Efficient cross-linking agent for recombinant collagen, cross-linked recombinant collagen gel and application

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a high-efficiency crosslinking agent of recombinant collagen, a crosslinked recombinant collagen hydrogel and application, and particularly provides a new use of cationic tetra(hydroxymethyl)phosphonium chloride (THPC) as a crosslinking agent in crosslinking of a recombinant collagen hydrogel, wherein the amino acid sequence of the recombinant collagen is shown as SEQ ID No. 1, the method for crosslinking the recombinant collagen hydrogel by THPC, and the prepared hydrogel has high biocompatibility, good mechanical properties and injectability, and significantly enhanced anti-enzymatic hydrolysis capacity; the method is simple, convenient and easy to operate, and can be applied to the fields of implant agents, artificial skin, hemostatic sponges, stent materials, medical devices and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a highly efficient cross-linking agent for recombinant collagen, a cross-linked recombinant collagen gel, and its applications. Background Technology

[0002] As a key structural protein in the skin, collagen levels gradually decrease with age, and it is considered a major cause of skin aging. Collagen implants have been found to effectively improve wrinkles and enhance skin health, leading to their increasingly widespread application in dermatology. Currently, collagen is primarily prepared through animal tissue extraction or recombinant gene expression. Recombinant collagen has advantages such as easy quality control, uniform molecular weight, and no risk of viral transmission, thus attracting considerable attention.

[0003] Compared to animal-derived collagen, recombinant collagen exhibits significant sequence differences and generally possesses significantly improved water solubility, but its gel-forming ability is greatly weakened. Therefore, chemical cross-linking has been attempted to prepare recombinant collagen hydrogels. Chinese patent CN114404667A discloses a method for cross-linking recombinant collagen using glutaraldehyde; Chinese patent CN114470333A discloses a method for cross-linking recombinant collagen using 1,4-butanediol diglycidyl ether. The residual cross-linking agents such as glutaraldehyde in these patents can cause cytotoxicity issues, highlighting the urgent need to develop cross-linked recombinant collagen hydrogels with high biocompatibility and long-lasting durability.

[0004] In their previous research, the inventors developed a recombinant collagen product (CN113520899B) for repairing photodamage to the skin. This recombinant collagen exhibits excellent repair effects on UV-damaged skin. However, this recombinant collagen possesses exceptionally high water solubility and cannot self-assemble into a gel. To prepare a hydrogel of this recombinant collagen and broaden its applications, the inventors added cross-linking agents such as glutaraldehyde to the recombinant collagen, but found that a hydrogel could not be prepared.

[0005] To address the aforementioned technical problems, the inventors unexpectedly discovered a highly efficient cross-linking agent for recombinant collagen, a cross-linked recombinant collagen hydrogel, and its applications. This invention provides a novel use of cationic tetra(hydroxymethyl)phosphorus chloride (THPC) as a cross-linking agent in cross-linked recombinant protein hydrogels. The method for cross-linking recombinant collagen hydrogels using THPC according to this invention produces hydrogels with high biocompatibility, good mechanical properties and injectability, and significantly enhanced resistance to enzymatic degradation. The method described in this invention is simple, convenient, and easy to operate, and can be applied to implants, artificial skin, hemostatic sponges, scaffold materials, medical devices, and other fields. Summary of the Invention

[0006] To address the aforementioned technical problems, the primary objective of this invention is to provide the application of cationic tetra(hydroxymethyl)phosphorus chloride (THPC) as a cross-linking agent for recombinant collagen, wherein the amino acid sequence of the recombinant collagen is shown in SEQ ID No. 1. Specifically:

[0007] GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTG

[0008] LAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGE

[0009] PGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDG

[0010] QNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP.

[0011] A second objective of this invention is to provide a method for cross-linking recombinant collagen, wherein the recombinant collagen is cross-linked with a cross-linking agent, cationic tetra(hydroxymethyl)phosphorus chloride (THPC), and the amino acid sequence of the recombinant collagen is shown in SEQ ID No. 1.

