E.coli recombinant expression).

The recombinant humanized collagen is prepared internally by Shanxi brocade biological medicine Co-Ltd. Can also be prepared as described above in the section "preparation of recombinant collagen".

Example 1 preparation of crosslinked recombinant humanized collagen hydrogels with different glutaraldehyde Cross-linker concentrations

30Mg 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) is fully dissolved in 1mL of 0.2M sodium phosphate buffer solution, and pH=7.0 and 7.5 are adjusted by using 0.5mol/L sodium hydroxide solution and 1M hydrochloric acid, so as to obtain a recombinant humanized collagen solution. Glutaraldehyde crosslinking agent is added into the recombinant humanized collagen solution according to the final concentration of 0.01%, 0.03%, 0.08%, 0.1%, 0.15% and 0.20%, and the mixture is placed into a constant temperature water bath kettle at a temperature of 4 ℃ for crosslinking reaction for 12 hours, so that the crosslinked recombinant humanized collagen gel is obtained (the morphology before and after crosslinking is shown in figures 3-6 and 8).

In order to reduce the glutaraldehyde crosslinking agent remained in the recombinant humanized collagen gel, the crosslinked recombinant humanized collagen may be freeze-dried, washed with 500mL of phosphate buffer solution with a concentration of 0.2M for 2 hours, and washed with 500mL of physiological saline for 8 hours to form glutaraldehyde crosslinked recombinant humanized collagen gel.

Example 2 preparation of crosslinked recombinant humanized collagen hydrogels under different pH conditions

30Mg of recombinant humanized collagen (type III recombinant humanized collagen T16) is fully dissolved in 1mL of phosphate and carbonate buffer solution, and the pH is adjusted to 5.7, 6, 6.5, 7, 7.5, 8.0, 9.5, 10.0 and 10.5 by adopting 0.5mol/L sodium hydroxide solution and 1M hydrochloric acid, so that a solution of the recombinant humanized collagen is obtained. Glutaraldehyde crosslinking agent is added into the recombinant humanized collagen solution according to 0.03%, and the mixture is placed into a constant-temperature water bath kettle at a temperature of 4 ℃ for crosslinking reaction for 12 hours.

The crosslinked recombinant humanized collagen is freeze-dried, washed for 2 hours by 500mL of phosphate buffer solution with the concentration of 0.2M, and washed for 8 hours by 500mL of normal saline, so as to form glutaraldehyde crosslinked recombinant humanized collagen gel.

EXAMPLE 3 circular dichroism spectrum of recombinant humanized collagen hydrogel

1) Sample preparation

Phosphate Buffer Solution (PBS) 1X was prepared by dissolving 8g NaCl, 0.2g KCl, 3.62g Na ₂HPO₄.12H₂ O and 0.24g KH ₂PO₄ in 800ml distilled water, adjusting the pH of the solution to 7.4 with HCl, adding water to a volume of 1L, autoclaving, and storing at room temperature. If the preservation time is longer than 1 week, the mixture is filtered by a 0.45 filter membrane if the mixture is needed to be used.

Sample dilution the crosslinked hydrogel samples were diluted with PBS to a final concentration of 0.8mg/mL.

Sample detection:

Gradient dilution sample configuration:

1) Ice was made with an ice box ice maker, and samples were subjected to two-fold gradient dilution (note gun head change) at 4 degrees celsius overnight, 300 μl of overnight samples were taken, and checked on-line.

2) Parameter setting of instrument (JASCO circular dichroism spectrometer, JASCO J-815)

Band width (Band width): 1.0nm

Step:1.0nm

Measuring range (Measurement range) 190-260nm

Time per point (Time-per-Point) 1s

Scanning speed (SCANNING SPEED) 50nm/min

Repeating (Repeats) 3 times

The temperature (Measurement temperature) was measured at 4 ℃.

3) Standard CD scan

Setting a blank buffer baseline test with a scanning wavelength of 190-260nm, and collecting circular dichroism absorption of a sample solution PBS in the 190-260nm range.

