CN116173291A - Chitosan recombinant humanized collagen gel and preparation method and application thereof - Google Patents
Chitosan recombinant humanized collagen gel and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0033—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0023—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/009—Materials resorbable by the body
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides chitosan recombinant humanized collagen gel, which comprises the following components in percentage by mass: III, 0.08 to 0.30 percent of recombinant humanized collagen; chitosan or its derivative 0.20-0.45%; 0.05 to 0.13 percent of cross-linking agent; 0.10-0.20% of humectant; the balance being water. The dressing provided by the invention can slowly release the humanized collagen, is nontoxic and nonirritating, has high biocompatibility and good stability, and can continuously promote the healing of the uterine incision.
Description
Technical Field
The invention relates to the technical field of dressing, in particular to chitosan recombinant humanized collagen gel and a preparation method and application thereof.
Background
Collagen is an important component of tissue, and type I, III collagen not only maintains the elastic function of tissue together with elastic fibers, but also provides effective tensile strength. When the wound surface injury is repaired, collagen fibers are deposited and participate in reconstructing the wound surface. Many studies have shown that recombinant humanized collagen has a promoting effect on wound healing.
The recombinant human source III type collagen (Rhc-III) is obtained by using a genetic engineering technology and using the original gene sequence of human collagen III to carry out optimized recombinant expression, and is highly consistent with the amino acid sequence of natural collagen of human body. Rhc-III is used for wound healing, on one hand, rhc-III has rich hydrophilic groups, can increase the adhesion and proliferation of fibroblasts, quicken the synthesis and deposition speed of collagen, and shortens the time of wound healing. On the other hand, the Rhc-III on the wound surface can be directly supplemented, and the occurrence probability of scars is reduced. The recombinant humanized collagen has lower immunogenicity and higher safety, realizes large-scale production along with the technical development, maintains higher biological activity, and has better performance than natural collagen. However, the water-soluble Rhc-III has the problems of easy degradation and short acting time.
Uterine incision restoration is a lengthy procedure, but there are few methods to continue to promote healing of the uterine incision and restore tissue function at the uterine incision. There is currently no collagen gel for uterine incision healing. Other common collagen gel is mostly used for epidermis injury, has lighter and thinner texture and strong irritation, can not be degraded in vivo, is suitable for acute wound recovery, and is not suitable for recovery of chronic wounds such as uterine incisions.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides chitosan recombinant humanized collagen gel and a preparation method and application thereof, and solves the problems that the collagen gel in the prior art is strong in irritation, can not be degraded in vivo and is easy to degrade.
In one aspect of the invention, a chitosan recombinant humanized collagen gel is provided, which comprises the following components in percentage by mass:
further, the chitosan derivative is carboxymethyl chitosan.
Further, the cross-linking agent is genipin.
Further, the humectant is xylitol.
In another aspect of the present invention, a method for preparing a chitosan recombinant humanized collagen gel is provided, which is characterized in that: the method comprises the following steps:
s1: adding III type recombinant humanized collagen into water for first stirring and dissolving to obtain solution 1;
s2: adding the chitosan derivative into water for second stirring and dissolving to obtain a solution 2;
s3: adding a cross-linking agent into the solution 2, and carrying out third stirring and dissolving to obtain a solution 3;
s4: adding the solution 1 into the solution 3, and stirring and uniformly mixing the solution; and adding a humectant, and carrying out fifth stirring dissolution to obtain the chitosan recombinant humanized collagen gel.
Further, the steps include one or more of the following conditions:
(1) In the step S1, the first stirring speed is 30r/min-50r/min, and the time is 10-20min;
(2) In the step S2, the second stirring speed is 40r/min-80r/min, and the time is 15-20min;
(3) In the step S3, the third stirring speed is 40r/min-80r/min, and the time is 10-20min;
(4) In the step S4, the fourth stirring speed is 50r/min-100r/min, and the time is 50min-90min; the fifth stirring speed is 50r/min-100r/min, and the time is 10min-20min.
In the steps S1-S5, the stirring temperature is 25-35 ℃.
Further, the method also comprises the step of freeze-drying the chitosan recombinant humanized collagen gel.
