Chitosan recombinant humanized collagen gel and its preparation method and application

Abstract

The present invention provides a chitosan recombinant humanized collagen gel, comprising the following components in mass percentage: 0.08-0.30% of type III recombinant humanized collagen; 0.20-0.45% of chitosan or its derivatives; 0.05-0.13% of a cross-linking agent; 0.10-0.20% of a moisturizing agent; and the balance is water. The dressing provided by the present invention can slowly release humanized collagen, is non-toxic, non-irritating, has high biocompatibility, good stability, and can continuously promote the healing of uterine incisions.

Description

Technical Field

[0001] The present invention relates to the technical field of dressings, and in particular to a chitosan recombinant humanized collagen gel and a preparation method and application thereof. Background Art

[0002] Collagen is an important component of tissues. Type I and III collagen not only work together with elastic fibers to maintain the elastic function of tissues, but also provide effective tension strength. When wounds are damaged and repaired, collagen fibers are deposited and participate in the reconstruction of the wound. Many studies have shown that recombinant humanized collagen promotes wound recovery.

[0003] Recombinant human type III collagen (Rhc-Ⅲ) is obtained by optimizing and recombinantly expressing the original gene sequence of human type III collagen using genetic engineering technology, and is highly consistent with the amino acid sequence of natural human collagen. Rhc-Ⅲ is used for wound healing. On the one hand, Rhc-Ⅲ has abundant hydrophilic groups, which can increase the adhesion and proliferation of fibroblasts, accelerate the synthesis and deposition of collagen, and shorten the wound healing time. On the other hand, it can directly supplement Rhc-Ⅲ in the wound surface to reduce the incidence of scars. Recombinant humanized collagen has lower immunogenicity and higher safety. With the development of technology, recombinant humanized collagen has achieved large-scale production and maintained high biological activity. Its performance is better than that of natural collagen. However, water-soluble Rhc-Ⅲ is easily degraded and has a short action time.

[0004] The recovery of uterine incision is a long process, but there are few methods that can continuously promote the healing of uterine incision and restore the function of tissues at the uterine incision. There is currently no collagen gel for the healing of uterine incision. Other common collagen gels are mostly used for epidermal injuries. They are relatively thin, highly irritating, and cannot be degraded in the body. They are suitable for the recovery of acute wounds, but not for the recovery of chronic wounds such as uterine incisions. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a chitosan recombinant humanized collagen gel and a preparation method and application thereof, which solves the problems of the prior art that the collagen gel is highly irritating, cannot be degraded in the body, and is easily degraded.

[0006] In one aspect, the present invention provides a chitosan recombinant humanized collagen gel, comprising the following components in percentage by weight:

[0007]

[0008] Furthermore, the chitosan derivative is carboxymethyl chitosan.

[0009] Furthermore, the cross-linking agent is genipin.

[0010] Furthermore, the moisturizing agent is xylitol.

[0011] Another aspect of the present invention provides a method for preparing chitosan recombinant humanized collagen gel, which is characterized by comprising the following steps:

[0012] S1: adding type III recombinant humanized collagen to water for a first stirring dissolution to obtain solution 1;

[0013] S2: adding the chitosan derivative into water for a second stirring dissolution to obtain solution 2;

[0014] S3: adding a cross-linking agent to solution 2, and performing a third stirring dissolution to obtain solution 3;

[0015] S4: adding solution 1 to solution 3, and performing a fourth stirring to mix; then adding a moisturizer, and performing a fifth stirring to dissolve, to obtain a chitosan recombinant humanized collagen gel.

[0016] Furthermore, the steps include one or more of the following conditions:

[0017] (1) In step S1, the first stirring speed is 30 r / min-50 r / min, and the time is 10-20 min;

[0018] (2) In step S2, the second stirring speed is 40 r / min-80 r / min, and the time is 15-20 min;

[0019] (3) In step S3, the third stirring speed is 40 r / min-80 r / min, and the time is 10-20 min;

[0020] (4) In step S4, the fourth stirring speed is 50 r / min-100 r / min, and the time is 50 min-90 min; the fifth stirring speed is 50 r / min-100 r / min, and the time is 10 min-20 min.

