Cross-linked recombinant humanized collagen gel, method of making and uses thereof

Abstract

This application provides a cross-linked recombinant humanized collagen gel, wherein the cross-linked recombinant humanized collagen gel comprises recombinant humanized collagen and a cross-linking agent. The cross-linked recombinant humanized collagen gel has rheological properties, with a Tanδ of 0.20-0.50 and a G' of 90-600 Pa at a frequency of 0.1 Hz. The cross-linked recombinant humanized collagen gel overcomes the problem of easy degradation of recombinant humanized collagen in vivo, and can maintain itself in vivo for a longer period of time, thus exerting ideal medical aesthetic effects.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to a cross-linked recombinant humanized collagen gel, its preparation method, and its application. Background Technology

[0002] As human skin ages (whether gradually over time or accelerated by prolonged sun exposure), matrix metalloproteinases increase, leading to damage and breakage of the extracellular matrix (ECM) in the dermis. This is attributed to reduced collagen production and the fragmentation of existing collagen fibrils. This, in turn, produces even more matrix metalloproteinases, resulting in less collagen and related proteins, further reducing dermal quality. Clinically, this damage typically manifests as fine lines, wrinkles, atrophy, irregular contours, and fragility.

[0003] Intradermal collagen injection can replenish collagen in situ, improving skin texture, reducing fine lines, improving skin laxity, and enhancing skin elasticity. It also stimulates the synthesis of new collagen and tissue repair and remodeling; the amino acids metabolized from the material can also serve as nutrients to continuously stimulate fibroblasts. Currently, most collagen products on the market are animal-derived. However, safety issues should be considered when using animal-derived collagen, including the risk of transmitting viruses and diseases, pathogenicity, and allergic reactions. With advancements in genetic engineering and synthetic biology, recombinant collagen has been prepared, retaining the basic characteristics of natural collagen and possessing advantages such as being pathogen-free and having low immunogenicity, showing potential as a starting material for surgical implants and regenerative medicine substrates. However, recombinant collagen often lacks certain rheological properties and is easily degraded in vivo. As a soft tissue filler, it is difficult to achieve ideal cosmetic effects. Cross-linking recombinant collagen may improve its rheological properties and in vivo maintenance time.

[0004] Chinese patent application CN115154665A discloses a lubricant, filler, and their application containing recombinant human collagen. By controlling the degree of cross-linking reaction between HA and BDDE, a low-cross-linked sodium hyaluronate gel is obtained. This gel is then homogenized and sterilized to obtain a sterile, micro-cross-linked sodium hyaluronate gel. This micro-cross-linked gel is then mixed with a lubricant containing recombinant human collagen tissue to obtain a micro-cross-linked sodium hyaluronate gel containing recombinant human collagen. This results in a final product with suitable rheological properties, allowing the micro-cross-linked gel to be injected into the dermis to repair shape and correct contours for satisfactory results. However, this patent application first cross-links BDDE with HA before mixing it with a recombinant human collagen solution. Its rheological properties depend on the cross-linking of BDDE and HA, and it does not actually prepare a cross-linked recombinant human collagen gel. Furthermore, the cross-linking reaction of BDDE and HA disclosed in this patent application is carried out in a 1.0%–1.2% sodium hydroxide solution, meaning the pH of the cross-linking reaction system is above 12. High pH conditions easily lead to collagen denaturation.

[0005] Therefore, the cross-linking method described in this patent application cannot be directly referenced in the preparation of cross-linked recombinant human collagen. Currently, there are no reports on whether cross-linked recombinant human collagen gels with suitable rheological properties can be prepared by cross-linking recombinant human collagen with a suitable cross-linking agent under appropriate reaction conditions. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this application provides a cross-linked recombinant humanized collagen gel, its preparation method, and its application. The cross-linked recombinant humanized collagen gel is prepared by a cross-linking reaction between recombinant humanized collagen and an epoxy compound cross-linking agent. The resulting cross-linked recombinant humanized collagen gel has suitable rheological properties, good diffusion after subcutaneous injection, and neither clumps nor is it rapidly lost, thus effectively improving skin texture, reducing fine lines, improving skin laxity, and enhancing skin elasticity.