In still another aspect, the invention provides an application of chitosan recombinant humanized collagen gel in preparing a chronic repair drug for treating a uterine incision.
In yet another aspect of the invention, there is provided the use of a chitosan recombinant humanized collagen gel for the preparation of a medicament for preventing uterine adhesion or promoting endometrial repair.
The technical principle of the invention is as follows: the chitosan recombinant humanized collagen gel provided by the invention adopts carboxymethyl chitosan with good compatibility with recombinant humanized collagen as a matrix, adopts low-toxicity natural biological material genipin as a cross-linking agent, and is added with humectant xylitol and water, so that the chitosan recombinant humanized collagen gel is nontoxic, non-irritant, degradable in vivo and capable of continuously repairing uterine incisions.
Carboxymethyl chitosan is a water-soluble chitosan derivative, not only can promote fibroblast proliferation, but also can improve phagocytic capacity of macrophages, and can effectively accelerate wound repair process. In addition, carboxymethyl chitosan also has the effects of inhibiting the secretion of type I collagen, reducing the proportion of type I/III collagen of keloid fibroblasts, and inhibiting scar hyperplasia; the carboxymethyl chitosan has good inhibition effect on escherichia coli, staphylococcus aureus and the like. Genipin is a natural biological cross-linking agent, the toxicity of the genipin is far lower than that of other chemical cross-linking agents such as glutaraldehyde, and under the neutral condition, amino groups on chitosan attack the ethylenic carbon atoms of genipin to form a network structure polymer taking genipin as a cross-linking bridge. Xylitol is a polyhydric alcohol with high stability, is rich in hydroxyl, and has antiinflammatory and antibacterial effects while keeping moisture.
Compared with the prior art, the invention has the following beneficial effects:
(1) The chitosan recombinant humanized collagen gel disclosed by the invention is nontoxic and nonirritating, has good biocompatibility, is expected to be used in the repair of a uterine incision, provides a moist healing environment for the uterine incision, promotes cell growth and collagen synthesis at the incision, promotes functional healing of the uterine incision, reduces scar formation, shortens the course of disease, can prevent uterine adhesion, and promotes the repair of endometrium.
(2) The chitosan recombinant humanized collagen gel is used as in-vivo medicine, can be completely degraded in vivo and is discharged out of the body. In the prior art, most of collagen gel is used in vitro, so that the formula is complex, most of the collagen gel is macromolecular compounds, such as glycerol, and the collagen gel cannot be completely degraded and discharged out of the body, so that the collagen gel can accumulate and cause irreversible damage to organs such as liver and kidney.
(3) The combined action of genipin and xylitol in the chitosan recombinant humanized collagen gel improves the stability of recombinant humanized collagen III, so that the drug effect is longer, the storage time is longer, and a synergistic effect is generated.
(4) The preparation method of the invention fully mixes the components at the temperature and the rotating speed without damaging the structure of the collagen, the operation is simple, and the obtained chitosan recombinant humanized collagen gel has good bioactivity of the recombinant humanized collagen.
Drawings
FIG. 1 is a graph showing the relative activity of cells in test example 1 according to the present invention.
FIG. 2 is a chart showing staining of the tissue HE and MASSON of the uterine incision in test example 2 of the present invention.
FIG. 3 is an external view showing the gel prepared in example 1 at 25℃in test example 3 according to the present invention.
FIG. 4 is an external view showing the comparative example prepared gel at 25℃in test example 3 according to the present invention.
FIG. 5 is an external view showing the gel prepared in example 1 at 4℃in test example 3 according to the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and examples. III recombinant humanized collagen is provided by Shanxi Bo biomedical Co., ltd.
EXAMPLE 1 preparation of recombinant humanized collagen gel of chitosan
The weight percentages of the raw materials are as follows: III recombinant humanized collagen 0.24% (0.24 g), carboxymethyl chitosan 0.40% (0.40 g), genipin 0.10% (0.10 g), xylitol 0.16% (0.16 g) and the balance of pure water (99.1 g).
The preparation method of the chitosan recombinant humanized collagen gel comprises the following steps:
s1: adding pure water with the prescription amount of 40% into a stirring container A, adding III type recombinant humanized collagen, stirring at the rotation speed of 40r/min until the pure water is fully dissolved, and stirring for 15min to obtain a solution 1.
