Enzyme crosslinking recombinant collagen hydrogel for injection and application thereof

Abstract

The invention discloses enzyme cross-linked recombinant collagen hydrogel for injection and application thereof. The recombinant collagen hydrogel is prepared by mixing recombinant collagen with an amino acid sequence as shown in SEQ ID NO: 1 with glutamine transaminase and carrying out cross-linking reaction. The recombinant collagen hydrogel disclosed by the invention has excellent biocompatibility and good mechanical properties, and can be applied to the fields of implants, artificial skins, organoids, hemostatic sponges, stent materials, cell culture media, medical instruments and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an enzyme-crosslinked recombinant collagen hydrogel for injection and application thereof. Background Art

[0002] Collagen is the most abundant protein in the human body and is widely distributed in human connective tissue. The content of collagen gradually decreases with age and is considered to be the main cause of skin aging. Collagen implants can effectively improve skin wrinkles, enhance skin brightness and health, and are increasingly used in medical beauty. Collagen can be divided into animal collagen and recombinant collagen. Animal collagen is mainly extracted from animal materials by hot water extraction, acid extraction and enzyme extraction. This method has a long history, mature technology, low threshold and cost, but there are problems such as raw materials being contaminated by microorganisms and viruses, allergies and immune rejection. Recombinant collagen is produced by fermentation expression after human collagen genes are designed with specific sequences, enzyme cleavage and splicing, and connected to vectors through genetic engineering technology. The recombinant method effectively avoids the influence of potential viruses and impurities in natural collagen, and the entire production process is controllable, so it has attracted much attention.

[0003] Recombinant collagen has excellent biocompatibility, efficient biodegradability, low immunogenicity, and coordination between collagen host and tissue, and is suitable for injection filling medical devices. However, its mechanical properties and anti-enzymatic rate are poor. By cross-linking collagen, its structural stability can be improved. The methods commonly used for collagen cross-linking are physical methods, chemical methods, and biological methods. As we all know, this process requires physical induction or chemical reagents to promote the formation of intramolecular or intermolecular chemical bonds. Physical cross-linking can prevent toxic substances from entering the collagen molecule, but the molecular structure formed is poorly stable and the degree of cross-linking is low. Chemical reagents can interact with collagen through functional groups (amino and carboxyl groups) to form cross-links and increase the degree of cross-linking. However, chemical reagents usually have problems such as toxicity or bioincompatibility. Biological cross-linked collagen has low cytotoxicity, excellent cell compatibility, and is not prone to collagen denaturation. At present, collagen cross-linking is developing in the direction of safety, environmental protection, and controllability. More and more new methods are gradually replacing traditional cross-linking methods, allowing collagen materials to be better used in tissue repair, wound hemostasis, tissue engineering, treatment of burns and scalds, and beauty. Summary of the invention

[0004] In order to enrich the types of recombinant collagen and expand the application scope of recombinant collagen in the field of medical beauty, the present invention provides the following technical solutions.

[0005] In a first aspect, the present invention provides a recombinant collagen protein, wherein the amino acid sequence of the recombinant collagen protein is shown in SEQ ID NO:1.

[0006] Specifically, the sequence shown in SEQ ID NO:1 is as follows:

[0007] GEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGS

[0008] PGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGP

[0009] PGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLP

[0010] GPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPA

[0011] GPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGDAGAKGDAGP

[0012] PGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGN

[0013] AGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGP

[0014] AGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGE

[0015] RGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGP

[0016] PGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRG

[0017] DKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPG

[0018] SAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPP

[0019] In a second aspect, the present invention provides a recombinant collagen hydrogel, wherein the recombinant collagen hydrogel is prepared by mixing a recombinant collagen solution containing the recombinant collagen described in the first aspect with glutamyl transaminase (TG) and subjecting the mixture to a cross-linking reaction.

[0020] Preferably, the concentration of the recombinant collagen solution is 2-3wt%, for example: 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%.

[0021] Preferably, the solvent of the recombinant collagen is purified water, water for injection, distilled water or physiological saline.

[0022] Preferably, the added amount of the glutamine aminotransferase is 0.05-0.5wt% of the recombinant collagen solution, for example: 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%.

[0023] Preferably, the temperature of the cross-linking reaction is 35-45°C, for example: 35°C, 6°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C.

[0024] Preferably, the cross-linking reaction time is 0.2 to 24 h, for example: 0.2 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, 20 h, 24 h.

[0025] Preferably, the recombinant collagen and glutamyl transaminase mixture is filtered and sterilized before the cross-linking reaction.

[0026] Furthermore, the pore size of the filter membrane for filtration and sterilization is 0.22 μm or 0.45 mm, more preferably 0.22 μm.