[0012] Preferably, the concentration of the crosslinking agent cationic tetra(hydroxymethyl)phosphorus chloride (THPC) is 0.02% v / v to 1% v / v.

[0013] Preferably, the recombinant collagen is dissolved in 0.02M PBS buffer solution.

[0014] Preferably, the reaction temperature of the crosslinking agent is 4-37°C and the reaction time is 0.1-24hrs.

[0015] A third objective of this invention is to provide a method for preparing recombinant collagen hydrogel, which involves mixing and reacting cationic tetra(hydroxymethyl)phosphorus chloride (THPC) with a recombinant collagen solution, wherein the amino acid sequence of the recombinant collagen is shown in SEQ ID No. 1.

[0016] A fourth objective of this invention is to provide a cross-linked recombinant collagen hydrogel prepared by the aforementioned preparation method.

[0017] A fifth objective of this invention is to provide a cross-linked recombinant collagen hydrogel implant prepared by the aforementioned preparation method.

[0018] The sixth objective of this invention is to provide the application of the cross-linked recombinant collagen hydrogel in the preparation of implants, artificial skin, hemostatic sponges, scaffold materials, and medical devices.

[0019] The beneficial effects of this invention are: ① This invention provides a novel application of cationic tetra(hydroxymethyl)phosphorus chloride (THPC) as a crosslinking agent in crosslinked recombinant protein hydrogels. THPC can crosslink recombinant collagen to prepare recombinant collagen hydrogels with good mechanical properties; ② The method for crosslinking recombinant collagen hydrogels using THPC according to this invention produces recombinant collagen gels with significantly enhanced resistance to enzymatic degradation and good injectability; ③ The THPC-crosslinked recombinant collagen gels of this invention have high biocompatibility and bioactivity, and can significantly promote fibroblast proliferation; ④ The THPC-crosslinked recombinant collagen gels of this invention are produced under mild conditions, are highly efficient, require small amounts, and have short crosslinking times; ⑤ The THPC-crosslinked recombinant collagen gels prepared by the method of this invention can be used in implants, artificial skin, hemostatic sponges, scaffold materials, medical devices, and other fields, and have broad application prospects. Attached Figure Description

[0020] Figure 1 Circular dichroism spectroscopy of the recombinant collagen prepared in Example 1 of this invention;

[0021] Figure 2 Rheological characterization of cross-linked recombinant collagen hydrogels with different amounts of THPC;

[0022] Note: Figure 2 In the physical graph in the lower right corner, the concentrations of each group from left to right are: blank, 0.005% v / v, 0.01% v / v, 0.02% v / v, 0.05% v / v, 0.1% v / v, 0.2% v / v, 0.25% v / v, 0.4% v / v, 0.5% v / v, 0.6% v / v, 1% v / v; in the rheological graph, the concentrations of each group are: blank, 0.02% v / v, 0.05% v / v, 0.1% v / v, 0.2% v / v, 0.25% v / v, 0.4% v / v, 0.5% v / v, 0.6% v / v, 1% v / v.

[0023] Figure 3 Physical images of THPC cross-linked recombinant collagen hydrogels at different temperatures;

[0024] Figure 4 Comparison of cross-linked recombinant collagen samples;

[0025] Figure 5 Scanning electron microscope image of THPC cross-linked recombinant collagen hydrogel;

[0026] Figure 6 Figure of the extrusion force experiment of THPC cross-linked recombinant collagen hydrogel;

[0027] Figure 7 Viscosity characterization diagram of THPC cross-linked recombinant collagen hydrogel;

[0028] Figure 8 Enzymatic hydrolysis experiment diagram of THPC cross-linked recombinant collagen hydrogel;

[0029] Figure 9 Schematic diagram of cytotoxicity and proliferation experiments of THPC cross-linked recombinant collagen hydrogel;

[0030] Figure 10 Live / dead cell staining images of THPC cross-linked recombinant collagen hydrogel. Detailed Implementation

[0031] The present invention will be described in detail below through specific embodiments. Any technical solutions that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art are within the protection scope of the present invention.