[0007] The specific technical solution of this application is as follows:

[0008] 1. A cross-linked recombinant humanized collagen gel, wherein the cross-linked recombinant humanized collagen gel comprises recombinant humanized collagen and a cross-linking agent, and the cross-linked recombinant humanized collagen gel has rheological properties, wherein Tanδ is 0.20-0.50 and G' is 90-600 Pa at a frequency of 0.1 Hz.

[0009] 2. The cross-linked recombinant humanized collagen gel according to item 1, wherein the cross-linked recombinant humanized collagen gel has cohesive properties and its cohesive force is 15-25 μL / drop.

[0010] 3. The cross-linked recombinant humanized collagen gel according to item 1 or 2, wherein the degree of cross-linking of the cross-linked recombinant humanized collagen gel is 15-100%.

[0011] 4. The cross-linked recombinant humanized collagen gel according to any one of items 1-3, wherein the content of recombinant humanized collagen in the cross-linked recombinant humanized collagen gel is 10-120 mg / mL, preferably 20-80 mg / mL.

[0012] 5. The cross-linked recombinant humanized collagen gel according to claim 1, wherein the recombinant humanized collagen includes recombinant humanized type XVII collagen.

[0013] 6. The cross-linked recombinant humanized collagen gel according to any one of items 1-5, wherein the cross-linking agent is an epoxy compound cross-linking agent, preferably butylene glycol diglycidyl ether.

[0014] 7. A method for preparing cross-linked recombinant humanized collagen gel, comprising the following steps:

[0015] Recombinant humanized collagen is dissolved to obtain a solution, and then cross-linked with a cross-linking agent to obtain a cross-linked recombinant humanized collagen gel. The cross-linked recombinant humanized collagen gel has rheological properties, with Tanδ of 0.20-0.50 and G' of 90-600 Pa at a frequency of 0.1 Hz.

[0016] Preferably, the pH value of the solution is 5-10;

[0017] Preferably, the crosslinking reaction takes 16-72 hours.

[0018] 8. The method according to item 7, wherein the cross-linked recombinant humanized collagen gel is the cross-linked recombinant humanized collagen gel involved in items 2-6.

[0019] 9. A filler comprising any one of claims 1-6 crosslinked recombinant humanized collagen gel or the crosslinked recombinant humanized collagen gel prepared by the method described in claim 7 or 8;

[0020] Preferably, the filler further comprises an anesthetic and / or excipients.

[0021] 10. A skin-lightening product comprising any one of claims 1-6, or the cross-linked recombinant humanized collagen gel prepared by the method described in claim 7 or 8;

[0022] Preferably, the water-light product further comprises an anesthetic and / or excipients.

[0023] The effects of the invention

[0024] The cross-linked recombinant humanized collagen gel described in this application overcomes the problem of easy degradation of recombinant humanized collagen in vivo, and can be maintained in vivo for a longer period of time to achieve ideal medical aesthetic effects.

[0025] The cross-linked recombinant humanized collagen gel described in this application has suitable rheological properties and good diffusion after subcutaneous injection. It does not clump or flow away rapidly, thus effectively improving skin texture, reducing fine lines, improving skin laxity, and enhancing skin elasticity.

[0026] The cross-linked recombinant humanized collagen gel described in this application has a low residual amount of cross-linking agent, which meets the safety requirements. Attached Figure Description

[0027] Figures 1A to 1B This is a schematic diagram showing the injection of the gels described in Example 1 and Comparative Example 4 into SD rats, wherein... Figure 1A This is a schematic diagram showing the gel described in Example 1 injected into SD rats. Figure 1B This is a schematic diagram showing the gel described in Comparative Example 4 injected into SD rats. Detailed Implementation

[0028] The present application will now be described in detail with reference to the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0029] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0030] This application provides a cross-linked recombinant humanized collagen gel, which includes recombinant humanized collagen and a cross-linking agent. The cross-linked recombinant humanized collagen gel has rheological properties, with a Tanδ of 0.20-0.50 at a frequency of 0.1 Hz.

[0031] The gel can have a Tanδ of 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, etc. at a frequency of 0.1 Hz.

[0032] This application does not impose any restrictions on the method for determining Tanδ; it can be determined using conventional methods in the art, such as using a DHR-HR rheometer.

[0033] In some embodiments, the cross-linked recombinant humanized collagen gel has rheological properties, with G' of 90-600 Pa at a frequency of 0.1 Hz.