[0021] In steps S1-S5, the stirring temperature is 25°C-35°C.

[0022] Furthermore, the method also includes the step of freeze-drying the chitosan recombinant humanized collagen gel.

[0023] In another aspect, the present invention provides a use of chitosan recombinant humanized collagen gel in the preparation of a chronic repair medicine for treating uterine incision.

[0024] In another aspect, the present invention provides a use of chitosan recombinant humanized collagen gel in the preparation of a drug for preventing uterine adhesion or promoting endometrial repair.

[0025] The technical principle of the present invention is: the chitosan recombinant humanized collagen gel of the present invention uses carboxymethyl chitosan with good compatibility with recombinant humanized collagen as a matrix, the low-toxic natural biological material genipin as a cross-linking agent, and the moisturizing agents xylitol and water. The formed chitosan recombinant humanized collagen gel is non-toxic, non-irritating, degradable in the body, and can continuously repair uterine incisions.

[0026] Carboxymethyl chitosan is a water-soluble chitosan derivative that can not only promote fibroblast proliferation, but also improve the phagocytic ability of macrophages. The levels of TGF-β1 and IL-6 are increased, which can effectively accelerate the wound repair process. In addition, carboxymethyl chitosan can inhibit the secretion of type I collagen, reduce the ratio of type I / III collagen in keloid fibroblasts, and inhibit scar hyperplasia; carboxymethyl chitosan has a good inhibitory effect on Escherichia coli and Staphylococcus aureus. Genipin is a natural biological cross-linking agent with much lower toxicity than other chemical cross-linking agents such as glutaraldehyde. Under neutral conditions, the amino groups on chitosan attack the olefinic carbon atoms of genipin to form a network structure polymer with genipin as the cross-linking bridge. Xylitol is a polyol with high stability and rich in hydroxyl groups. It has anti-inflammatory and antibacterial effects while moisturizing.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The chitosan recombinant humanized collagen gel of the present invention is non-toxic, non-irritating, and has good biocompatibility. Its intended use is in the repair of uterine incisions, providing a moist healing environment for the uterine incisions, promoting cell growth and collagen synthesis at the incisions, promoting functional healing of uterine incisions, reducing scar formation, shortening the course of the disease, preventing uterine adhesions, and promoting the repair of the endometrium.

[0029] (2) The chitosan recombinant humanized collagen gel of the present invention is for in vivo use and can be completely degraded in the body and excreted from the body. However, the collagen gels in the prior art are mostly for in vitro use and not only have complex formulas, but also contain mostly high molecular weight compounds, such as glycerol, which cannot be completely degraded and excreted from the body, resulting in drug accumulation and irreversible damage to organs such as the liver and kidneys.

[0030] (3) The combined effect of genipin and xylitol in the chitosan recombinant humanized collagen gel of the present invention improves the stability of recombinant human type III collagen, making the drug effect more lasting and the storage time longer, thereby producing a synergistic effect.

[0031] (4) The preparation method of the present invention fully mixes the components at a temperature and rotation speed that does not destroy the collagen structure, is simple to operate, and the recombinant humanized collagen in the obtained chitosan recombinant humanized collagen gel still has good biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the relative cell viability in Experimental Example 1 of the present invention.

[0033] Figure 2 HE and MASSON staining images of uterine incision tissue in Experimental Example 2 of the present invention.

[0034] Figure 3 This is the appearance of the gel prepared in Example 1 at 25°C in Test Example 3 of the present invention.

[0035] Figure 4 This is the appearance of the gel prepared in the comparative example at 25°C in Test Example 3 of the present invention.

[0036] Figure 5 This is the appearance of the gel prepared in Example 1 at 4°C in Test Example 3 of the present invention. DETAILED DESCRIPTION

[0037] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and examples. Type III recombinant humanized collagen is provided by Shanxi Jinbo Biopharmaceutical Co., Ltd.

[0038] Example 1 Preparation of chitosan recombinant humanized collagen gel

[0039] The weight percentages of the raw materials are as follows: 0.24% (0.24 g) of type III recombinant humanized collagen, 0.40% (0.40 g) of carboxymethyl chitosan, 0.10% (0.10 g) of genipin, 0.16% (0.16 g) of xylitol and the balance of pure water (99.1 g).