4) And (5) carrying out scanning map processing. The data was processed using machine self-contained software, exported as Excel, and plotted using graphpad.

Example 4 detection of bioactivity of crosslinked recombinant humanized collagen hydrogels

The method for detecting the adhesion activity of the collagen can be specifically implemented by the following method according to reference 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).:

(1) The concentration of the protein sample to be detected is detected by utilizing an ultraviolet absorption method, and the protein sample to be detected comprises bovine type I collagen (China food and drug verification institute, number: 38002) and the crosslinked collagen provided by the invention (crosslinked XVII type collagen, crosslinked type II collagen and crosslinked type III collagen T16). Specifically, the ultraviolet light absorption of the sample at 215nm and 225nm was measured, and the protein concentration was calculated using the empirical formula C (μg/mL) =144 x (a 215-a 225), taking care of detection under a condition of a215< 1.5. The principle of the method is that the characteristic absorption of peptide bonds under far ultraviolet light is measured, the method is not influenced by chromophore content, has few interfering substances and simple and convenient operation, and is suitable for detecting the human collagen and analogues thereof which are not developed by coomassie brilliant blue. (reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After the protein concentration was detected, all the protein concentrations to be tested were adjusted to 0.5mg/mL with PBS.

(2) To a 96-well plate, 100. Mu.L of each protein solution (bovine type I collagen or crosslinked collagen) was added.

(3) 10 ⁵ Well-cultured 3T3 cells were added to each well and incubated at 37℃for 60min.

(4) Each well was washed 4 times with PBS.

(5) The absorbance at OD492nm was measured using LDH detection kit (Roche, 04744926001) according to the manufacturer's instructions. The cell attachment rate can be calculated from the values of the blank.

The calculation formula of the adherence rate is as follows:

the adhesion rate of the cells can reflect the adhesion activity of each protein. The higher the activity of the protein, the better the environment can be provided for the cells in a short time, and the cell attachment is assisted.

Experimental results

Figure 1 shows circular dichroism spectrum of recombinant humanized collagen T16 cross-linked at different glutaraldehyde concentrations. Unexpectedly, most glutaraldehyde-crosslinked recombinant collagen cannot guarantee its structural characteristics and can damage its protein structure. The results of this example demonstrate that 0.03% glutaraldehyde concentration is capable of maintaining the triple helix structure of recombinant humanized collagen while achieving crosslinking. As can be seen from FIG. 1, the circular dichroism spectrum has a negative peak around 195nm and a positive peak around 221nm, which are typical of collagen, and has a triple helix structure. At 0.03% glutaraldehyde concentration, the recombinant humanized collagen T8, type II recombinant humanized collagen and type XVII recombinant humanized collagen also have similar triple helix structures.

FIG. 2 shows circular dichroism spectrum of recombinant humanized collagen glutaraldehyde crosslinks in buffers of different pH. To determine the optimum pH crosslinking conditions for the buffers (phosphate and carbonate), crosslinking experiments were performed at different pH conditions of the buffer system using the same glutaraldehyde concentration (0.03%) and circular dichroism analysis was used on crosslinked samples. The results in FIG. 2 show that recombinant humanized collagen T16 can maintain a triple helix structure having a negative peak around 195nm, a positive peak around 221nm, and a triple helix structure in the pH range of 5.7 to 8.0. The triple helix structure cannot be maintained in the pH range of 9.5 to 10.5. In the pH range of 5.7-8.0, the recombinant humanized collagen T8, the recombinant humanized collagen type II and the recombinant humanized collagen type XVII also have similar triple helix structures.

FIG. 3 shows the morphological changes of recombinant humanized collagen type III T8 expressed by the yeast system (pH 7.0) before and after crosslinking. The crosslinked recombinant humanized collagen has a typical hydrogel morphology. Compared with the recombinant humanized collagen before crosslinking, the crosslinked recombinant humanized collagen can have a certain morphological support.