[0034] For example, at a frequency of 0.1Hz, G' can be 90Pa, 100Pa, 120Pa, 150Pa, 180Pa, 200Pa, 220Pa, 250Pa, 280Pa, 300Pa, 320Pa, 350Pa, 380Pa, 400Pa, 420Pa, 450Pa, 480Pa, 500Pa, 520Pa, 550Pa, 580Pa, 600Pa, etc.

[0035] In this application, no restrictions are placed on the method for measuring G', which can be measured using conventional methods in the art, such as using a DHR-HR rheometer.

[0036] In some embodiments, the cross-linked recombinant humanized collagen gel exhibits cohesiveness with a cohesive force of 15-25 μL / drop. For example, the cohesive force can be 15 μL / drop, 16 μL / drop, 17 μL / drop, 18 μL / drop, 19 μL / drop, 20 μL / drop, 21 μL / drop, 22 μL / drop, 23 μL / drop, 24 μL / drop, 25 μL / drop, etc.

[0037] In this application, no restrictions are placed on the method for determining cohesive force. Those skilled in the art can use conventional methods in the field to perform the determination, such as the method described in Physical properties of a novel small-particle hyaluronic acid filler: In vitro, in vivo, and clinical studies (DOI: 10.1111 / jocd.12560).

[0038] In some embodiments, the degree of crosslinking of the crosslinked recombinant humanized collagen gel is 15%-100%.

[0039] For example, the degree of crosslinking of the crosslinked recombinant humanized collagen gel can be 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc.

[0040] In this application, the degree of crosslinking refers to the ratio of the content of recombinant humanized collagen participating in the crosslinking reaction to the total content of recombinant humanized collagen participating in the crosslinking reaction and recombinant humanized collagen not participating in the crosslinking reaction. This application does not impose any restrictions on the method for determining the degree of crosslinking. Those skilled in the art can determine it based on conventional methods in the field, such as using the TNBS method to determine the degree of crosslinking.

[0041] In some embodiments, the recombinant humanized collagen content in the cross-linked recombinant humanized collagen gel is 10-120 mg / mL, preferably 20-80 mg / mL.

[0042] For example, the recombinant humanized collagen content is 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL, 60mg / mL, 70mg / mL, 80mg / mL, 90mg / mL, 100mg / mL, 110mg / mL, 120mg / mL, etc.

[0043] In this application, no restrictions are placed on the method for determining the content of recombinant humanized collagen. Those skilled in the art can use conventional methods in the field to determine it, such as the biuret method (Chinese Pharmacopoeia, 0731).

[0044] In some embodiments, the recombinant humanized collagen includes recombinant humanized type XVII collagen.

[0045] In some embodiments, the recombinant humanized type XVII collagen described in this application contains or is composed of the amino acid sequence shown in SEQ ID NO:1.

[0046]

[0047]

[0048] The amino acid sequence shown in SEQ ID NO:1 can be prepared by conventional methods in the art, or by the method disclosed in patent application CN202411675097.6, for example, by the method used to prepare SEQ ID NO:4 in that patent application.

[0049] Those skilled in the art will also understand that the amino acid sequence of the recombinant humanized type XVII collagen is not limited to the specific sequences listed above. The amino acid sequence of the recombinant humanized type XVII collagen should encompass amino acid sequences that, compared to the amino acid sequence shown in SEQ ID NO:1, contain one, two, or three or more amino acid mutations but are substantially functionally identical to it, and also include sequences that have 95%, 96%, 97%, 98%, or 99% sequence identity compared to the amino acid sequence shown in SEQ ID NO:1.

[0050] In some embodiments, the crosslinking agent is an epoxy compound crosslinking agent, preferably butanediol diglycidyl ether, and most preferably 1,4-butanediol diglycidyl ether (BDDE).

[0051] In some embodiments, the cross-linked recombinant humanized collagen gel is prepared by a method comprising the following steps:

[0052] Recombinant humanized collagen is dissolved to obtain a solution, which is then subjected to a cross-linking reaction with a cross-linking agent to obtain a cross-linked recombinant humanized collagen gel. In some embodiments, the recombinant humanized collagen is dissolved in an aqueous solution with a pH of 5-10 to obtain the solution.

[0053] For example, the pH value can be 5, 6, 7, 8, 9, 10, etc.

[0054] In some embodiments, the amount of the crosslinking agent added is 0.01-1 wt%.

