Preparation method of recombinant collagen dressing

Abstract

The invention provides a preparation method of a recombinant collagen dressing, and belongs to the technical field of medical dressings. According to the technical scheme, stacked short sequences and continuous human collagen amino acid short sequences are repeatedly combined for multiple times to form recombinant collagen with specificity, and high similarity of the recombinant collagen with human collagen is ensured. On the basis, 4-FPBA is used as a cross-linking agent to respectively form a Schiff base bond responsive to pH and a boric acid ester bond responsive to glucose with CMCS and two kinds of HA with different molecular weights, and then the double-dynamic covalent cross-linked network adhesive hydrogel is synthesized. The triple-helix structure of the recombinant collagen is combined with a specific receptor on a cell membrane, and cell behaviors are regulated and controlled by inducing signal transduction through a cytoskeleton, so that skin microcirculation can be effectively improved, and a skin barrier can be repaired; the hydrogel dressing matrix has the effects of resisting inflammation, resisting oxidation, resisting bacteria, promoting healing and the like, the biological performance of the dressing is remarkably improved, and a better curative effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical dressings, and particularly to a preparation method of a recombinant collagen dressing. Background Art

[0002] Medical dressings are a class of medical supplies used to cover and protect wounds and ulcers, and promote wound healing. They can be in direct contact with the wound, with diverse materials including natural materials, synthetic materials, or a combination of both, and come in various types in terms of design and function to meet the needs of different wound conditions. The functions of medical dressings include protecting the wound surface, absorbing exudates, promoting healing, relieving pain, and having a fixing effect, etc.

[0003] Medical dressings can form a physical barrier on the wound surface to prevent external pollutants such as bacteria, dust, and liquids from entering the wound and avoid wound infection. For example, using a suitable dressing at the surgical incision can effectively block pathogens in the external environment and create a clean environment for wound healing. During the wound healing process, exudates are produced, including blood, tissue fluid, etc. Medical dressings can absorb these exudates and keep the skin around the wound dry. Some dressings with high water absorption can effectively manage exudates for chronic wounds or large - area traumas with more exudation, preventing skin maceration and inflammation of the tissues around the wound. Some medical dressings contain special components such as growth factors, collagen, etc. These components can stimulate cell proliferation, promote granulation tissue growth, and accelerate the wound healing process. For example, dressings containing recombinant human epidermal growth factor have a positive promoting effect on the healing of burns, ulcers and other wounds. Certain dressings have the characteristics of soothing and relieving pain. They can relieve pain by maintaining a moist environment of the wound and reducing the stimulation of nerve endings. For example, the moist environment formed after the hydrocolloid dressing comes into contact with the wound can relieve the pain of patients to a certain extent, especially for wounds with obvious pain such as abrasions and superficial burns. Medical dressings can be used to fix drugs, instruments, etc. at the wound. For example, using a dressing to wrap outside the small splint for fracture fixation can not only protect the skin but also keep the splint in place, ensuring the fixation effect and being beneficial to the recovery of the fracture. After applying medicine to some wounds, the dressing can prevent the drug from shifting and ensure the continuous effect of the drug.

[0004] Collagen dressing is a material used for wound healing and skin repair. It is mainly composed of collagen, which is a structure with good ductility, tensile strength and strength. Collagen dressing utilizes the properties of collagen to adhere to the wound surface or skin, forming a protective layer to promote tissue regeneration and repair. The working principle of collagen dressing is based on the properties and active ingredients of collagen. Collagen can bind to the wound surface or skin and absorb moisture to promote tissue healing and repair. Collagen dressing can also continuously release the active ingredients of collagen to accelerate the wound healing and skin recovery process. By moisturizing, repairing and promoting regeneration. Recombinant human collagen is prepared by DNA recombination technology. Its amino acid sequence can be designed according to needs, and its gene sequence is highly consistent with the human gene sequence, with good tissue compatibility. This kind of collagen can be directly absorbed by the human body, participate in the construction of collagen, help repair damaged tissues and fill sunken parts. Using recombinant collagen to construct medical dressings is a very promising research direction. However, at present, due to the limitations of the performance of collagen, the curative effects of such medical dressings generally need to be improved. Summary of the Invention