[0027] Preferably, the non-cross-linked transglutaminase is inactivated after the cross-linking reaction.

[0028] Furthermore, the inactivation of glutamine aminotransferase is carried out in a high temperature water bath.

[0029] Furthermore, the temperature of the water bath is 60-90°C, for example, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C.

[0030] Furthermore, the water bath time is 20 to 40 min, for example: 20 min, 22 min, 25 min, 27 min, 30 min, 32 min, 35 min, 37 min, 40 min.

[0031] In a third aspect, the present invention provides a method for preparing a recombinant collagen hydrogel, the preparation method comprising the following steps:

[0032] (1) An aqueous solution of recombinant collagen having an amino acid sequence as shown in SEQ ID NO: 1 is mixed with transglutaminase (TG), and the mixture is filtered and sterilized.

[0033] (2) subjecting the filtered and sterilized mixture to a cross-linking reaction at 35-45° C. for 0.2-24 h, and inactivating it in a water bath at 60-90° C. for 20-40 min to obtain the recombinant collagen hydrogel.

[0034] In a fourth aspect, the present invention provides a hydrogel implant, wherein the hydrogel implant comprises the recombinant collagen described in the first aspect or the recombinant collagen hydrogel described in the second aspect.

[0035] In a fifth aspect, the present invention provides the use of the recombinant collagen described in the first aspect or the recombinant collagen hydrogel described in the second aspect in the preparation of implants, artificial skins, organoids, hemostatic sponges, scaffold materials, cell culture media or medical devices.

[0036] In a sixth aspect, the present invention provides use of the recombinant collagen described in the first aspect or the recombinant collagen hydrogel described in the second aspect in the preparation of a product for promoting the proliferation of in vivo extracellular fibroblasts.

[0037] Beneficial effects of the present invention:

[0038] The present invention designs a new recombinant collagen, which is cross-linked with glutamine aminotransferase to obtain an injectable hydrogel, which has excellent biocompatibility and good mechanical properties and can be used in the fields of implants, artificial skin, organoids, hemostatic sponges, scaffold materials, cell culture media, medical devices, etc. The present invention expands the application range of recombinant collagen and provides a method for preparing a gel with simple process and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Shown is the circular dichroism spectrum of the recombinant collagen prepared in Example 1;

[0040] Figure 2 Shown are the actual pictures of recombinant collagen hydrogels with different amounts of recombinant collagen added;

[0041] Figure 3 The figure shows a schematic diagram of a cross-linked recombinant collagen hydrogel syringe in Example 2, wherein the left figure is a schematic diagram of loading the hydrogel into the syringe, and the right figure is a schematic diagram of injecting the hydrogel;

[0042] Figure 4The figure shows the pushing force test results of the cross-linked recombinant collagen hydrogel in Example 2, wherein A is a recombinant collagen hydrogel with a concentration of 2wt%, B is a recombinant collagen hydrogel with a concentration of 2.5wt%, and C is a recombinant collagen hydrogel with a concentration of 3.0wt%;

[0043] Figure 5 Shown is a scanning electron micrograph of the recombinant collagen freeze-dried sponge in Example 1;

[0044] Figure 6 Shown is a scanning electron micrograph of the cross-linked recombinant collagen hydrogel in Example 2;

[0045] Figure 7 Shown is a photograph of the cytotoxicity results of the cross-linked recombinant collagen hydrogel in Example 2;

[0046] Figure 8 Shown are the test results of the relative cell proliferation rate of the cross-linked recombinant collagen hydrogel in Example 2. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention.

[0048] Example 1 Preparation and characterization of recombinant collagen

[0049] 1.1 Preparation of recombinant collagen

[0050] The recombinant collagen was synthesized using the amino acid sequence of the recombinant collagen shown in SEQ ID NO: 1. The nucleotide sequence encoding the amino acid sequence was synthesized and codon optimized to construct the pPIC9k expression plasmid. The plasmid synthesis was confirmed to be accurate by DNA sequencing. The plasmid was then linearized and electroporated into the Pichia pastoris GS115 strain. After resistance screening, a genetically engineered bacterium with stable expression was obtained. The genetically engineered bacterium was fermented and cultured, and the recombinant collagen was obtained after purification and freeze-dried for storage.

[0051] 1.2 Circular dichroism characterization of recombinant collagen

[0052] The recombinant collagen prepared in 1.1 was dissolved in a PBS buffer solution at pH 7.0 to a final concentration of 1 mg / mL. The recombinant collagen solution was spectrally scanned using a circular dichroism spectrometer at a scanning wavelength of 190-260 nm and a scanning temperature of 4°C.