[0032] It should be noted that the recombinant collagen used in this invention is disclosed in invention patent CN113520899B, a recombinant collagen product for repairing photodamage to the skin.

[0033] Example 1: Preparation and Characterization of Recombinant Collagen

[0034] 1. Preparation of recombinant collagen

[0035] Recombinant collagen was synthesized using the amino acid sequence of recombinant collagen as shown in SEQ ID No. 1; nucleotide sequences encoding the above amino acid sequences were synthesized, and corresponding expression plasmids were constructed. After confirming the successful synthesis of the plasmid by DNA sequencing, the plasmid was transformed into Escherichia coli BL21-DE3 strain. The successfully transformed cloned strain was added to glycerol and stored at -80℃; finally, a small amount of the recombinant collagen expression strain was induced to grow by IPTG, and after treatment with trypsin, the recombinant collagen was purified and lyophilized for storage.

[0036] 2. Circular dichroism characterization of recombinant collagen

[0037] The recombinant collagen prepared in Example 1 was dissolved in PBS buffer solution at pH 7.0 to a final concentration of 1 mg / mL. The parameters were set as follows: scanning wavelength 210-260 nm, scanning temperature 4 °C, wavelength step 0.5 nm, and averaging time 0.5 s. The above recombinant collagen solution was spectrally scanned using a circular dichroism chromatograph.

[0038] The results are as follows Figure 1 As shown, the recombinant collagen prepared in Example 1 exhibits a characteristic absorption peak around 220 nm, indicating that it forms the classic triple helix structure of collagen.

[0039] Example 2: Preparation and characterization of THPC cross-linked recombinant collagen hydrogels with different addition amounts

[0040] 1. Preparation of THPC cross-linked recombinant collagen hydrogel

[0041] The lyophilized recombinant collagen was dissolved in 0.02M PBS buffer solution (pH 7.4) to make the recombinant collagen concentration 10 mg / mL. 0.005% v / v to 1% v / v THPC solution was added to the solution and crosslinked at room temperature for 1 h to obtain THPC crosslinked recombinant collagen hydrogel.

[0042] 2. Rheological and mechanical characterization of THPC cross-linked recombinant collagen hydrogels

[0043] The sample prepared in Example 2 was used. When the amount of THPC added was less than 0.02% v / v, the sample was in a solution state and no gel was formed; when the amount of THPC added was between 0.02% and 1% v / v, the sample was in a hydrogel state. THPC crosslinked recombinant collagen hydrogel and recombinant collagen with an addition amount of 0.02% v / v to 1% v / v were placed on the sample stage of the rheometer. The scanning frequency of the rheometer was fixed, and the storage modulus G' (Pa) and loss modulus G' (Pa) of the gel as a function of strain were tested.

[0044] Experimental results are as follows Figure 2As shown, the storage modulus G' (Pa) and loss modulus G' (Pa) before crosslinking were 116 Pa and 15 Pa, respectively. With the increase of THPC addition, the storage moduli of the THPC-crosslinked recombinant protein hydrogel were 145 Pa (0.02% v / v), 249 Pa (0.05% v / v), 427 Pa (0.1% v / v), 474 Pa (0.2% v / v), 527 Pa (0.25% v / v), 539 Pa (0.4% v / v), 614 Pa (0.5% v / v), and 615 Pa (0.6% v / v), respectively. The loss moduli of the THPC-crosslinked recombinant collagen hydrogels were 14 Pa (0.02% v / v), 23 Pa (0.05% v / v), 26 Pa (0.1% v / v), 30 Pa (0.2% v / v), 33 Pa (0.25% v / v), 32 Pa (0.4% v / v), 32 Pa (0.5% v / v), 30 Pa (0.6% v / v), and 27 Pa (1% v / v). These results indicate that THPC crosslinking successfully prepared recombinant collagen hydrogels with good mechanical strength.