FIG. 4 shows morphological characteristics of E.coli system expressing purified recombinant humanized collagen type III T16 before and after cross-linking. FIG. 4 shows the morphology change at pH7.0 and 7.5, and the cross-linked recombinant collagen formed at pH7.0 and 7.5 maintained a very well mechanically supported morphology.

Figure 5 shows the morphological changes of the recombinant humanized collagen type II before and after cross-linking. The recombinant humanized type II collagen is subjected to liquid-to-solid conversion under the crosslinking of glutaraldehyde buffer solution with pH of 7.0, and the morphological characteristics of mechanical support are maintained.

Figure 6 shows the morphological changes of the recombinant humanized collagen of type XVII before and after cross-linking. The recombinant humanized collagen of type XVII can be converted from liquid to solid by cross-linking with glutaraldehyde buffer at pH 7.0.

FIG. 7 shows that the crosslinked recombinant humanized collagen hydrogels of the present invention retain their bioadhesive activity as compared to bovine type I collagen (PC group, 0.5 mg/ml). The crosslinked recombinant humanized collagen is capable of maintaining cell adhesion activity unexpectedly.

FIG. 8 shows the morphology of the type I recombinant humanized collagen after the crosslinking treatment. Type I recombinant humanized collagen does not possess a triple helix structure. After treatment by the method of example 1, the type I recombinant humanized collagen was still in a fluid state and failed to form a gel.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A method for crosslinking recombinant humanized collagen comprises the steps of 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%,

Preferably, the recombinant humanized collagen has a triple helix structure,

Preferably, the recombinant humanized collagen has a negative peak at 175-220nm and a positive peak at 200-240nm on a circular dichroism spectrum,

Preferably, the recombinant humanized collagen has a negative peak at about 195nm and a positive peak at about 221nm on a 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 crosslinking 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 to 5.5-8.7 or 5.7-8;

preferably, the buffer solution is a phosphate buffer solution or a carbonate buffer solution, preferably a phosphate buffer solution or a carbonate buffer solution of 0.1M-0.4M or 0.1M-0.3M;

Preferably, the buffer salt is selected from the group consisting of sodium dihydrogen phosphate and disodium hydrogen phosphate;

preferably, the concentration of recombinant humanized collagen is 10-100mg/mL,10-70mg/mL,20-40mg/mL or 30-40mg/mL;

preferably, the crosslinked recombinant humanized collagen retains a triple helix structure;

Preferably, the recombinant humanized collagen comprises the sequence of (Gly-X-Y) n, n being 2-300;

Preferably, the recombinant humanized collagen has one or more repeat units 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 to any one of SEQ ID NOs 1-3 or an amino acid sequence of any one of SEQ ID NOs 1-3 in which one or more amino acid residues are substituted, added, deleted or inserted, preferably the recombinant humanized collagen has 2-50 repeat units;

Preferably, the recombinant humanized collagen has an amino acid sequence of any one of SEQ ID NOs 4 to 7 or 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 to 7 or an amino acid sequence of any one of SEQ ID NOs 4 to 7 in which one or more amino acid residues are substituted, added, deleted or inserted;

Preferably, the recombinant humanized collagen is expressed by E.coli or by yeast, such as Pichia pastoris, e.g., pichia pastoris;

Preferably, the method comprises reacting the recombinant humanized collagen with a dialdehyde crosslinking agent at a temperature of-20 ℃ to 50 ℃,0 to 25 ℃,0 to 10 ℃,0 to 8 ℃ or 0 to 4 ℃ for a period of 6 to 24 hours, 12 to 20 hours or 12 to 16 hours to form a crosslinked recombinant collagen gel;

Preferably, the crosslinked recombinant collagen gel is lyophilized to a powder.