[0040] The preparation steps of the chitosan recombinant humanized collagen gel are as follows:

[0041] S1: Add 40% of the prescribed amount of pure water into a stirring container A, add type III recombinant humanized collagen, stir at a speed of 40 r / min until fully dissolved, stir for 15 minutes, and obtain solution 1.

[0042] S2: Add 60% of the prescribed amount of pure water into stirring container B, add carboxymethyl chitosan, stir at a speed of 60 r / min until fully dissolved, stir for 20 minutes, and obtain solution 2.

[0043] S3: Add the cross-linking agent genipin into solution 2 at a rotation speed of 60 r / min, stir until fully dissolved, and stir for 15 minutes to obtain solution 3.

[0044] S4: Add solution 1 to solution 3 while stirring at a speed of 80 r / min until the mixture is evenly mixed, stir for 70 minutes, then add moisturizer xylitol at a speed of 80 r / min, stir until fully dissolved, stir for 15 minutes to obtain a chitosan recombinant humanized collagen gel semi-finished product.

[0045] The solution temperature in the above steps was controlled within 35°C.

[0046] S5: After standing, filling and sterilization, chitosan recombinant humanized collagen gel is obtained.

[0047] Example 2 Preparation of chitosan recombinant humanized collagen gel

[0048] The weight percentages of the raw materials are as follows: 0.16% (0.16 g) of type III recombinant humanized collagen, 0.32% (0.32 g) of carboxymethyl chitosan, 0.12% (0.12 g) of genipin, 0.16% (0.16 g) of xylitol and the balance of pure water (99.24 g).

[0049] The preparation method is the same as Example 1.

[0050] Example 3 Preparation of chitosan recombinant humanized collagen gel

[0051] The weight percentages of the raw materials are as follows: 0.08% (0.08 g) of type III recombinant humanized collagen, 0.24% (0.24 g) of carboxymethyl chitosan, 0.06% (0.06 g) of genipin, 0.16% (0.16 g) of xylitol and the balance of pure water (99.46 g).

[0052] The preparation method is the same as Example 1.

[0053] Example 4 Preparation of chitosan recombinant humanized collagen gel

[0054] The weight percentages of the raw materials are as follows: 0.30% (0.30 g) of type III recombinant humanized collagen, 0.20% (0.20 g) of carboxymethyl chitosan, 0.05% (0.05 g) of genipin, 0.10% (0.10 g) of xylitol and the balance of pure water (99.35 g).

[0055] The preparation method is the same as Example 1.

[0056] Example 4 Preparation of chitosan recombinant humanized collagen gel

[0057] The weight percentages of the raw materials are as follows: 0.12% (0.12 g) of type III recombinant humanized collagen, 0.45% (0.45 g) of carboxymethyl chitosan, 0.13% (0.13 g) of genipin, 0.20% (0.20 g) of xylitol and the balance of pure water (99.1 g).

[0058] The preparation method is the same as Example 1.

[0059] Comparative Example

[0060] Similar to Example 1, except that no xylitol was added.

[0061] Test Example 1 Cytotoxicity Test

[0062] The chitosan recombinant humanized collagen gel freeze-dried extract prepared in Example 1 with different concentrations was co-cultured with endometrial stromal cells for 24 hours. The relative viability of the obtained cells was as follows: Figure 1 As shown in the figure, it can be seen that the chitosan recombinant humanized collagen gel prepared in Example 1 has basically no toxic effect on endometrial stromal cells.

[0063] Test Example 2 Healing Effect Test

[0064] A SD rat model after cesarean section was established to observe the repair effect of the chitosan recombinant humanized collagen gel prepared in Example 1 on the uterine incision after cesarean section.

[0065] Experimental groups: model group, test group 1 (Example 1), test group 2 (Comparative example)

[0066] Experimental method: A cesarean section model was established in SD rats, and uterine tissue pathology was observed.

