Enzyme cross-linked recombinant collagen hydrogel for injection and application thereof

Abstract

The application discloses an enzyme cross-linked recombinant collagen hydrogel for injection and application thereof, and the recombinant collagen hydrogel is prepared by mixing and cross-linking reaction of recombinant collagen with an amino acid sequence as shown in SEQ ID NO:1 and glutamine transaminase. The recombinant collagen hydrogel has excellent biocompatibility and good mechanical properties, and can be applied to the fields of implant, artificial skin, organoid, hemostatic sponge, stent material, cell culture medium, medical device and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an injectable enzyme-crosslinked recombinant collagen hydrogel and its applications. Background Technology

[0002] Collagen is the most abundant protein in the human body, widely distributed in connective tissues. Collagen levels gradually decrease with age, and it is considered a major cause of skin aging. Collagen implants can effectively improve wrinkles, enhance skin radiance and overall health, leading to their increasingly widespread use in aesthetic medicine. Collagen can be divided into animal-derived collagen and recombinant collagen. Animal-derived collagen is mainly extracted from animal materials through methods such as hot water extraction, acid extraction, and enzymatic extraction. These methods have a long history, mature technology, and relatively low barriers to entry and costs; however, they suffer from issues such as contamination of raw materials by microorganisms and viruses, allergies, and immune rejection. Recombinant collagen is produced through genetic engineering. Human collagen genes are designed with specific sequences, digested and spliced ​​with enzymes, ligated into vectors, and then transferred into engineered cells for fermentation expression. The recombinant method effectively avoids the potential influence of viruses and other proteins in natural collagen, and the entire production process is controllable, thus attracting significant attention.

[0003] Recombinant collagen possesses excellent biocompatibility, high biodegradability, low immunogenicity, and good coordination between the collagen host and tissues, making it suitable for use in injectable filler medical devices. However, its mechanical properties and resistance to enzymatic degradation are relatively poor. Cross-linking collagen can improve its structural stability. Common methods for collagen cross-linking include physical, chemical, and biological methods. It is well known that 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 resulting molecular structure has poor stability and a low degree of cross-linking. Chemical reagents can interact with collagen through functional groups (amino and carboxyl groups) to form cross-links, increasing the degree of cross-linking. However, chemical reagents often have problems such as toxicity or biocompatibility. Biologically cross-linked collagen has low cytotoxicity, excellent cell compatibility, and is less prone to collagen denaturation. Currently, collagen cross-linking is developing towards safety, environmental friendliness, and controllability. More and more new methods are gradually replacing traditional cross-linking methods, enabling adhesive raw materials to be better applied in fields such as tissue repair, wound hemostasis, tissue engineering, burn treatment, and cosmetics. Summary of the Invention

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

[0005] In a first aspect, the present invention provides a recombinant collagen protein, the amino acid sequence of which 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 gamma-glutamyl transferase (TG) and performing a cross-linking reaction.

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

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

[0022] Preferably, the amount of glutamyl transaminase added is 0.05 to 0.5 wt% 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%, or 0.5%.

[0023] Preferably, the temperature of the crosslinking 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 crosslinking reaction time is 0.2 to 24 hours, for example: 0.2 hours, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, and 24 hours.

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

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

[0027] Preferably, the uncrosslinked transglutaminase is inactivated after the crosslinking reaction.

[0028] Furthermore, the inactivation of transglutaminase is performed using a high-temperature water bath.

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

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

[0031] Thirdly, the present invention provides a method for preparing recombinant collagen hydrogel, the method comprising the following steps:

[0032] (1) Mix the recombinant collagen aqueous solution with glutamine transaminase (TG) as shown in SEQ ID NO:1 and filter to remove bacteria.

[0033] (2) After filtering and sterilizing, the mixture is cross-linked at 35-45°C for 0.2-24 h, and then inactivated in a water bath at 60-90°C for 20-40 min to obtain the recombinant collagen hydrogel.

[0034] Fourthly, the present invention provides a hydrogel implant comprising the recombinant collagen described in the first aspect or the recombinant collagen hydrogel described in the second aspect.

[0035] Fifthly, 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 skin, organoids, hemostatic sponges, scaffold materials, cell culture media or medical devices.

[0036] In a sixth 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 products that promote the proliferation of in vivo extracellular fibroblasts.

[0037] The beneficial effects of this invention are:

[0038] This invention designs a novel recombinant collagen protein, which is cross-linked with transglutaminase to obtain an injectable hydrogel with excellent biocompatibility and good mechanical properties. It can be applied in implants, artificial skin, organoids, hemostatic sponges, scaffold materials, cell culture media, and medical devices. This invention expands the application scope of recombinant collagen and provides a simple and convenient gel preparation method. Attached Figure Description

[0039] Figure 1 The image shown is a circular dichroism chromatogram of the recombinant collagen prepared in Example 1;

[0040] Figure 2 The image shown is a photograph of recombinant collagen hydrogels with different amounts of recombinant collagen added.