[0055] For example, the amount of crosslinking agent added can be 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, etc.

[0056] In some embodiments, the solution obtained by dissolving recombinant humanized collagen refers to a solution with a concentration of 10-120 mg / mL, preferably a solution with a concentration of 20-80 mg / mL.

[0057] For example, the concentration of the recombinant humanized collagen is 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, etc.

[0058] In some embodiments, the crosslinking reaction is carried out at a temperature of 20-60°C and / or for a time of 16-72 hours.

[0059] For example, the temperature for the crosslinking reaction can be 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc.

[0060] The cross-linking reaction times are 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, and 72h.

[0061] The cross-linked recombinant humanized collagen gel obtained in this application possesses suitable rheological properties, which can be maintained for a relatively long time, effectively improving skin texture, reducing fine lines, improving skin laxity, and enhancing skin elasticity. For example, it can be used for skin improvement and rejuvenation treatments on the face, neck, chest, back of hands, and upper arms (including improving dry skin, rough skin, skin laxity, and decreased elasticity), as well as for filling fine lines on the cheeks, forehead, between the eyebrows, around the eyes, and around the mouth.

[0062] This application provides a method for preparing cross-linked recombinant humanized collagen gel, which includes the following steps:

[0063] Recombinant humanized collagen is dissolved to obtain a solution, and then cross-linked with a cross-linking agent to obtain a cross-linked recombinant humanized collagen gel; wherein the cross-linked recombinant humanized collagen gel has rheological properties, with Tanδ of 0.20-0.50 and G' of 90-600 Pa at a frequency of 0.1 Hz.

[0064] Preferably, the pH value of the solution is 5 to 10.

[0065] For example, the Tanδ of the gel can be 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, etc. at a frequency of 0.1Hz;

[0066] pH values ​​can be 5, 6, 7, 8, 9, 10, etc.

[0067] In some embodiments, dissolving recombinant humanized collagen to obtain a solution refers to dissolving recombinant humanized collagen to obtain a solution with a recombinant humanized collagen concentration of 10-120 mg / mL, preferably 20-80 mg / mL.

[0068] For example, the concentration of the recombinant humanized collagen is 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, etc.

[0069] In some embodiments, the amount of the crosslinking agent added is 0.01-1 wt%.

[0070] For example, the amount of crosslinking agent added can be 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, etc.

[0071] In some embodiments, the cross-linked recombinant humanized collagen gel is the cross-linked recombinant humanized collagen gel described above.

[0072] This application provides a filler comprising the cross-linked recombinant humanized collagen gel described above or the cross-linked recombinant humanized collagen gel prepared by the method described above.

[0073] In this application, no restrictions are placed on the content of cross-linked recombinant humanized collagen gel in the filler; those skilled in the art can select the content based on actual needs. For example, the content of cross-linked recombinant humanized collagen gel can be 10-120 mg / mL, preferably 20-80 mg / mL.

[0074] For example, the recombinant humanized collagen content is 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL, 60mg / mL, 70mg / mL, 80mg / mL, 90mg / mL, 100mg / mL, 110mg / mL, 120mg / mL, etc.

[0075] The filler described in this application can be used for filling fine lines.

[0076] In some embodiments, the filler also comprises an anesthetic and / or excipients.

[0077] In this application, no restrictions are placed on the content of anesthetics and / or excipients. Those skilled in the art can make conventional selections based on actual needs. For example, the anesthetic can be lidocaine, lidocaine hydrochloride, prilocaine, etc., and the excipient can be phosphate buffer solution, borate buffer solution, sodium chloride solution, etc.

[0078] This application provides a water-light product comprising the cross-linked recombinant humanized collagen gel described above or the cross-linked recombinant humanized collagen gel prepared by the method described above.

[0079] The aforementioned water-light products refer to products that utilize mesotherapy, using specialized instruments to precisely inject nutrients into the dermis layer of the skin to solve skin problems, such as water-light injections and microneedling products.

[0080] In this application, no restrictions are placed on the content of cross-linked recombinant humanized collagen gel in the hydrating product; those skilled in the art can select the content based on actual needs. For example, the content of cross-linked recombinant humanized collagen gel can be 10-120 mg / mL, preferably 20-80 mg / mL.

[0081] For example, the recombinant humanized collagen content is 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL, 60mg / mL, 70mg / mL, 80mg / mL, 90mg / mL, 100mg / mL, 110mg / mL, 120mg / mL, etc.