[0067] Test results: The test results are as follows: Figure 2 As shown, Figure 2 It is the pathological change of the uterus after cesarean section in rats. Compared with the model group and test group 2, the uterine incision structure of test group 1 recovered more thoroughly and collagen deposition was more. The uterine incision structure recovery of test group 2 was lower than that of test group 1.

[0068] Test Example 3 Stability Test

[0069] Test purpose: To observe the stability of the gel at 4°C and 25°C

[0070] Test groups: Test group 1 (Example 1), test group 2 (Comparative example)

[0071] Test method:

[0072] 1. Place the gel on a glass slide and filter paper at 25°C and observe the gel's appearance, content and degradation.

[0073] 2. Store the gel in a sealed container at 4°C. Take samples after 5 and 10 days to observe its appearance, content and degradation.

[0074] Test results:

[0075] 1. Gel at 25℃, whether on glass slide or filter paper, the matrix of test group 1 gel did not precipitate, the gel results were basically stable, and the water in the gel was precipitated. However, the matrix of the control group gel was partially precipitated. The results are as follows Figure 3 (Example 1), Figure 4 (Comparative Example), as shown in Table 1.

[0076] Table 1 Gel stability test at 25℃

[0077] Example 1 Comparative Example 0min No matrix precipitation No matrix precipitation 15min No matrix precipitation Matrix analysis 30min No matrix precipitation Matrix analysis 45min No matrix precipitation Matrix analysis

[0078] 2. In the refrigerator at 4°C, on the 5th and 10th days, the gel matrix of test group 1 did not precipitate, the gel results were basically stable, and the gel was partially degraded. Figure 4 (Example 1), as shown in Table 2.

[0079] Table 2 4℃ gel stability test

[0080] Collagen content (%) Example 1 Comparative Example 0d 100 100 1d 100 96 5d 95 90 10d 92 83

[0081] It can be seen that the addition of xylitol can make the gel structure more stable and prolong the shelf life. Xylitol and genipin have a synergistic effect in promoting gel stability.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A chitosan recombinant humanized collagen gel, characterized in that: The components include the following mass percentages: Type III recombinant humanized collagen 0.24%; Chitosan derivative 0.40%; Cross-linking agent 0.10%; Moisturizer 0.16%; The balance is water; The chitosan derivative is carboxymethyl chitosan; the cross-linking agent is genipin; and the moisturizing agent is xylitol.

2. The method for preparing a chitosan recombinant humanized collagen gel according to claim 1, characterized in that: The following steps are involved: S1: adding type III recombinant humanized collagen to water for a first stirring dissolution to obtain solution 1; S2: adding the chitosan derivative into water for a second stirring dissolution to obtain solution 2; S3: adding a cross-linking agent to solution 2, and performing a third stirring dissolution to obtain solution 3; S4: adding solution 1 to solution 3, and performing a fourth stirring to mix; then adding a moisturizer, and performing a fifth stirring to dissolve, to obtain a chitosan recombinant humanized collagen gel.

3. The method for preparing a chitosan recombinant humanized collagen gel according to claim 2, characterized in that: The steps include one or more of the following conditions: (1) In step S1, the first stirring speed is 30 r / min-50 r / min, and the time is 10-20 min; (2) In step S2, the second stirring speed is 40 r / min-80 r / min, and the time is 15-20 min; (3) In step S3, the third stirring speed is 40 r / min-80 r / min, and the time is 10-20 min; (4) In step S4, the fourth stirring speed is 50 r / min-100 r / min, and the time is 50 min-90 min; the fifth stirring speed is 50 r / min-100 r / min, and the time is 10 min-20 min.

4. The method for preparing a chitosan recombinant humanized collagen gel according to claim 2, characterized in that: In steps S1-S4, the stirring temperature is 25°C-35°C.

5. The method for preparing a chitosan recombinant humanized collagen gel according to claim 2, characterized in that: The method also includes the step of freeze-drying the chitosan recombinant humanized collagen gel.

6. Use of the chitosan recombinant humanized collagen gel according to claim 1 in the preparation of a chronic repair drug for treating uterine incision.

7. Use of the chitosan recombinant humanized collagen gel according to claim 1 in the preparation of a drug for preventing uterine adhesion or promoting endometrial repair.

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