[0041] Figure 3 The diagram shown is a schematic diagram of the cross-linked recombinant collagen hydrogel syringe in Example 2. The left diagram is a schematic diagram of the syringe loading hydrogel, and the right diagram is a schematic diagram of the hydrogel being injected.

[0042] Figure 4The following is the extrusion force test result of the cross-linked recombinant collagen hydrogel in Example 2, where A is a 2wt% recombinant collagen hydrogel, B is a 2.5wt% recombinant collagen hydrogel, and C is a 3.0wt% recombinant collagen hydrogel.

[0043] Figure 5 The image shown is a scanning electron microscope image of the recombinant collagen freeze-dried sponge in Example 1;

[0044] Figure 6 The image shown is a scanning electron microscope image of the cross-linked recombinant collagen hydrogel in Example 2;

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

[0046] Figure 8 The results shown are the relative cell proliferation rate test results of the cross-linked recombinant collagen hydrogel in Example 2. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0048] Example 1: Preparation and Characterization of Recombinant Collagen

[0049] 1.1 Preparation of recombinant collagen

[0050] Recombinant collagen was synthesized using the amino acid sequence of recombinant collagen shown in SEQ ID NO:1. The nucleotide sequence encoding the above amino acid sequence was synthesized and codon optimized to construct the pPIC9k expression plasmid. The accuracy of plasmid synthesis was confirmed by DNA sequencing. Subsequently, the plasmid was linearized and electroporated into Pichia pastoris strain GS115. After resistance selection, a stably expressing genetically engineered strain was obtained. The genetically engineered strain was fermented and cultured, and the recombinant collagen was obtained after purification and lyophilized for storage.

[0051] 1.2 Circular dichroism characterization of recombinant collagen

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

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

[0054] Example 2: Preparation and Characterization of TG Crosslinked Recombinant Collagen Gels with Different Recombinant Collagen Concentrations

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

[0056] The recombinant collagen lyophilized sponge prepared in 1.1 was dissolved in physiological saline to prepare solutions with recombinant collagen concentrations of 1wt%, 2wt%, 2.5wt%, 3wt%, 4wt%, and 5wt%, respectively. Then, 0.1wt% of the recombinant collagen solution mass of gamma-glutamyl transferase (TG) was added to each solution. After mixing, the mixture was filtered through a 0.22μm filter membrane for sterilization, crosslinked at 37℃ for 1h, and then incubated in a water bath at 80℃ for 30min to obtain TG crosslinked recombinant collagen hydrogel.

[0057] 2.2 Characterization of TG Crosslinked Recombinant Collagen Gel

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

[0059] like Figure 2 As shown, the 1 wt% recombinant collagen sample did not form a gel. Recombinant collagen samples at concentrations of 2 wt%, 2.5 wt%, 3 wt%, 4 wt%, and 5 wt% all formed gels, but the 4 wt% and 5 wt% concentrations were too firm and unsuitable for injection. Therefore, at a recombinant collagen concentration of 2%–3 wt%, injectable recombinant collagen hydrogels were prepared using gamma-glutamyl transferase (TG) as a cross-linking agent.

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

[0061] 3.1 Injectability testing

[0062] Take 2wt%, 2.5wt%, and 3wt% TG crosslinked recombinant collagen hydrogel samples prepared in Example 2, respectively, and place them in a 1mL syringe. Fix the syringe on the sample stage of the syringe plunger and perform an extrusion force test at an extrusion speed of 10mm / min (see [link to sample stage]). Figure 3 and Figure 4 ).

[0063] like Figure 4As shown, the maximum pushing force of the 2wt% TG cross-linked recombinant collagen hydrogel was 1.4 N, the minimum pushing force was 0.76 N, and the average pushing force was 0.82 N; the maximum pushing force of the 2.5wt% TG cross-linked recombinant collagen hydrogel was 1.9 N, the minimum pushing force was 0.87 N, and the average pushing force was 0.95 N; and the maximum pushing force of the 3wt% TG cross-linked recombinant collagen hydrogel was 2.2 N, the minimum pushing force was 1.26 N, and the average pushing force was 1.3 N. This indicates that the TG cross-linked recombinant collagen hydrogels possess excellent injection properties. Among them, the 3wt% TG cross-linked recombinant collagen hydrogel exhibited moderate pushing force and was 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, sputter-coated with gold, and characterize by SEM. Figure 5 As shown, recombinant collagen has a fibrous structure, similar to the morphology of collagen in the human body, and can play a supporting role.