[0005] The present invention aims to provide a preparation method of recombinant collagen dressing for the technical defects of the prior art, so as to solve the technical problem that the curative effect of conventional medical dressings needs to be improved.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solutions: A preparation method of recombinant collagen dressing, comprising: 1) Using stacked short sequences and human collagen short sequences as repeating units, repeating 8 times to form recombinant collagen; wherein, the stacked short sequence is composed of the G-X-Y type triple amino acid sequence in human collagen, and the human collagen is any one or more of type I, III, IV, V, VII and XVII human collagen; the human collagen short sequence is composed of the amino acid sequence of (G-X-Y)10 consecutive in the α1 chain of type III human collagen; wherein, G is a glycine residue, and X or Y represents any one amino acid residue; 2) Respectively take high and low molecular weight hyaluronic acid, mix them and dissolve them in PBS to obtain an HA solution, take carboxymethyl chitosan and dissolve it completely in PBS to obtain a CMCS solution, take 4-formylphenylboronic acid and dissolve it completely in absolute ethanol to obtain a 4-FPBA solution; successively mix the HA solution, 4-FPBA solution and CMCS solution in sequence, mix and stir to form a CFHh hydrogel, and freeze-dry to obtain a hydrogel freeze-dried powder; 3) Take carbomer, sodium propylparaben, and the recombinant collagen obtained in step 1), and soak them separately in purified water until dissolved; add the dissolved materials into a stirring container, then add menthol, disodium ethylenediaminetetraacetate, and the hydrogel lyophilized powder obtained in step 2), and stir; then add triethanolamine and stir the materials until they become gel-like to obtain the recombinant collagen dressing.

[0007] Preferably, in step 1), use the pPIC9K expression vector and Pichia pastoris to construct a recombinant Pichia pastoris genetic engineering bacterium; the genetic engineering bacterium is induced to express by methanol, and recombinant human collagen is secreted into the fermentation broth; the collected fermentation broth is separated and purified by chromatography and then lyophilized to obtain recombinant collagen.

[0008] Preferably, in step 1), for the recombinant collagen obtained by lyophilization, use an ultrafiltration column membrane with a cut-off molecular weight of 10 kDa to separate the supernatant, remove small molecular weight proteins and salts, and then purify the recombinant collagen by molecular sieve chromatography. Use pure water as the mobile phase and elute at a flow rate of 0.5 mL / min. Collect the samples in the elution peak and lyophilize to obtain the finished product.

[0009] Preferably, in step 2), the molecular weight range of low molecular weight hyaluronic acid is 2000 - 5000.

[0010] Preferably, in step 2), the molecular weight range of high molecular weight hyaluronic acid is 100,000 - 1,500,000.

[0011] Preferably, in step 2), the concentration of the CMCS solution is 5 wt%; the concentration of the 4-FPBA solution is 3 wt%; the concentration of the HA solution is 8 wt%, and the concentration of high and low molecular weight HA is 4 wt% each; when mixing, the volume ratio of the HA solution, 4-FPBA solution, and CMCS solution is 2:0.5:2.

[0012] Preferably, in step 2), the rotation speed of the mixing and stirring is 300 - 500 rpm, the temperature of the mixing and stirring is 45 - 55 °C, and during the mixing and stirring process, the system is continuously in an ultrasonic oscillation environment.

[0013] Preferably, in step 3), the mass ratio of carbomer, sodium propylparaben, recombinant collagen, menthol, disodium ethylenediaminetetraacetate, hydrogel lyophilized powder, and triethanolamine is 25 - 30:8 - 11:15 - 17:2 - 4:6 - 10:10 - 12:2 - 3.

[0014] Preferably, in step 3), the temperature of the purified water soaking is 50 - 60 °C, and the soaking time is not less than 1 h.

[0015] Preferably, in step 3), the stirring duration in the first stage is 2 to 3 h, and the stirring duration in the second stage is 30 to 60 min.

[0016] The present invention provides a preparation method of a recombinant collagen dressing. This technical solution combines a stacked short sequence with a continuous short sequence of human collagen amino acids repeated multiple times to form a specific recombinant collagen, ensuring its high similarity to human collagen. On this basis, using 4-FPBA as a crosslinking agent, Schiff base bonds responsive to pH and borate ester bonds responsive to glucose are respectively formed with CMCS and two different molecular weight HAs, and then a dual-dynamic covalent crosslinked network adhesive hydrogel is synthesized. Based on the recombinant collagen and hydrogel constructed above, the present invention prepares a medical dressing. The triple helix structure of the recombinant collagen binds to specific receptors on the cell membrane, and regulates cell behavior by inducing signal transduction through the cytoskeleton, which can effectively improve skin microcirculation and repair the skin barrier; the hydrogel dressing matrix has anti-inflammatory, antioxidant, antibacterial, and wound healing-promoting effects, significantly improving the biological properties of the dressing, contributing to promoting wound healing, and having better curative effects. Detailed implementation manners