[0045] Comparative Example 1: Preparation of Cross-linked Recombinant Collagen with 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS)

[0046] The recombinant collagen prepared in Example 1 was dissolved in 0.02M PBS buffer solution (pH 7.4) at a concentration of 10 mg / mL. 0.5% v / v EDC / NHS solution was added to the solution, and the mixture was cross-linked at 25°C for 1 h to obtain EDC / NHS cross-linked recombinant collagen.

[0047] Comparative Example 2: Preparation of 1,4-Butanediol diglycidyl ether (BDDE) cross-linked recombinant collagen

[0048] The recombinant collagen prepared in Example 1 was dissolved in 0.02M PBS buffer solution (pH 7.4) at a concentration of 10 mg / mL. 0.5% v / v BDDE solution was added to it, and crosslinking was carried out at 25°C for 1 h. The BDDE then yielded crosslinked recombinant collagen.

[0049] Preparation of comparative triglutaraldehyde (GA) cross-linked recombinant collagen

[0050] The recombinant collagen prepared in Example 1 was dissolved in 0.02M PBS buffer solution (pH 7.4) at a concentration of 10 mg / mL. 0.5% v / v GA solution was added to it, and crosslinking was carried out at 25°C for 1 h to obtain GA crosslinked recombinant collagen.

[0051] Subsequent experiments used 0.5% v / v THPC cross-linked recombinant collagen from Example 2, cross-linked at 25°C for 1 hour, to obtain THPC cross-linked recombinant collagen hydrogel.

[0052] The samples of uncrosslinked, comparative examples 1, 2, and 3, and crosslinked recombinant collagen from Example 2 were photographed, as follows: Figure 3 As shown, EDC / NHS, GA, and BDDE cross-linked recombinant collagen did not form hydrogels, while THPC cross-linked recombinant collagen did form a hydrogel. These results indicate that THPC is a highly efficient cross-linking agent for recombinant collagen and can form recombinant collagen hydrogels.

[0053] Example 3: Preparation of THPC crosslinked recombinant collagen hydrogels at different temperatures

[0054] Crosslinking at 1.4℃

[0055] The recombinant collagen prepared in Example 1 was dissolved in 0.02M PBS buffer solution (pH 7.4) at a concentration of 10 mg / mL. 0.5% v / v THPC solution was added to it, and crosslinking was carried out at 4°C for 1 h to obtain THPC crosslinked recombinant collagen.

[0056] Crosslinking at 2.37℃

[0057] The recombinant collagen prepared in Example 1 was dissolved in 0.02M PBS buffer solution (pH 7.4) at a concentration of 10 mg / mL. 0.5% v / v THPC solution was added to it, and crosslinking was carried out at 37°C for 1 h to obtain THPC crosslinked recombinant collagen.

[0058] Samples of recombinant collagen that underwent THPC cross-linking at 4℃ and 37℃ were photographed. Figure 4 As shown, hydrogels were formed at both 4℃ and 37℃. The results indicate that THPC is a highly efficient recombinant collagen cross-linking agent that can form hydrogels over a wide cross-linking temperature range.

[0059] Example 4: Characterization of THPC Crosslinked Recombinant Collagen Hydrogel

[0060] 1. Scanning electron microscopy characterization

[0061] The THPC cross-linked recombinant collagen hydrogel sample prepared in Example 2 was sliced, sputtered with gold, and then characterized by SEM.

[0062] Experimental results are as follows Figure 5 As shown, THPC cross-linked recombinant collagen hydrogels form a uniformly distributed three-dimensional network structure, providing a beneficial environment for cell growth.