[0053] like Figure 1 As shown, the recombinant collagen has a negative peak at a wavelength near 195 nm and a positive peak at a wavelength near 221 nm, which is consistent with the triple helix structure characteristics of collagen.

[0054] Example 2 Preparation and characterization of TG cross-linked recombinant collagen gels with different recombinant collagen concentrations

[0055] 2.1 Preparation of TG cross-linked recombinant protein gels with different recombinant collagen concentrations

[0056] The recombinant collagen freeze-dried sponge prepared in 1.1 was dissolved in physiological saline, and solutions with recombinant collagen concentrations of 1wt%, 2wt%, 2.5wt%, 3wt%, 4wt%, and 5wt% were prepared respectively. Then, 0.1wt% of the mass of the recombinant collagen solution of glutamyl transaminase (TG) was added, and after mixing, the mixture was sterilized by filtering with a 0.22μm filter membrane, cross-linked at 37°C for 1h, and bathed in a water bath at 80°C for 30min to obtain TG cross-linked recombinant collagen hydrogel.

[0057] 2.2 Characterization of TG cross-linked recombinant collagen gel

[0058] Take the recombinant collagen gel samples with recombinant collagen concentrations of 1wt%, 2wt%, 2.5wt%, 3wt%, 4wt%, and 5wt% prepared in 2.1, and the uncross-linked recombinant collagen solution sample with a concentration of 2wt%, and take pictures.

[0059] like Figure 2 As shown in the figure, the recombinant collagen sample with a concentration of 1wt% did not form a gel. The recombinant collagen samples with concentrations of 2wt%, 2.5wt%, 3wt%, 4wt%, and 5wt% all formed a gel, but the recombinant collagen samples with concentrations of 4wt% and 5wt% were relatively hard and not suitable for injection. Therefore, at a recombinant collagen concentration of 2% to 3wt%, an injectable recombinant collagen hydrogel was prepared using glutamyl transaminase (TG) as a crosslinking agent.

[0060] Example 3 Characterization of TG cross-linked recombinant collagen hydrogel

[0061] 3.1 Injectability determination

[0062] Take 2wt%, 2.5wt%, and 3wt% TG cross-linked recombinant collagen hydrogel samples prepared in Example 2, respectively, place them in 1mL syringes, fix the syringes on the sample stage of the needle instrument, and perform the extrusion force test at an extrusion speed of 10mm / min (see Figure 3 and Figure 4 ).

[0063] like Figure 4As shown, the maximum pushing force of 2wt% TG cross-linked recombinant collagen hydrogel is 1.4N, the minimum pushing force is 0.76N, and the average pushing force is 0.82N; the maximum pushing force of 2.5wt% TG cross-linked recombinant collagen hydrogel is 1.9N, the minimum pushing force is 0.87N, and the average pushing force is 0.95N; the maximum pushing force of 3wt% TG cross-linked recombinant collagen hydrogel is 2.2N, the minimum pushing force is 1.26N, and the average pushing force is 1.3N. This shows that TG cross-linked recombinant collagen hydrogel has excellent injection performance. Among them, the 3wt% TG cross-linked recombinant collagen hydrogel has a moderate pushing force, which is used for subsequent hydrogel characterization.

[0064] 3.2 Scanning electron microscopy characterization

[0065] Take 0.5 g of the 3 wt% recombinant collagen freeze-dried sponge sample prepared in Example 1, spray gold and perform SEM characterization. Figure 5 As shown, the recombinant collagen has a fibrous structure, which is similar to the shape of collagen in the human body and can play a supporting role.

[0066] The 3 wt% cross-linked recombinant collagen hydrogel sample prepared in Example 2 was freeze-dried, and 0.5 g of the freeze-dried sample was taken and subjected to SEM characterization after gold spraying. Figure 6 As shown, 3wt% TG cross-linked recombinant collagen hydrogel presents a uniformly distributed network structure, which can carry cells and provide favorable growth conditions for cells.

[0067] 3.3 Viscosity determination

[0068] The 3 wt % TG cross-linked recombinant collagen hydrogel and the uncross-linked 3 wt % recombinant collagen sample prepared in Example 2 were placed on the sample stage of the rheometer, respectively, and the rotation speed was set to 60 rpm to test the viscosity of different samples.

[0069] The test results show that the viscosity of 3wt% TG cross-linked recombinant collagen hydrogel is 26299mPa·s at a rotation speed of 60rpm. The viscosity of uncross-linked 3wt% recombinant collagen is 6.6mPa·s at a rotation speed of 60rpm. This shows that compared with uncross-linked recombinant collagen, the viscosity of TG cross-linked recombinant collagen hydrogel is significantly improved. The good viscosity ensures that TG cross-linked recombinant collagen hydrogel is not easily displaced after implantation, making it more suitable for tissue filling products.

