High-stability recombinant collagen gel and preparation method thereof

Abstract

The invention discloses a high-stability recombinant collagen gel and a preparation method thereof. The recombinant collagen gel is prepared from the following components in percentage by weight: 1-50% of recombinant I-type collagen, 1-20% of polyethylene glycol or a derivative thereof and the balance of a gel matrix material. Recombinant I-type collagen which is modified by a genetic engineering technology and has temperature-sensitive characteristic and high thermal stability is preferably selected as a raw material, polyethylene glycol and a derivative thereof are screened from multiple substances and matched with the recombinant I-type collagen, and the recombinant I-type collagen can be self-assembled to form micelles. Meanwhile, better thermodynamic properties, degradation resistance and biological properties are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and more particularly, to a highly stable recombinant collagen gel and a preparation method thereof. Background Art

[0002] In daily life or battlefield environments, non-self-healing wounds frequently occur, including but not limited to accidental injuries, combat injuries, burns, scalds, and chronic ulcers. Common wound care methods include gauze dressing after debridement or using medical dressings to adhere to the wound. Among them, collagen dressings have received extensive attention due to their excellent biocompatibility, tissue repair promotion ability, and anti-infection performance. However, conventional collagen dressings have poor mechanical properties and are easily degraded in body surface wounds and in vitro high-temperature environments, resulting in problems such as accelerated dressing replacement frequency and inconvenient use that are difficult to solve. Therefore, through technologies such as polymer materials and high-efficiency preparations, to maximize the efficacy of collagen, the selection of the dressing formula is crucial.

[0003] Recombinant collagen is currently widely used in the medical field. It can overcome the inherent xenogeneic rejection and virus-carrying risks of animal-derived proteins, has good biocompatibility, can be used as a scaffold material for the repair of various cells and tissues, has good biosafety, and can be absorbed by the human body. Depending on the solvent and environment, it can self-assemble to form uniform, thermodynamically and mechanically stable collagen micelles.

[0004] Polyethylene glycol and its derivatives, as a synthetic polymer material with excellent biocompatibility, have characteristics such as colorless, odorless, low toxicity, low immunogenicity, and biocompatibility. Combining click chemistry and functional group modification can connect a variety of functional molecules, and it can be widely used in aspects such as biomaterials, effectively improving the physical and chemical stability and biocompatibility of recombinant proteins. Currently, common collagen dressing products generally have disadvantages such as the need for low-temperature cold chain preservation and being easily degraded in body surface wounds and in vitro high-temperature environments, with a high replacement frequency and being difficult to be applied in harsh environments such as the battlefield. Therefore, it is of great significance to develop a recombinant collagen dressing that can maintain the stability of protein properties, is convenient to use, and has excellent therapeutic effects in harsh environments such as the battlefield. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant collagen dressing that can maintain the biological properties of recombinant proteins in harsh environments such as the battlefield. The present invention prepares recombinant collagen micelles based on a self-assembly strategy, and specifically selects protectants and optimizes the ratio of polyethylene glycol and its derivatives to combine with recombinant collagen. The recombinant collagen dressing provided by the present invention can maintain the stability of recombinant collagen properties and is convenient to use in hot environments, and can effectively improve the quality of wound healing, shorten the healing time, and improve the quality of life of patients after healing.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A recombinant collagen gel is made from the following components in weight percentages:

[0008] Recombinant type I collagen 1-50%, polyethylene glycol or its derivative 1-20%, and the balance is a gel matrix material.

[0009] Preferably, in the above recombinant collagen gel, the gel matrix material is made from the following components in weight percentages:

[0010] Sodium carboxymethylcellulose 1-20%, sucrose 1-5%, glycerol 1-5%, carbomer 0.1-10%, and the balance is ultrapure water.

[0011] Preferably, in the above recombinant collagen gel, the polyethylene glycol or its derivative is one or a mixture of PEG400, tetra-arm polyethylene glycol azide, and tetra-arm monomethoxy ether polyethylene glycol, and the weight-average molecular weight of the polyethylene glycol is 400-20000.

[0012] Preferably, in the above recombinant collagen gel, the recombinant type I collagen is the recombinant type I collagen described in the Chinese invention patent with the application number 202310670144.7.

