CN115944775A - Recombinant humanized collagen mucosa repair preparation and preparation method thereof - Google Patents
Recombinant humanized collagen mucosa repair preparation and preparation method thereof Download PDFInfo
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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
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Abstract
The invention discloses a recombinant humanized collagen mucosa repair preparation and a preparation method thereof. The recombinant humanized collagen mucosa repair preparation comprises the following components in percentage by mass: 0.05-1 percent of recombinant humanized collagen, 0.6-2 percent of thickening agent, 2-5 percent of humectant, 0.05-1 percent of sodium hyaluronate, 0.02-0.05 percent of methyl hydroxybenzoate, 0.02-0.05 percent of phenoxyethanol, 0.01-0.2 percent of pH regulator and the balance of water. The gel type mucosa repair preparation is prepared by taking recombinant humanized collagen and sodium hyaluronate as raw materials and adding pharmaceutic adjuvants, and when the gel type mucosa repair preparation is applied to the non-chronic wound repair of mucosa, the gel type mucosa repair preparation cannot be absorbed by a human body, warmly moistens and nurses the wound, creates a moist healing environment, isolates harmful substances, and promotes the repair of the wound.
Description
Technical Field
The invention belongs to the technical field of mucosa repair biomedical materials, and relates to a recombinant humanized collagen mucosa repair preparation and a preparation method thereof.
Background
Collagen is used as attachment and a scaffold for cell growth, can induce proliferation, differentiation and migration of epithelial cells and the like, and interaction of collagen and cells is an essential characteristic of cell activity during wound healing and tissue remodeling, and collagen plays a role in promoting and even inducing formation of epithelium. The recombinant collagen is one of the biological materials approved by FDA and NMPA earlier, has the excellent characteristics of no virus hidden danger, high biocompatibility, excellent water solubility and the like, and is widely used in a plurality of fields of tissue engineering, biomedicine, drug release and the like. Chinese patent ZL201110327865.5 discloses a recombinant humanized collagen, which is prepared by fermentation of pichia pastoris genetic engineering bacteria, has stable molecular weight (55 KD), meets the raw material standard of YY/T recombinant collagen, and can be applied to development and application of medical instruments.
The mucous membrane is a membrane-like structure composed of epithelial tissue and connective tissue in living body (inside organs such as oral cavity, organ, stomach, intestine, urethra, etc.). The mucosa injury is relatively difficult to repair, and meanwhile, the pain feeling brought to people is larger than the injury of common skin surgery, and the corresponding postoperative repair is also a great trouble. The use of traditional gauze type dressings easily causes secondary damage to damaged tissues, and seriously affects the repair of mucosa. Chinese patent ZL201611062703 discloses a human-like collagen gynecological mucosa repair gel which comprises, by weight, 0.1-1% of human-like collagen, 0.9-1.1% of carbomer, 20-25% of glycerol, 0.001-0.01% of sodium hyaluronate, 0.05-0.1% of sodium hydroxide, 0.1-0.5% of ethylparaben, 0.2-1% of chitosan, and water. When the collagen is applied to the epidermis of a gynecological part with damaged mucosa, the human-like collagen can permeate the human epidermis and be absorbed by the human body, and cannot be used as the requirement of non-transdermal absorption of II-type medical instruments.
Disclosure of Invention
The invention aims to provide a recombinant humanized collagen mucosa repair preparation and a preparation method thereof. The mucosa repair preparation adopts recombinant humanized collagen prepared by microbial fermentation and sodium hyaluronate as raw materials, and has no immunogenicity and no virus. The recombinant humanized collagen has remarkable repair effect, can provide a moist and mild healing environment for the non-chronic wound of a mucosal tissue by matching with the moisture retention of hyaluronic acid, provides better use experience for patients, and promotes the repair of the wound. Meanwhile, the preservative combination formula of methylparaben and phenoxyethanol is selected, so that the biocompatibility of the mucosa repair preparation is improved.
The technical scheme for realizing the purpose of the invention is as follows:
the recombinant humanized collagen mucosa repair preparation comprises the following components in percentage by mass: 0.05-1% of recombinant humanized collagen, 0.6-2% of thickening agent, 2-5% of humectant, 0.05-1% of sodium hyaluronate, 0.02-0.05% of methyl hydroxybenzoate, 0.02-0.05% of phenoxyethanol, 0.01-0.2% of pH regulator and the balance of water, wherein the recombinant humanized collagen is produced by fermentation of Pichia pastoris with the preservation number of CGMCC No. 5021.