[0017] The following will describe the detailed implementation manners of the present invention in detail. To avoid excessive unnecessary details, the well-known structures or functions will not be described in detail in the following embodiments. The approximate language used in the following embodiments can be used for quantitative expression, indicating that a certain change in quantity is allowed without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0018] A preparation method of a recombinant collagen dressing, comprising: 1) Using a stacked short sequence and a short sequence of human collagen as repeating units, repeating 8 times to form a recombinant collagen; wherein, the stacked short sequence is composed of a G-X-Y type triple amino acid sequence in human collagen, and the human collagen is any one or more of type I, III, IV, V, VII, and XVII human collagen; the short sequence of human collagen is composed of a continuous (G-X-Y)10 amino acid sequence in the α1 chain of type III human collagen; wherein, G is a glycine residue, and X or Y represents any amino acid residue; 2) Respectively take high- and low-molecular-weight hyaluronic acids, mix them, and dissolve in PBS to obtain an HA solution. Take carboxymethyl chitosan and completely dissolve it in PBS to obtain a CMCS solution. Take 4-formylphenylboronic acid and completely dissolve it in absolute ethanol to obtain a 4-FPBA solution. Sequentially mix and stir the HA solution, 4-FPBA solution, and CMCS solution in this order to form a CFHh hydrogel, and obtain a freeze-dried powder of the hydrogel after freeze-drying. 3) Take carbomer, sodium propylparaben, and the recombinant collagen obtained in step 1), and soak them in purified water until dissolved. Add the dissolved materials to a stirring container, and then add menthol, disodium ethylenediaminetetraacetate, and the freeze-dried powder of the hydrogel obtained in step 2) thereto, and stir. Then add triethanolamine and stir the materials until they become gel-like to obtain the recombinant collagen dressing.

[0019] In step 1), use the pPIC9K expression vector and Pichia pastoris to construct a recombinant Pichia pastoris genetic engineering bacterium. The genetic engineering bacterium is induced to express by methanol, and recombinant human collagen is secreted into the fermentation broth. The collected fermentation broth is subjected to chromatographic separation and purification, and after freeze-drying, recombinant collagen is obtained. In step 1), for the recombinant collagen obtained by freeze-drying, use an ultrafiltration column membrane with a cut-off molecular weight of 10 kDa to separate the supernatant, remove small-molecular-weight proteins and salts, and then purify the recombinant collagen by molecular sieve chromatography. Use pure water as the mobile phase and elute at a flow rate of 0.5 mL / min. Collect the samples in the elution peak and freeze-dry to obtain the finished product.

[0020] In step 2), the molecular weight range of the low-molecular-weight hyaluronic acid is 2000 - 5000. In step 2), the molecular weight range of the high-molecular-weight hyaluronic acid is 100,000 - 1,500,000. In step 2), the concentration of the CMCS solution is 5 wt%. The concentration of the 4-FPBA solution is 3 wt%. The concentration of the HA solution is 8 wt%, and the concentrations of the high- and low-molecular-weight HAs are both 4 wt%. When mixing, the volume ratio of the HA solution, 4-FPBA solution, and CMCS solution is 2:0.5:2. In step 2), the rotation speed of the mixing and stirring is 300 - 500 rpm, and the temperature of the mixing and stirring is 45 - 55 °C. During the mixing and stirring process, the system is continuously in an ultrasonic oscillation environment.

[0021] In step 3), the mass ratio of carbomer, sodium propylparaben, recombinant collagen, menthol, disodium ethylenediaminetetraacetate, freeze-dried powder of the hydrogel, and triethanolamine is 27:10:16:3:8:11:2.5. In step 3), the temperature of the soaking in purified water is 55 °C, and the soaking duration is 2 h. The duration of the first-stage stirring in step 3) is 2.5 h, and the duration of the second-stage stirring is 50 min.

[0022] Using a commercially available collagen dressing as a comparative example, the biological properties and efficacy of the recombinant collagen dressing of the present invention (i.e., the above examples) and the comparative example were evaluated as follows.

[0023] Macrophages (RAW264.7) were cultured in high-glucose medium containing 25 mM glucose and 10% fetal bovine serum with 100 ng / mL LPS for 24 h. RAW264.7 was seeded in 24-well plates and divided into 5 groups. Equal amounts of the dressings of the examples and the comparative example were added to the RAW264.7 medium, with 3 replicate wells in each group. The expression of CD86 and CD206 molecules was detected by cell immunofluorescence. The experimental results showed that only the dressing of the example had a significant effect of promoting the polarization of M1 macrophages into M2 macrophages.