[0013] A preparation method of a recombinant collagen gel includes the following steps:

[0014] Weigh each component according to the ratio, disperse it in distilled water, homogenize and stir, prepare it by the direct dissolution method or the emulsification method, seal it, and sterilize it by irradiation to obtain the gel product.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The present invention preferably uses recombinant type I collagen, which is genetically engineered, has thermosensitive properties and strong thermal stability, as a raw material. At the same time, polyethylene glycol and its derivatives are screened from many substances to be matched with recombinant type I collagen, which can form micelles by self-assembly of recombinant type I collagen, and has better thermodynamic properties, degradation resistance and biological properties.

[0017] (2) By preferably selecting the ratio and composition of different gel matrices, the present invention can effectively increase the thermal stability and radiation resistance of recombinant collagen, and preferably maintain its protein content, purity and activity, which will have very important significance in the field of medical materials.

[0018] (3) The preparation process of the recombinant collagen gel prepared by the present invention is simple and can be industrialized.

[0019] (4) The recombinant collagen gel prepared by the present invention is beneficial to the adhesion and migration of fibroblasts, thereby promoting the synthesis and secretion of collagen by wound fibroblasts and facilitating wound healing. At the same time, recombinant collagen is also beneficial to the adhesion and migration of keratinocytes and conducive to the regeneration of the skin barrier. Proven by in vitro and in vivo clinical trials, this recombinant collagen gel has good in vitro and in vivo biological safety, effectively improves the healing quality of burn and scald wounds in rats and the wounds of the patient's skin donor area, greatly shortens the wound healing time, and improves the quality of life of patients after healing, which has important clinical and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Scanning electron microscope data of the recombinant collagen gel in Example 1;

[0021] Figure 2 Normal temperature stability data of the recombinant collagen gel;

[0022] Figure 3 Stability data of the recombinant collagen gel after irradiation and accelerated aging for 5 days;

[0023] Figure 4 Record cases of the wound healing time of the clinical control group;

[0024] Figure 5 Record cases of the wound healing time of the clinical experimental group. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will be further described below in conjunction with the embodiments. The following embodiments are illustrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0026] The formula components of each embodiment and comparative example are shown in Table 1.

[0027] The preparation method is as follows:

[0028] (1) Preparation of the gel matrix material: Each component is made into the following components by weight percentage: sodium carboxymethylcellulose 10%, sucrose 2%, glycerol 3%, carbomer 2%, and ultrapure water 83%. Weigh according to the product material ratio, add the weighed carbomer and glycerol to the mixing tank of the vacuum mixer, heat for 40 minutes, turn on homogenization and stirring, and homogenize to obtain a transparent and uniform liquid. Slowly add sodium carboxymethylcellulose to the mixing tank, keep heating and stirring for dispersion for 10 minutes. Then stop stirring and heating, cool with circulating water to 25 ± 5 °C, and stand still for more than 3 hours to allow sodium carboxymethylcellulose to fully swell. Subsequently, add sucrose, add sodium hydroxide to adjust the pH, turn on homogenization and stirring, and homogenize to obtain a transparent and uniform gel matrix.

[0029] (2) Preparation of recombinant collagen gel: Add polyethylene glycol and its derivatives and recombinant type I collagen into a clean container, adjust the pH value to 8, control the temperature at 37°C, stir for 2 h for self-assembly to form collagen micelles, and then add them to a stirring tank. Carry out homogenizing stirring at a constant speed for 40 min to obtain a uniform recombinant collagen gel. The rotation speed of the gel matrix during homogenizing stirring is 1500 r / min, and the heating temperature is 90°C.

[0030] The preparation methods of Comparative Examples 1-3 are as follows: Add sodium hyaluronate and collagen into a clean container, adjust the pH value to 8, control the temperature at 37°C, stir, and homogenize.

[0031] Table 1 shows the formula components of each example and comparative example.

[0032]

[0033] After gradient cooling at 4°C, -20°C, and -80°C, remove the moisture by freeze-drying with a vacuum freeze-dryer for 24-72 h. Subsequently, observe the morphology of the sample of Example 1 under a scanning electron microscope and take pictures for recording. Figure 1 It is the scanning electron micrograph of the recombinant collagen gel. Figure 1 The results show that the present invention successfully prepares recombinant collagen micelles by self-assembly means and uniformly disperses them into the recombinant collagen dressing product.

[0034] Stability test: Place the recombinant collagen gel dressings of Examples 1-7 at room temperature (25°C) for 5 months for the stability test, and detect the protein stability after being placed at room temperature for 5 months by SDS-PAGE. The results are as Figure 2 shown. By adjusting the synergistic ratio of recombinant type I collagen and polyethylene glycol derivatives in Examples 1-7, the thermal stability of the protein is significantly improved. The SDS-PAGE results show that Example 5 has the best effect, with darker protein band color and less degradation, fully proving the scientificity and effectiveness of the formula.