S2: adding 60% pure water into stirring container B, adding carboxymethyl chitosan, stirring at rotation speed of 60r/min until it is fully dissolved, and stirring for 20min to obtain solution 2.
S3: adding a cross-linking agent genipin into the solution 2, stirring until the genipin is fully dissolved at the rotating speed of 60r/min, and stirring for 15min to obtain a solution 3.
S4: adding the solution 1 into the solution 3, stirring at the rotation speed of 80r/min, stirring to be uniform, stirring for 70min, adding xylitol as a humectant, stirring at the rotation speed of 80r/min, stirring to be fully dissolved, and stirring for 15min to obtain the chitosan recombinant humanized collagen gel semi-finished product.
The solution temperature in the steps is controlled within 35 ℃.
S5: standing, filling and sterilizing to obtain the chitosan recombinant humanized collagen gel.
EXAMPLE 2 preparation of recombinant humanized collagen gel of chitosan
The weight percentages of the raw materials are as follows: III recombinant humanized collagen 0.16% (0.16 g), carboxymethyl chitosan 0.32% (0.32 g), genipin 0.12% (0.12 g), xylitol 0.16% (0.16 g) and the balance of pure water (99.24 g).
The preparation method is the same as in example 1.
EXAMPLE 3 preparation of recombinant humanized collagen gel of chitosan
The weight percentages of the raw materials are as follows: 0.08 percent (0.08 g) of III recombinant humanized collagen, 0.24 percent (0.24 g) of carboxymethyl chitosan, 0.06 percent (0.06 g) of genipin, 0.16 percent (0.16 g) of xylitol and the balance of pure water (99.46 g).
The preparation method is the same as in example 1.
EXAMPLE 4 preparation of recombinant humanized collagen gel of chitosan
The weight percentages of the raw materials are as follows: 0.30 percent (0.30 g) of III recombinant humanized collagen, 0.20 percent (0.20 g) of carboxymethyl chitosan, 0.05 percent (0.05 g) of genipin, 0.10 percent (0.10 g) of xylitol and the balance of pure water (99.35 g).
The preparation method is the same as in example 1.
EXAMPLE 4 preparation of recombinant humanized collagen gel of chitosan
The weight percentages of the raw materials are as follows: 0.12 percent (0.12 g) of III recombinant humanized collagen, 0.45 percent (0.45 g) of carboxymethyl chitosan, 0.13 percent (0.13 g) of genipin, 0.20 percent (0.20 g) of xylitol and the balance of pure water (99.1 g).
The preparation method is the same as in example 1.
Comparative example
Similar to example 1, the difference is that: xylitol was not added.
Test example 1 cytotoxicity test
The chitosan recombinant humanized collagen gel prepared in example 1 with different concentrations is co-cultured with endometrial stromal cells, and the relative activity of the cells obtained by culturing for 24 hours is shown in figure 1, and the chitosan recombinant humanized collagen gel prepared in example 1 has no toxic effect on endometrial stromal cells.
Test example 2 healing Effect test
An SD rat post-caesarean section model is established, and the repairing effect of the chitosan recombinant humanized collagen gel prepared in the example 1 on post-caesarean section uterine incisions is observed.
Test grouping: model group, test group 1 (example 1), test group 2 (comparative example)
The test method comprises the following steps: and establishing an SD rat post-cesarean section model for uterine histopathological observation.
Test results: the results of the test are shown in fig. 2, and fig. 2 shows pathological changes of uterus after the caesarean section of rats, and the incision structure of uterus in the test group 1 is recovered more thoroughly and collagen deposition is more than that in the model group and the test group 2. The uterine incision structure recovery was lower for test group 2 than for test group 1.
Test example 3 stability test
Test purpose: the stability of the gel at 4℃and 25℃was observed
Test grouping: test group 1 (example 1), test group 2 (comparative example)
The test method comprises the following steps:
1. the gel was placed on a slide glass and filter paper at 25℃to observe the appearance, content and degradation of the gel.