[0082] In some embodiments, the water-light product also contains anesthetics and / or excipients.

[0083] In this application, no restrictions are placed on the content of anesthetics and / or excipients. Those skilled in the art can make conventional selections based on actual needs. For example, the anesthetic can be lidocaine, lidocaine hydrochloride, prilocaine, etc., and the excipient can be phosphate buffer solution, borate buffer solution, sodium chloride solution, etc.

[0084] The gel described in this application has suitable rheological properties, and when used as an injection, it has good diffusivity, neither clumping nor rapid loss.

[0085] Example

[0086] This application provides a general and / or specific description of the materials and methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. The preparation of recombinant humanized type XVII collagen is as follows:

[0087] 1. Construction of genetically engineered bacteria:

[0088] Table 1

[0089]

[0090] The nucleic acid molecules of recombinant humanized XVII collagen designed in Table 1 (which can be obtained by those skilled in the art based on the amino acid sequence) were ligated into the BamH1 and Xho1 sites of the vector pET32a to obtain the recombinant plasmid pET32a-XVII. The recombinant plasmid was then transformed into E. coli BL21(DE3) host to obtain the genetically engineered strain T-XVII. See Table 2 for details.

[0091] Table 2. Strains Design and Target Protein Size

[0092] strain plasmid Theoretical size of the target protein (kDa) T-XVII pET32a-XVII 48

[0093] 2. Expression of recombinant humanized type XVII collagen:

[0094] Single colonies of the engineered strain were picked from the plate and inoculated into 5 mL of LB medium (containing 100 μg / ml Amp) and cultured overnight at 37°C and 220 rpm. The above culture was then transferred 1:100 to 20 mL of LB medium (containing 100 μg / ml Amp) and cultured at 37°C and 220 rpm. When the OD reached 0.6-0.8, 0.2 mM isopropyl thiogalactoside (IPTG) was added, and the mixture was cultured at 16°C and 220 rpm for 20 h. 10 OD cells were collected, resuspended in 1 mL of buffer (100 mM Tris-HCl, pH 7.0), and the cells were sonicated. 20 μL of the supernatant was collected, and 20 μL of 2× loading buffer was added. The mixture was boiled for 10 min, centrifuged, and the supernatant was collected for SDS-PAGE. Simultaneously, bovine serum albumin (BSA) was used for quantification. The results are shown in Table 3.

[0095] Table 3 Protein expression of strains

[0096] strain Expression level T-XVII 0.19g / L

[0097] As shown in Table 3, the designed recombinant humanized XVII collagen can be expressed in host Escherichia coli at an expression level of 0.19 g / L.

[0098] 3. Purification of recombinant humanized type XVII collagen:

[0099] Single colonies of the T-XVII engineered strain were picked from the plate and inoculated into 5 mL of LB medium (containing 100 μg / ml Amp), and cultured overnight at 37°C and 220 rpm. The above culture was then transferred 1:100 to 100 mL of LB medium (containing 100 μg / ml Amp), and cultured at 37°C and 220 rpm. When the OD reached 0.6-0.8, 0.2 mM isopropyl thiogalactoside (IPTG) was added, and the mixture was cultured at 16°C and 220 rpm for 20 h. 10 OD of bacterial cells were collected, suspended in 1 ml buffer (100 mM Tris-HCl, pH 7.0), and the cells were sonicated to disrupt the cell structure. The cells were centrifuged at 12000 rpm for 60 min to remove the precipitate. The supernatant was used for enzyme protein purification. The supernatant was loaded onto a Ni2+ agarose affinity chromatography column and washed for 3–4 column volumes with binding buffer (50 mmol / L Tris-HCl, 100 mmol / L NaCl, and 25 mmol / L imidazole, pH 7.0). Finally, the cells were eluted with elution buffer (50 mmol / L Tris-HCl, 400 mmol / L NaCl, and 250 mmol / L imidazole, pH 7.0) to obtain the purified enzyme protein. The purified enzyme protein was digested with TEV protease overnight at 4°C, and then flow-through through a nickel column to obtain the unlabeled purified protein.

[0100] Example 1

[0101] Step (1): Dissolve 2.5g of the recombinant humanized type XVII collagen prepared above in 50mL of phosphate buffer, adjust the pH to 7.4, filter sterile, and obtain the recombinant humanized collagen solution.