[0035] Place the irradiated and sterilized recombinant collagen gel dressings of Examples 1-7 in an accelerated aging environment (50°C - 60°C) for 5 days for the accelerated aging test. Detect the protein stability after 5 days of accelerated aging by SDS-PAGE. The results are as Figure 3As shown. The results show that after the accelerated aging test, there are no flocculants or obvious appearance changes in each example observed by the naked eye. The SDS-PAGE results show that the protein stability of Examples 1-7 is significantly better than that of Comparative Example 1. Among them, Example 5 has the best effect. Without being limited to any specific theory, these properties may be due to polyethylene glycol and its derivatives with different concentrations, different molecular weights and different branched chain structures forming numerous intermolecular hydrogen bonds with a part of the amino and carboxyl groups of proteins in the aqueous phase. In addition, as an ampholyte, collagen also has a strong electrostatic interaction with negatively charged carboxymethyl cellulose, coupled with the interaction of hydrogen bonds, van der Waals forces and other networks throughout the network. Therefore, the above preparations effectively improve the thermal stability of recombinant collagen and form a stable complex.

[0036] In vitro cytotoxicity experiment:

[0037] According to the standard requirements of GBT16886.5-2017 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test", the non-specific cytotoxicity generated after the contact of the sample extract with cells was evaluated. The specific operation is as follows, and the experimental results are shown in Table 2.

[0038] L929 cells in the exponential growth phase were configured into a cell suspension with a concentration of 1x10 5 cells / mL with growth medium. The cell suspension was inoculated into a 96-well plate, 100 μL per well. All culture plates were placed in an environment containing 5% volume fraction of carbon dioxide at 37 °C and cultured for 24 h until the cells grew to a confluent state of 70%-80%. Take out the 96-well plate with growing cells, add 100 μL of the test sample and control sample of the recombinant collagen dressing respectively, repeat 5 wells in parallel, and continue to culture at 37 °C in an environment containing 5% volume fraction of carbon dioxide for 24 h. Then discard the original medium, add 50 μL of MTT solution with a concentration of 1 mg / mL to each well, and incubate at 37 °C for 2 h. Take out the 96-well plate, discard the MTT solution, add 100 μL of isopropanol to each well, and mix well on a shaker for 10 min. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value of each well at a detection wavelength of 570 nm. Calculate the cell survival rate % of each group according to: (OD value of the experimental group - OD value of the control group) / OD value of the control group × 100%.

[0039] The results in Table 2 show that all examples have no cytotoxicity, proving that the recombinant collagen dressing prepared by the present invention has good cell compatibility.

[0040] Table 2 is the result of the in vitro cytotoxicity test

[0041]

[0042] Skin sensitization experiment:

[0043] According to the standard requirements of GB / T 16886.10-2017 "Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization", the potential risk of the sample causing skin sensitization reaction in guinea pigs under test conditions was evaluated. The specific operations are as follows, and the experimental results are shown in Table 3.

[0044] 1) Local induction: On the (7±1)th day after intradermal induction, a sterile gauze with an area of 8 cm² was soaked with the extract and locally applied to the inner side of the scapula of each guinea pig, covering the induction injection site. It was fixed with a closed dressing band, and the dressing band and gauze block were removed after (48±2) h. If no irritation reaction occurred after intradermal induction, 10% sodium dodecyl sulfate was used for pretreatment of the test area (24±2) h before local induction, and it was massaged and introduced into the skin. The control group animals were operated in the same way with the blank extraction medium.

[0045] 2) Challenge: (14±1) days after local induction, absorbent gauzes were soaked in the extract of the recombinant collagen dressing test sample and the control solution respectively, and were applied to the depilated areas on the ventral and dorsal parts of each animal (the areas not tested in the induction stage). It was fixed with a closed dressing band, and the dressing band and the patch were removed after (24±2) h.

[0046] 3) Observation: The skin reactions at the challenge sites of the animals in the recombinant collagen dressing test group and the control group were observed (24±2) h and (48±2) h after removing the patch. The skin erythema and edema reactions at each challenge site and each observation time were described and graded according to the Magnusson and Kligman grading criteria.

[0047] The results in Table 3 show that no skin sensitization reaction exists in all examples, which proves that the recombinant collagen dressing prepared by the present invention has good in vivo safety.