2. A method of preparing a crosslinked recombinant humanized collagen gel comprising the method of claim 1 and comprising removing residual dialdehyde crosslinking agent from the crosslinked recombinant collagen gel, preferably by dialysis;

Placing the crosslinked recombinant collagen powder in a dialysis bag, or directly placing the crosslinked recombinant collagen gel in a dialysis bag, stirring with buffer and/or physiological saline to remove residual dialdehyde crosslinking agent, preferably stirring with physiological saline for 5-48 hours, preferably 10-24 hours to prepare collagen gel, preferably stirring with phosphate buffer for dialysis, for example stirring for 1-5 hours, preferably 2-4 hours, before stirring with physiological saline to obtain crosslinked recombinant collagen gel with reduced residual dialdehyde crosslinking agent content;

preferably, the crosslinked recombinant humanized collagen gel is a hydrogel;

Preferably, the volume ratio of phosphate buffer or physiological saline to the crosslinked recombinant collagen gel is 10:1-50:1.

3. A crosslinked recombinant humanized collagen prepared by the method according to claim 1.

4. A crosslinked recombinant humanized collagen gel comprising the crosslinked recombinant humanized collagen of claim 3 or prepared by the method according to claim 2, preferably the crosslinked recombinant humanized collagen gel is a hydrogel.

5. A composition comprising the crosslinked recombinant humanized collagen of claim 3 or the crosslinked 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 having anti-wrinkle efficacy, skin oil control efficacy, skin repair efficacy and/or skin soothing efficacy, preferably, the skin oil control efficacy is facial skin oil control efficacy;

preferably, the composition is a kit, preferably comprising physiological saline;

preferably, the composition is one or more of medical devices, biological dressings, human biomimetic materials, cosmetic materials for plastic surgery, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecologic biological materials, nerve repair regeneration materials, liver tissue materials and vascular repair regeneration materials, 3D printing artificial organ biological materials, cosmetic raw materials, pharmaceutical excipients and food additives;

preferably, the composition is a solid, liquid or gel composition;

preferably, the composition is an orally and/or topically applied composition;

Preferably, the topical application is to the skin, preferably the composition is a transdermal composition;

preferably, the collagen hydrogel further comprises physiological saline.

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

7. A method of adhering or attaching cells or promoting cell migration, the method comprising contacting cells with the crosslinked recombinant humanized collagen according to claim 3, the crosslinked recombinant humanized collagen gel according to claim 4, or the composition of claim 5.

8. Cosmetic or cosmesis method comprising applying to the skin the crosslinked recombinant humanized collagen according to claim 3, the crosslinked recombinant humanized collagen gel according to claim 4 or the composition according to claim 5, and optionally applying to the skin other cosmesis and/or make-up products;

preferably, the cosmetic or cosmesis method of anti-wrinkle, oil control, repair and/or soothing of the skin;

Preferably, the administration is topical;

Preferably, the administration is transdermal.

9. Use of the crosslinked recombinant humanized collagen according to claim 3, the crosslinked recombinant humanized collagen gel according to claim 4 or the composition according to claim 5 for the preparation of a pharmaceutical, cosmetic or cosmetical product,

Preferably, wherein the product is a topical product,

Preferably, wherein the cosmetic or cosmesis product is a dermatological anti-wrinkle, oil control, repair and/or soothing product;

Preferably, the product is selected from one or more of medical devices, biological dressings, human biomimetic materials, cosmetic materials for plastic, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrical and gynecological biomaterials, nerve repair regeneration materials, liver tissue materials and vascular repair regeneration materials, 3D printing artificial organ biomaterials.

10. Use of the crosslinked recombinant humanized collagen according to claim 3, the crosslinked recombinant humanized collagen gel according to claim 4 or the composition according to claim 5 for the preparation of dermal implants, drug carriers, medical device coatings, implant coatings, shape forming substances, adhesive surgical materials, vascular sealants, spongy substances such as for three-dimensional cell cultures, tissue and organ engineering substances, hemostatic agents and wound healing agents, fibers such as for surgical sutures, tissue implants, cornea protective films or matrices for cell cultures, and membranous substances such as for anti-adhesive films or artificial skin.