[0070] 3.4 Cell proliferation experiment

[0071] L929 cells in the logarithmic growth phase were taken, digested, counted, centrifuged, and resuspended in DMEM complete medium to a cell density of 10 5cells / mL, and the cell suspension was transferred to a 96-well plate after being blown and mixed, with 100 μL per well. Cultured in a 37°C, 5% CO2 incubator. The 3wt% recombinant collagen solution (uncrosslinked group) and the 3wt% TG cross-linked recombinant collagen hydrogel (Example 2 group) samples prepared in Example 2 were placed in DMEM high-glucose culture medium, respectively, and soaked at 37°C for 72h to prepare the extract. When the cell density in the 96-well plate was close to confluence, the original culture medium in the well was aspirated and replaced with 3wt% recombinant collagen solution and hydrogel extraction culture medium, respectively. A 0.64% (w / v) phenol solution was added to the positive control group, and an equal volume of DMEM complete culture medium was added to the blank control group. Cultured in a 37°C, 5% CO2 incubator for 24h, the culture medium was removed, and 100 μL of DMEM complete culture medium containing 10% CCK-8 solution was added to each well. Incubate in a 37°C, 5% CO2 incubator for 2h. After incubation, the cells treated in each experimental group were photographed, and the absorbance at a wavelength of 450 nm (reference wavelength 630 nm) was detected using an ELISA instrument. The relative growth rate (RGR) of cells in each group was calculated according to the following formula to determine the cytotoxicity of the hydrogel.

[0072] RGR (%) = sample absorbance / blank control absorbance × 100%

[0073] like Figure 7 As shown, the cells in the non-crosslinked group and Example 2 group were in good condition, and the cell morphology was similar to that of the blank control group, indicating that the 3wt% TG cross-linked recombinant collagen hydrogel had no obvious cytotoxicity.

[0074] like Figure 8 As shown, the cell survival rate of the positive control group was 8.67%, which was significantly different from the blank control group, the cell survival rate of the non-crosslinked group was 124.29%, and the cell survival rate of Example 2 group was 127.65%. Compared with the blank control group, the cell survival rates of the non-crosslinked group and Example 2 group increased by more than 24%, indicating that 3wt% TG cross-linked recombinant collagen hydrogel can enhance the proliferation and survival ability of cells.

[0075] The above experimental results show that TG cross-linked recombinant collagen gel is not only non-cytotoxic, but also can significantly promote the proliferation of fibroblasts.

[0076] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A recombinant collagen, characterized in that: The amino acid sequence of the recombinant collagen is shown in SEQ ID NO:

1.

2. A recombinant collagen hydrogel, characterized in that: The recombinant collagen hydrogel is prepared by mixing a recombinant collagen solution containing the recombinant collagen according to claim 1 with glutamyl aminotransferase and subjecting the mixture to a cross-linking reaction.

3. The recombinant collagen hydrogel according to claim 2, characterized in that: The concentration of the recombinant collagen solution is 2wt% to 3wt%.

4. The recombinant collagen hydrogel according to claim 3, characterized in that: The added amount of the glutamine aminotransferase is 0.05-0.5 wt % of the recombinant collagen solution.

5. The recombinant collagen hydrogel according to claim 2, characterized in that: The cross-linking reaction temperature is 35-45°C, and / or The cross-linking reaction time is 0.2 to 24 hours.

6. The recombinant collagen hydrogel according to claim 2, characterized in that: The recombinant collagen and glutamyl transaminase mixture is filtered and sterilized before the cross-linking reaction. Preferably, the pore size of the filter membrane for filtration and sterilization is 0.22 μm or 0.45 μm.

7. The recombinant collagen hydrogel according to claim 2, characterized in that: After the cross-linking reaction, the uncross-linked transglutaminase is inactivated. Preferably, the transglutaminase is inactivated by water bathing at 60-90° C. for 20-40 min.

8. A hydrogel implant, characterized in that: The hydrogel implant comprises the recombinant collagen described in claim 1 or the recombinant collagen hydrogel described in any one of claims 2-7.

9. Use of the recombinant collagen according to claim 1 or the recombinant collagen hydrogel according to any one of claims 2 to 7 in the preparation of implants, artificial skins, organoids, hemostatic sponges, scaffold materials, cell culture media or medical devices.

10. Use of the recombinant collagen according to claim 1 or the recombinant collagen hydrogel according to any one of claims 2 to 7 in the preparation of a product for promoting the proliferation of extracorporeal fibroblasts.

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