[0048] Table 3 is the result of the skin sensitization test

[0049]

[0050] Intradermal reaction and pyrogen test:

[0051] For the intradermal reaction test, according to the standard requirements of GBTI6886.10-2017 "Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization", the potential risk of the sample's polar and non-polar extracts causing intradermal reactions in rabbits under test conditions was evaluated. For the pyrogen test, according to the standard requirements of Section 1142, Part IV of the Chinese Pharmacopoeia 2020 Edition, the potential risk of the sample extract causing pyrogenic effects in rabbits under test conditions was evaluated. The specific operations are as follows, and the experimental results are shown in Table 4.

[0052] The injection dose for rabbits with normal body temperature is 10 mL / kg. Before injection, disinfect the marginal ear vein of the rabbit with 75% ethanol, and slowly inject the test article of the recombinant collagen dressing at the specified dose and warmed to about 38°C from the marginal ear vein. After injection, measure the body temperature once every 30 minutes for a total of 6 times.

[0053] The results in Table 4 show that there are no abnormal reactions in all examples, proving that the recombinant collagen dressing prepared by the present invention has good in vivo safety.

[0054] Table 4 shows the results of intradermal reaction and pyrogen test

[0055]

[0056] Wound repair indicators:

[0057] 1) Burn and scald healing experiment: In this Example 5, the wound healing promotion effect and immunogenicity of the recombinant collagen dressing were demonstrated through a circular abrasion and scald model of the back skin of rats (purchased from Guangdong Medical Experimental Animal Center). The specific operation is as follows, and the experimental results are shown in Table 5.

[0058] After debriding the back skin of the rats, four circular abrasion and scald wounds with a diameter of 12 mm were formed by applying mechanical friction and scalding at 65 - 70°C for 10 - 15 s, and drugs were administered respectively. Regularly measure the changes in the wounds, as well as the subsidence of scabbing, redness and swelling.

[0059] 2) Clinical trial: Wound repair of the donor site

[0060] The recombinant collagen gel of Example 5 of the present invention, as shown by the results of in vitro tests, animal experiments and clinical trials, has excellent in vitro thermal stability, biocompatibility and wound healing promotion effect, and does not show immunogenicity.

[0061] This example lists the results of wound repair on the back of rats. The HE and MASSON staining results show that the control group showed more inflammatory cell infiltration, a more obvious bleeding center, and the connective tissue was looser than other groups. In addition, compared with the control group, the recombinant collagen dressing group had more new collagen deposition, and the regenerated epithelial tissue was thicker and more. These results indicate that the recombinant collagen gel of Example 5 helps to promote wound healing. Clinical trials such as Figure 4 and Figure 5 show that the experimental results indicate that compared with the control group (Urgotul, LABORATOIRES URGO) using only imported dressings, the recombinant collagen gel dressing of Example 5 can significantly shorten the healing time of the donor site wound while increasing the generation of new granulation.

[0062] Table 5 shows the results of the rat burn and scald wound healing test

[0063] Wound repair index The recombinant collagen dressing of the present invention Control of commercially available dressings Healing time 14 ± 2 days 28 ± 2 days Wound healing rate 95±2.4％ 78±3.1％

Claims

1. A recombinant collagen gel, characterized in that Made of the following components in weight percentage: 1-50% recombinant type I collagen, 1-20% polyethylene glycol or its derivatives, and the remainder is gel matrix material.

2. The recombinant collagen gel according to claim 1, characterized in that The gel matrix material is made of the following components in weight percentage: Sodium carboxymethylcellulose 1-20%, sucrose 1-5%, glycerol 1-5%, carbomer 0.1-10%, and the balance is ultrapure water.

3. The recombinant collagen gel according to claim 1, characterized in that The polyethylene glycol or its derivative is one of PEG400, four-arm polyethylene glycol azide, four-arm monomethoxy ether polyethylene glycol or a mixture thereof, and the weight average molecular weight of the polyethylene glycol is 400-20000.

4. The recombinant collagen gel according to claim 1, characterized in that The recombinant type I collagen is the recombinant type I collagen recorded in the Chinese invention patent with application number 202310670144.

7.

5. A method for preparing the recombinant collagen gel according to claim 1, characterized in that: The steps include: The components are weighed according to the proportion, dispersed in distilled water, homogenized and stirred, prepared by direct dissolution method or emulsification method, sealed and sterilized by irradiation to obtain the gel product.

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