[0024] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were respectively spread on agar plates at 1×10 7 CFU / mL. The dressings of the examples and the comparative example (sterile, 10 mm in diameter and 500 μm in thickness) were carefully attached to the agar plates evenly coated with bacteria and cultured at 37 °C for 24 h. The experimental results showed that only the example had obvious antibacterial properties against E. coli, and the diameter of the inhibition zone was significantly larger than that of the comparative example; similarly, it also had obvious antibacterial properties against S. aureus.

[0025] A clean glass slide (3 cm long and 1 cm wide) was attached to the tensile testing machine platform with double-sided tape. Different volumes of ultrapure water were evenly distributed on the glass slide. The dressing in the example (5 cm long, 1 cm wide and 500 μm thick) was pressed on the glass substrate coated with water with a pre-pressure of 5 N, and then the 90° peeling method was used to investigate the adhesion strength of the dressing in the example on surfaces with different water contents. The experimental results showed that when the water content of the substrate was 15 μL, the dressing had a relatively high peeling strength, about 1.5 N / cm. As the water content of the substrate increased, the peeling strength of the dressing gradually decreased, indicating that the dressing had a non-irritating and controllable adhesion effect on demand.

[0026] C57 mice were anesthetized with 1 wt% sodium pentobarbital, and a full-thickness skin defect with a diameter of 10 mm was cut out on the back of the mice using ophthalmic scissors. The dressing of the example was taken, and the middle part was attached to the wound surface. After standing for 30 s, the dressing was gradually lifted, and the mice were not fixed otherwise. The height of the mice being lifted was observed. Then, a large amount of physiological saline was dropped on the wound surface, and the dressing peeling process was observed. The experimental results showed that the dressing could be easily changed by spraying physiological saline on the wound surface without damaging the wound and the surrounding tissues, reducing the secondary injury during dressing change.

[0027] Thirty 7-week-old female diabetic mice were randomly divided into 5 groups. After anesthesia with 3% sodium pentobarbital by mass concentration, two full-thickness skin defect wounds with a diameter of 10 mm were constructed on the backs of the mice. Then, 100 μL of Staphylococcus aureus bacterial solution with a density of 1×10 7 CFU / mL was dropped on the wounds to create an infectious diabetic wound model. After that, the dressings of the examples and comparative examples were respectively covered on the wounds, and the mice were raised in the same environment. The wound healing rate was statistically analyzed at 0 / 3 / 7 / 10 / 14 days. And at 3 and 7 days after injury of the mice, wound tissues were taken, ground into tissue homogenates, and the number of bacteria present in the wounds was analyzed by the agarose plate method. The experimental results showed that the wound healing rate of the infectious diabetic mice with the example was the fastest, indicating that the dressing of the example had a significant effect on promoting the healing of diabetic wounds.

[0028] Fifty healthy male SD rats weighing 180 - 220 g were selected. After 1 week of adaptive feeding, they were randomly divided into 5 groups, namely the blank group, the model group, the positive group, the example group, and the comparative example group, with 10 rats in each group. Except for the blank group, the hair on the backs of the rats in the other groups was shaved off, and a square piece of skin with a side length of 1.5 cm was cut off with a scalpel, and a wound with a length of 1.5 cm and reaching the muscle layer was cut at the wound. In the positive group, mupirocin ointment was applied on a common wound dressing and pasted on the wound; in the example dressing group, the dressing prepared in the example was pasted on the wound; in the comparative example group, the comparative example dressing was pasted on the wound; the model group was not treated. The dressings of each group were changed once a day for 3 consecutive days. After the administration was completed, the wounds of the rats in each group were observed and the mortality rate was statistically analyzed, and the peripheral blood picture of the rats was detected. At the same time, the levels of inflammatory factors IL-6 and TNF-α in the serum were detected by the Elisa method.

[0029] The experimental results showed that the wounds of the rats in the model group suppurated and the infection deepened, secondary infection occurred at the wound, and the animal mortality rate was 40%. The wounds of the rats in the positive group began to heal, there was no secondary infection at the wound, and no animals died. The wounds of the rats in the example dressing group began to heal, there was no secondary infection at the wound, and no animals died. The wounds of the rats in the comparative example dressing group healed slowly, secondary infection occurred at the wound, and the animal mortality rate was 20%.