[0102] Step (2): Add 0.25g of sterile filtered BDDE to the solution of recombinant humanized collagen, place it in a 25℃ constant temperature water bath, mix evenly, and cross-link for 48 hours to obtain gel.

[0103] Step (3): Cut the gel obtained in step (2) into 1cm pieces. 3 The two small pieces were washed with 1L of phosphate buffer (pH 7.4) for 2 hours with stirring. The eluent was then removed, and the washing process was repeated 3 times to obtain the purified gel.

[0104] Step (4): Homogenize and fill the gel obtained in step (3) to obtain a cross-linked recombinant humanized collagen product.

[0105] Example 2-12

[0106] The difference between Examples 2-12 and Example 1 lies in the content of recombinant humanized collagen, the amount of crosslinking agent, the pH value of the crosslinking reaction, the temperature or time of the crosslinking reaction, and the resulting crosslinked recombinant humanized collagen gels, as detailed in Table 4.

[0107] Comparative Examples 1-7

[0108] The difference between Comparative Examples 1-7 and Example 1 lies in the content of recombinant humanized collagen, the type of recombinant humanized collagen, the amount of crosslinking agent, the pH value, or the time obtained, which are different from those in Example 1. The specific details are shown in Table 4.

[0109] Table 4

[0110]

[0111] Note: The yak collagen in Comparative Example 6 was prepared according to... https: / / doi.org / 10.1088 / 1748-605X / In the article ad6548 The publicly disclosed method for extracting yak collagen was used to prepare it.

[0112] Experimental Example 1: Rheological Testing and Determination of Crosslinking Degree

[0113] Rheological test:

[0114] The test was conducted using a DHR-HR rheometer with the following parameters: gap size 1.0 mm, 25℃, strain 1%, and frequency range 0.01-1 Hz. Rheometer data: The storage modulus G' and loss modulus G” at 0.1 Hz were taken, and the loss factor Tanδ = G” / G' was used to obtain Tanδ and G'. The results are shown in Table 5.

[0115] Crosslinking degree determination:

[0116] The degree of cross-linking of collagen by BDDE was assessed using the TNBS method.

[0117] Collagen samples before and after cross-linking were freeze-dried. 11 mg of each sample was dissolved in a solution containing 1 mL of 4% Na₂CO₃ and 1 mL of 0.5% TNBS. The reaction was carried out at 40°C for 4 hours with a stirring speed of 220 rpm. Then, 3 mL of 6M HCl was added, and the mixture was incubated at 60°C for 1.5 hours. After incubation, any remaining unreacted TNBS was removed using anhydrous diethyl ether. The degree of cross-linking of the collagen was then quantified by measuring the UV absorbance at 345 nm using a UV spectrophotometer.

[0118]

[0119] α: degree of cross-linking; W0: background absorbance; W1: absorbance of the collagen freeze-dried sample before cross-linking; W2: absorbance of the collagen freeze-dried sample after cross-linking. The results are shown in Table 5.

[0120] Table 5

[0121]

[0122]

[0123] NA indicates that it is not applicable.

[0124] Experiment Example 2: Cohesion Test

[0125] Reference: Physical properties of a novel small-particle hyaluronic acidfiller: In vitro, in vivo, and clinical studies (DOI: 10.1111 / jocd.12560). Cohesion was determined using the falling weight method, specifically as follows: The average weight of each drop of gel fragment was measured by passing it through a specified vertical orifice at a constant speed. The gel was loaded into a 1 mL BD glass syringe and air was removed by centrifugation. An 18G cannula with a flat orifice was attached to the syringe, and the gel was extruded using a tensile testing machine at a constant speed of 7.5 mm / min. When constant force was reached, at least 10 drops of gel fragments were collected and weighed, and the average weight of each drop was calculated. A result of 15-25 μL / drop was considered to indicate suitable cohesion. Specific test results are shown in Table 6.

[0126] Table 6

[0127]

[0128]

[0129] Comparative Examples 5 and 7 are in solution state, so cohesive force cannot be measured.