2. The gel is put in a refrigerator at the temperature of 4 ℃ for sealing and preservation, sampling is carried out for 5 days and 10 days, and the appearance, the content and the degradation condition are observed.
Test results:
1. the gel was not precipitated on the substrate of the gel of the test group 1, either on a slide or on a filter paper, at 25℃and the gel results were substantially stable, with water in the gel precipitated. Whereas the matrix of the comparative gel is partially precipitated. The results are shown in FIG. 3 (example 1), FIG. 4 (comparative example), and Table 1.
TABLE 1 gel stability test at 25℃
| Example 1 | Comparative example | |
| 0min | The matrix is not precipitated | The matrix is not precipitated |
| 15min | The matrix is not precipitated | Precipitation of matrix part |
| 30min | The matrix is not precipitated | Precipitation of matrix part |
| 45min | The matrix is not precipitated | Precipitation of matrix part |
2. In the case of the refrigerator at 4 ℃, the gel matrix of the test group 1 is not precipitated at the 5 th day and the 10 th day, the gel result is basically stable, and the gel is partially degraded. The results are shown in FIG. 4 (example 1) and Table 2.
Table 24 ℃ gel stability assay
| Collagen content (%) | Example 1 | |
| 0d | ||
| 100 | 100 | |
| |
100 | 96 |
| 5d | 95 | 90 |
| 10d | 92 | 83 |
Therefore, the addition of xylitol can make the gel structure more stable, the preservation time is prolonged, and the xylitol and the genipin have a synergistic effect in promoting the gel stability.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (10)
1. A chitosan recombinant humanized collagen gel, characterized in that: comprises the following components in percentage by mass:
III, 0.08 to 0.30 percent of recombinant humanized collagen;
0.20-0.45% of chitosan derivative;
0.05 to 0.13 percent of cross-linking agent;
0.10-0.20% of humectant;
the balance being water.
2. A chitosan recombinant humanized collagen gel according to claim 1, wherein: the chitosan derivative is carboxymethyl chitosan.
3. A chitosan recombinant humanized collagen gel according to claim 1, wherein: the cross-linking agent is genipin.
4. A chitosan recombinant humanized collagen gel according to claim 1, wherein: the humectant is xylitol.
5. A method for preparing a chitosan recombinant humanized collagen gel according to any one of claims 1 to 4, characterized in that: the method comprises the following steps:
s1: adding III type recombinant humanized collagen into water for first stirring and dissolving to obtain solution 1;
s2: adding the chitosan derivative into water for second stirring and dissolving to obtain a solution 2;
s3: adding a cross-linking agent into the solution 2, and carrying out third stirring and dissolving to obtain a solution 3;
s4: adding the solution 1 into the solution 3, and stirring and uniformly mixing the solution; and adding a humectant, and carrying out fifth stirring dissolution to obtain the chitosan recombinant humanized collagen gel.
6. The method for preparing the chitosan recombinant humanized collagen gel according to claim 5, wherein the method comprises the following steps: the steps include one or more of the following conditions:
(1) In the step S1, the first stirring speed is 30r/min-50r/min, and the time is 10-20min;
(2) In the step S2, the second stirring speed is 40r/min-80r/min, and the time is 15-20min;
(3) In the step S3, the third stirring speed is 40r/min-80r/min, and the time is 10-20min;
(4) In the step S4, the fourth stirring speed is 50r/min-100r/min, and the time is 50min-90min; the fifth stirring speed is 50r/min-100r/min, and the time is 10min-20min.
7. The method for preparing the chitosan recombinant humanized collagen gel according to claim 5, wherein the method comprises the following steps: in the steps S1-S5, the stirring temperature is 25-35 ℃.
8. The method for preparing the chitosan recombinant humanized collagen gel according to claim 5, wherein the method comprises the following steps: the method also comprises the step of freeze-drying the chitosan recombinant humanized collagen gel.
9. Use of a chitosan recombinant humanized collagen gel according to any one of claims 1-4 in the manufacture of a medicament for the chronic repair of uterine incisions.
10. Use of a chitosan recombinant humanized collagen gel according to any one of claims 1 to 4 for the preparation of a medicament for preventing uterine adhesion or promoting endometrial repair.
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