[0063] 2. Injectability testing

[0064] The THPC cross-linked recombinant collagen hydrogel prepared in Example 2 was placed in a 1 mL sterile syringe. The syringe was fixed on the sample stage of the syringe and the extrusion speed was 10 mm / min to perform the extrusion force test.

[0065] Experimental results are as follows Figure 6 As shown, the THPC cross-linked recombinant collagen hydrogel prepared in Example 3 has a maximum extrusion force of 2.2 N, a minimum extrusion force of 1.1 N, and an average extrusion force of 1.5 N, indicating that the THPC cross-linked recombinant collagen hydrogel has excellent injectability.

[0066] 3. Viscosity Measurement

[0067] The THPC cross-linked recombinant collagen hydrogel prepared in Example 2 and the uncross-linked recombinant collagen samples were placed on the sample stage of a rheometer and the shear rates were tested from 0.001 to 1000 s⁻¹. -1 The change in viscosity, experimental results are as follows Figure 7 As shown, at shear rates of 0.001-1000 s... -1 Within the specified range, the viscosity of THPC cross-linked recombinant collagen hydrogel is much higher than that of uncross-linked recombinant collagen. The good viscosity of THPC cross-linked recombinant collagen hydrogel will ensure that it is not easily displaced after implantation, resulting in a good implantation effect.

[0068] 4. Enzymatic hydrolysis experiment determination

[0069] Take 2 mg of each of the uncrosslinked recombinant collagen and the THPC-crosslinked collagen prepared in Example 2 after lyophilization, and record the initial dry weight (approximately 2 mg). Prepare collagenase by dissolving it in a buffer solution (TES, 1 mM CaCl2, pH 7.4) at a concentration of 0.5 U / mL. Perform enzymatic hydrolysis at 37°C and 220 rpm. After a certain period of hydrolysis, wash the samples with water and centrifuge them. Lyophilize the centrifuged samples and weigh them to calculate the hydrolysis rate.

[0070] AW% = (Wo - W) / W × 100%

[0071] Where: AW%: enzymatic hydrolysis rate; Wo: initial weight of sample before enzymatic hydrolysis; W: weight of sample after enzymatic hydrolysis.

[0072] Experimental results are as follows Figure 8 As shown, the uncrosslinked recombinant collagen achieved a 100% enzymatic hydrolysis rate on day 1; the THPC crosslinked recombinant collagen hydrogel prepared in Example 2 had enzymatic hydrolysis rates of 51.2%, 59.1%, and 63.7% on days 1, 4, and 7, respectively. The results indicate that the THPC crosslinked recombinant collagen hydrogel exhibits significantly enhanced resistance to enzymatic hydrolysis.

[0073] 5. Cytotoxicity and proliferation assays

[0074] To investigate the cytotoxicity and proliferation of recombinant collagen and THPC-crosslinked recombinant collagen, HFF-1 human fibroblasts were cultured in high-glucose DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin antibiotics at 37°C and 5% CO2. Lyophilized samples of recombinant collagen and THPC-crosslinked recombinant collagen prepared in Example 2 were placed in DMEM high-glucose medium and soaked at 37°C for 72 h to prepare extracts. Cells were digested with 0.25% (w / w) trypsin and seeded at a density of 7000 cells per well in 96-well plates. The plates were incubated at 37°C and 5% CO2 for 24 h. Then, 100 μL of different samples were added to the corresponding wells and incubated for 24 h, 48 h, and 72 h, respectively. Afterward, 10 μL of CCK8 was added to each well and incubated at 37°C for 1–3 h. The basal medium served as a blank control (n=6), and the OD value at 450 nm was recorded.

[0075] Cell viability is calculated as follows: Cell viability (%) = (AC) / (BC) x 100%.

[0076] A: OD values ​​of different samples; B: OD value of the positive control group; C: OD value of the blank control group.