[0130] Fillers with suitable cohesiveness and G' are ideal for improving skin texture and filling fine lines in terms of physical properties. If the cohesiveness is too high, the gel has difficulty migrating to other tissues; if the G' is too low, the gel diffuses too quickly after injection, spreading to a wider area before it can exert its effects on improving skin texture and filling fine lines, thus failing to achieve the desired cosmetic results. In the embodiments of this application, the cohesiveness is in the range of 15-25 μL / drop, and the G' is in the range of 90-600 Pa, which is conducive to both gel migration after injection and the exertion of the gel's cosmetic effects. The Tanδ in the embodiments of this application is in the range of 0.2-0.5, indicating that the fluidity is greater than the elasticity, which helps with diffusion after injection and prevents the formation of lumps under the skin.

[0131] Experimental Example 3: Crosslinking Agent Residue

[0132] Referring to the "Gas Chromatography Method" in the determination of residual 1,4-butanediol diglycidyl ether (BDDE) crosslinking agent in YY / T 1571-2017 "Tissue Engineering Medical Device Products Sodium Hyaluronate", the residual crosslinking agent in the gel samples of Examples 1-12 was determined. The results showed that the residual crosslinking agent was less than or equal to 1 ppm, which meets the safety requirements for this type of product.

[0133] Experiment Example 4: Animal Experiment

[0134] Twenty-four SD rats were used, divided into groups of two. The backs of each rat were shaved, and two injection points were made on each side of the back, with the spine as the midline. Each group had four injection points. Samples from Examples 1, 5, 6, 9-12, and Comparative Examples 1-5 were injected intradermally at each point. The injection volume at each point was 0.2 ml. Skin samples were harvested one day after injection to observe gel diffusivity. The results are shown in Table 7.

[0135] Table 7

[0136]

[0137]

[0138] The gel diffusion effects after injection in Example 1 and Comparative Example 4 are as follows: Figure 1A and Figure 1B As shown, it can be seen that the gel in Example 1 has a uniform diffusion state after injection and has not formed lumps, while the gel in Comparative Example 4 has poor diffusion and obvious lumps after injection.

[0139] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A crosslinked recombinant humanized collagen gel, wherein, The cross-linked recombinant humanized collagen gel comprises recombinant humanized collagen and a cross-linking agent. The cross-linked recombinant humanized collagen gel has rheological properties, with Tanδ of 0.20-0.50 and G' of 90-600 Pa at a frequency of 0.1 Hz.

2. The cross-linked recombinant humanized collagen gel according to claim 1, wherein, The cross-linked recombinant humanized collagen gel has cohesive properties, with a cohesive force of 15-25 μL / drop.

3. The cross-linked recombinant humanized collagen gel according to claim 1 or 2, wherein, The degree of crosslinking of the crosslinked recombinant humanized collagen gel is 15-100%.

4. The cross-linked recombinant humanized collagen gel according to any one of claims 1-3, wherein the content of recombinant humanized collagen in the cross-linked recombinant humanized collagen gel is 10-120 mg / mL, preferably 20-80 mg / mL.

5. The cross-linked recombinant humanized collagen gel according to claim 1, wherein the recombinant humanized collagen includes recombinant humanized type XVII collagen.

6. The cross-linked recombinant humanized collagen gel according to any one of claims 1-5, wherein the cross-linking agent is an epoxy compound cross-linking agent, preferably butylene glycol diglycidyl ether.

7. A method for preparing cross-linked recombinant humanized collagen gel, comprising the following steps: Recombinant humanized collagen is dissolved to obtain a solution, and then cross-linked with a cross-linking agent to obtain a cross-linked recombinant humanized collagen gel. The cross-linked recombinant humanized collagen gel has rheological properties, with Tanδ of 0.20-0.50 and G' of 90-600 Pa at a frequency of 0.1 Hz. Preferably, the pH value of the solution is 5-10; Preferably, the crosslinking reaction takes 16-72 hours.

8. The method according to claim 7, wherein the cross-linked recombinant humanized collagen gel is the cross-linked recombinant humanized collagen gel according to claims 2-6.

9. A filler comprising the cross-linked recombinant humanized collagen gel according to any one of claims 1-6 or the cross-linked recombinant humanized collagen gel prepared by the method according to claim 7 or 8; Preferably, the filler further comprises an anesthetic and / or excipients.

10. A skin-lightening product comprising the cross-linked recombinant humanized collagen gel according to any one of claims 1-6 or the cross-linked recombinant humanized collagen gel prepared by the method according to claim 7 or 8; Preferably, the water-light product further comprises an anesthetic and / or excipients.

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