[0030] The results of blood routine tests showed that in the dressing group of the examples, the WBC and PLT increased, the HGB content increased significantly (P < 0.01), the RBC decreased significantly (P < 0.01), and the HGB content increased significantly (P < 0.05); there were no significant differences in each index of the dressing group of the comparative examples. The results of the detection of inflammatory factor levels showed that compared with the model group, the IL-6 level and TNF-α level in the positive group and the dressing group of the examples decreased significantly (P < 0.01), while the IL-6 and TNF-α levels in the dressing group of the comparative examples decreased but there were no significant differences (P > 0.05). Compared with the dressing group of the comparative examples, the TNF-α level in the dressing group of the examples decreased significantly (P < 0.05).

[0031] The above experimental results indicate that the recombinant collagen dressing of the present invention has excellent antibacterial and immunomodulatory effects, can significantly accelerate wound healing, and has good safety.

[0032] The embodiments of the present invention have been described in detail above, but the above content is only the preferred embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the scope of the application of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a recombinant collagen dressing, characterized in that: include: 1) The stacked short sequence and the human collagen short sequence are used as repeating units, which are repeated 8 times to form a recombinant collagen; wherein the stacked short sequence is composed of a GXY type triplet amino acid sequence in human collagen, and the human collagen is any one or more of type I, type III, type IV, type V, type VII and type XVII human collagen; the human collagen short sequence is composed of a continuous (GXY)10 amino acid sequence in the α1 chain of type III human collagen; wherein G is a glycine residue, and X or Y represents any amino acid residue; 2) High and low molecular weight hyaluronic acid are mixed and then dissolved in PBS to obtain HA solution, carboxymethyl chitosan is completely dissolved in PBS to obtain CMCS solution, and 4-formaldehyde phenylboronic acid is completely dissolved in anhydrous ethanol to obtain 4-FPBA solution; the HA solution, 4-FPBA solution, and CMCS solution are mixed and stirred in sequence to obtain CFHh hydrogel, and freeze-dried to obtain hydrogel freeze-dried powder; 3) Carbomer, sodium propylparaben and the recombinant collagen obtained in step 1) are soaked in purified water respectively until dissolved; the dissolved material is added to a stirring container, and menthol, disodium ethylenediaminetetraacetic acid and the hydrogel freeze-dried powder obtained in step 2) are added thereto, and stirred; and triethanolamine is then added to stir the material until it is in a gel state, thereby obtaining the recombinant collagen dressing.

2. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 1), pPIC9K expression vector and Pichia pastoris are used to construct recombinant Pichia pastoris genetically engineered bacteria; the genetically engineered bacteria are induced to express by methanol, and recombinant human collagen is secreted into the fermentation broth; the collected fermentation broth is separated and purified by chromatography, and then freeze-dried to obtain recombinant collagen.

3. The method for preparing a recombinant collagen dressing according to claim 2, characterized in that: In step 1), for the recombinant collagen obtained by freeze-drying, the supernatant is separated by an ultrafiltration column membrane with a molecular weight cutoff of 10 kDa to remove small molecular weight proteins and salts, and then the recombinant collagen is purified by molecular sieve chromatography, using pure water as the mobile phase and eluting at a flow rate of 0.5 mL / min, collecting samples in the elution peak, and freeze-drying to obtain the finished product.

4. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 2), the molecular weight of the low molecular weight hyaluronic acid ranges from 2000 to 5000.

5. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 2), the molecular weight of the high molecular weight hyaluronic acid ranges from 100,000 to 1.5 million.

6. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 2), the concentration of the CMCS solution is 5wt%; the concentration of the 4-FPBA solution is 3wt%; the concentration of the HA solution is 8wt%, wherein the concentrations of the high and low molecular weight HA are both 4wt%; when mixed, the volume ratio of the HA solution, the 4-FPBA solution, and the CMCS solution is 2:0.5:

2.

7. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 2), the rotation speed of the mixing and stirring is 300-500 rpm, the temperature of the mixing and stirring is 45-55° C., and during the mixing and stirring process, the system is continuously in an ultrasonic oscillation environment.

8. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 3), the mass ratio of carbomer, sodium propylparaben, recombinant collagen, menthol, disodium edetate, hydrogel freeze-dried powder, and triethanolamine is 25-30:8-11:15-17:2-4:6-10:10-12:2-3.

9. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 3), the temperature of the purified water immersion is 50-60° C., and the immersion time is not less than 1 hour.

10. The method for preparing a recombinant collagen dressing according to claim 1, characterized in that: In step 3), the first stirring period is 2 to 3 hours, and the second stirring period is 30 to 60 minutes.