[0066] The 3wt% cross-linked recombinant collagen hydrogel sample prepared in Example 2 was lyophilized. 0.5g of the lyophilized sample was sputter-coated with gold and characterized by SEM. Figure 6 As shown, the 3wt% TG cross-linked recombinant collagen hydrogel exhibits a uniformly distributed network structure that can support cells and provide favorable growth conditions for them.

[0067] 3.3 Viscosity Measurement

[0068] Take the 3wt% TG crosslinked recombinant collagen hydrogel prepared in Example 2 and the uncrosslinked 3wt% recombinant collagen sample, place them on the sample stage of the rheometer, set the rotation speed to 60 rpm, and test the viscosity of different samples.

[0069] Test results show that the viscosity of 3wt% TG cross-linked recombinant collagen hydrogel is 26299 mPa·s at a rotation speed of 60 rpm. The viscosity of uncross-linked 3wt% recombinant collagen at the same rotation speed is 6.6 mPa·s. This indicates that compared to uncross-linked recombinant collagen, TG cross-linked recombinant collagen hydrogel has significantly improved viscosity. This superior viscosity ensures that the TG cross-linked recombinant collagen hydrogel is less prone to displacement after implantation, making it more suitable for tissue filler products.

[0070] 3.4 Cell Proliferation Experiment

[0071] L929 cells in logarithmic growth phase were digested, counted, centrifuged, and then resuspended in DMEM complete medium to a cell density of 102. 5Cells / mL were mixed by pipetting and then the cell suspension was transferred to 96-well plates, 100 μL per well. The plates were incubated at 37°C in a 5% CO2 incubator. Samples of 3 wt% recombinant collagen solution (uncrosslinked group) and 3 wt% TG crosslinked recombinant collagen hydrogel prepared in Example 2 (Example 2 group) were placed in DMEM high-glucose medium and soaked at 37°C for 72 h to prepare extraction solutions. When the cell density in the 96-well plates was close to confluence, the original culture medium was aspirated and replaced with 3 wt% recombinant collagen solution and hydrogel extraction medium, respectively. The positive control group was supplemented with 0.64% (w / v) phenol solution, and the blank control group was supplemented with an equal volume of DMEM complete medium. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. The medium was then removed, and 100 μL of DMEM complete medium containing 10% CCK-8 solution was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 2 h. After incubation, the cells in each experimental group were photographed, and the absorbance at 450 nm (reference wavelength 630 nm) was measured using a microplate reader. The relative cell growth rate (RGR) for each group was calculated using the following formula. The cytotoxicity of the hydrogel was then assessed.

[0072] RGR (%) = Sample absorbance / Blank control absorbance × 100%

[0073] like Figure 7 As shown, the cells in the non-crosslinked group and the 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 crosslinked recombinant collagen hydrogel did not have significant cytotoxicity.

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

[0075] The above experimental results show that TG cross-linked recombinant collagen gel not only has no cytotoxicity, but can also significantly promote the proliferation of fibroblasts.

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

Claims

1. A recombinant collagen protein, 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 of claim 1 with gamma-glutamyl transaminase and cross-linking reaction, wherein the concentration of the recombinant collagen solution is 2.5wt%~3wt%.

3. The recombinant collagen hydrogel according to claim 2, characterized in that: The amount of glutamyl transaminase added is 0.05~0.5wt% of the recombinant collagen solution.

4. The recombinant collagen hydrogel according to claim 2, characterized in that: The crosslinking reaction is carried out at a temperature of 35-45°C, and / or The cross-linking reaction takes 0.2 to 24 hours.

5. The recombinant collagen hydrogel according to claim 2, characterized in that: The mixture of recombinant collagen and transglutaminase was filtered and sterilized before the cross-linking reaction.

6. The recombinant collagen hydrogel according to claim 5, characterized in that, The filter membrane used for filtration and sterilization has a pore size of 0.22 μm or 0.45 μm.

7. The recombinant collagen hydrogel according to claim 2, characterized in that: Uncrosslinked transglutaminase is inactivated after the crosslinking reaction.

8. The recombinant collagen hydrogel according to claim 7, characterized in that, The method for inactivating transglutaminase is to incubate it in a water bath at 60-90°C for 20-40 minutes.

9. A hydrogel implant, characterized in that: The hydrogel implant comprises the recombinant collagen hydrogel according to any one of claims 2-8.

10. The use of the recombinant collagen of claim 1 or the recombinant collagen hydrogel of any one of claims 2-8 in the preparation of medical devices.

11. The use of the recombinant collagen of claim 1 or the recombinant collagen hydrogel of any one of claims 2-8 in the preparation of hydrogel implants, artificial skin, organoids, hemostatic sponges, scaffold materials or cell culture media.

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