[0077] Experimental results are as follows Figure 9 As shown, at 24h, 48h, and 72h, the cell viability of recombinant collagen was 97%, 99%, and 100%, respectively, while the cell viability of THPC-crosslinked recombinant collagen was 102%, 105%, and 106%, respectively. The results indicate that THPC-crosslinked recombinant collagen hydrogel is non-cytotoxic and exhibits good biocompatibility.

[0078] 6. Cell survival experiment

[0079] To investigate the cell viability of recombinant collagen and THPC-crosslinked recombinant collagen, HFF-1 human fibroblasts were cultured in high-glucose DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin at 37°C and 5% CO2. Recombinant collagen and THPC-crosslinked recombinant collagen hydrogel samples (Example 2) were lyophilized in DMEM high-glucose medium and soaked at 37°C for 72 h. Cells were digested with 0.25% (w / w) trypsin and seeded at a density of 40,000 cells per well in laser confocal culture dishes. The cells were incubated at 37°C and 5% CO2 for 24 h. Then, 100 μL of different samples were added to the corresponding wells and incubated for 24 h each. After 72 h, the cells were stained with CA / PI and photographed using an Olympus laser confocal microscope.

[0080] Experimental results are as follows Figure 10As shown, fibroblasts survived well in recombinant collagen and THPC-crosslinked recombinant collagen hydrogels for 24 h and 72 h. The results indicate that THPC-crosslinked recombinant collagen hydrogels provide a favorable growth environment for fibroblasts.

[0081] In summary, this invention provides a novel application of cationic tetra(hydroxymethyl)phosphorus chloride (THPC) as a crosslinking agent in crosslinked recombinant protein hydrogels. The THPC can crosslink recombinant collagen to prepare a recombinant collagen hydrogel with excellent mechanical properties. The method for crosslinking recombinant collagen hydrogels using THPC according to this invention produces a recombinant collagen gel with significantly enhanced resistance to enzymatic degradation and good injectability. The THPC-crosslinked recombinant collagen gel exhibits high biocompatibility and bioactivity, and can significantly promote fibroblast proliferation. The THPC-crosslinked recombinant collagen hydrogels prepared by this invention utilize mild conditions, high efficiency, low dosage, and short crosslinking time. The THPC-crosslinked recombinant collagen gels prepared by this method have broad application prospects in fields such as implants, artificial skin, hemostatic sponges, scaffold materials, and medical devices.

Claims

1. The application of cationic tetra(hydroxymethyl)phosphorus chloride (THPC) as a recombinant collagen cross-linking agent, characterized in that, The amino acid sequence of the recombinant collagen is shown in SEQ ID No.

1.

2. A method for cross-linking recombinant collagen, characterized in that, Recombinant collagen was cross-linked with the cross-linking agent cationic tetra(hydroxymethyl)phosphorus chloride (THPC), and the amino acid sequence of the recombinant collagen is shown in SEQ ID No.

1.

3. The crosslinking method as described in claim 2, characterized in that, The concentration of the crosslinking agent, cationic tetra(hydroxymethyl)phosphorus chloride (THPC), is 0.02% v / v to 1% v / v.

4. The crosslinking method as described in claim 2, characterized in that, The recombinant collagen was dissolved in 0.02 M PBS buffer solution.

5. The crosslinking method as described in claim 2, characterized in that, The crosslinking reaction temperature is 4-37 ℃, and the reaction time is 0.1-24 hrs.

6. A method for preparing a recombinant collagen hydrogel, characterized in that, The cationic tetra(hydroxymethyl)phosphorus chloride (THPC) was mixed with a recombinant collagen solution and reacted. The amino acid sequence of the recombinant collagen is shown in SEQ ID No.

1.

7. The recombinant collagen hydrogel prepared by the preparation method according to claim 6.

8. The application of the recombinant collagen hydrogel as described in claim 7 in the preparation of medical devices.

9. The application as described in claim 8, characterized in that, The medical device in question is an implant.

10. The application as described in claim 9, characterized in that, The implant is an artificial skin, hemostatic sponge, or scaffold material.

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