Preferably, the recombinant humanized collagen mucosa repair preparation consists of the following components in percentage by mass: 0.05-0.1 percent of recombinant humanized collagen, 1 percent of thickening agent, 2-5 percent of humectant, 0.05-0.1 percent of sodium hyaluronate, 0.02-0.05 percent of methyl hydroxybenzoate, 0.02-0.05 percent of phenoxyethanol, 0.01-0.02 percent of pH regulator and the balance of water.
The thickener according to the invention is a thickener conventionally used in the art, preferably carbomer.
The moisturizer of the present invention is a moisturizer conventionally used in the art, and preferably glycerin.
The pH adjuster according to the present invention is a pH adjuster conventionally used in the art, and preferably sodium hydroxide.
Further preferably, the recombinant humanized collagen mucosa repair preparation consists of the following components in percentage by mass: 0.1% of recombinant humanized collagen, 1% of carbomer, 5% of glycerol, 0.1% of sodium hyaluronate, 0.05% of methylparaben, 0.05% of phenoxyethanol, 0.02% of pH regulator and the balance of water.
Further, the invention provides a preparation method of the recombinant humanized collagen mucosa repair preparation, which comprises the following specific steps:
step 1: heating purified water to 70-80 ℃ according to the proportion, adding carbomer, stirring until the mixture is uniformly mixed, and cooling to below 35-40 ℃;
and 2, step: pre-dissolving methylparaben in purified water at the temperature of 80 ℃ according to the proportion, then adding the dissolved methylparaben into the solution obtained in the step 1, and cooling the solution to the temperature of below 35-40 ℃;
and 3, step 3: pre-dissolving phenoxyethanol with glycerol according to a ratio, uniformly stirring, and then adding into the solution in the step (2);
and 4, step 4: dissolving recombinant humanized collagen and sodium hyaluronate in purified water in a pre-dissolving manner according to a ratio, and then adding the solution into the solution 3;
and 5: and (4) adding a pH regulator into the solution obtained in the step (4) according to the proportion, uniformly stirring, discharging after constant volume of purified water, filling and sterilizing to obtain the recombinant humanized collagen mucosa repair preparation.
Preferably, in step 1, the amount of the purified water is 60% to 80% of the total amount of water.
Preferably, in the steps 1 to 5, the stirring speed is 20 to 40r/min.
Preferably, in step 5, the sterilization temperature is 105 ℃ and the sterilization time is 60min.
Compared with the prior art, the invention has the following advantages:
(1) The invention selects the recombinant humanized collagen fermented by microorganism and sodium hyaluronate as raw materials, and the recombinant humanized collagen and sodium hyaluronate are used as skin repair agents, and have the advantages of stable molecular weight, good water solubility, no immunogenicity and no virus hidden trouble. Meanwhile, the repairing effect of the recombinant humanized collagen is combined with the high moisture retention of the sodium hyaluronate, a mild and moist healing environment is provided for the non-chronic wound of the mucosa, the use experience of a patient is improved, the probability of secondary injury is reduced, and the repair of the wound is promoted. In addition, the molecular weight of the recombinant humanized collagen selected by the invention is 55kDa, and when the recombinant humanized collagen is used as the epidermal non-chronic wound dressing, the recombinant humanized collagen cannot permeate through a skin structure and cannot be absorbed by a human body, thereby meeting the basic application requirements of II-class medical dressings.
(2) According to the invention, methyl hydroxybenzoate and phenoxyethanol are used as the composite preservative, and the proportion of the methyl hydroxybenzoate and the phenoxyethanol is regulated, so that the prepared gel type mucosa repair preparation has good biocompatibility.
Drawings
FIG. 1 is a sample object diagram of a recombinant humanized collagen mucosa repair preparation;
FIG. 2 is a graph showing the results of cell proliferation assays for human epidermal cells using different preservative formulations;
FIG. 3 is a graph of the results of cell proliferation assays of human fibroblasts with different preservative formulations;
FIG. 4 is a graph showing the results of cell proliferation assays in examples 1-3 and a commercially available control sample;
FIG. 5 is a photograph of a rectal mucosa stimulated tissue section (10X) wherein a is a non-polar leach (sesame oil) negative control, b is a non-polar leach sample, c is a polar leach negative control, and d is a polar leach (sodium chloride) sample.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings.
Example 1
Research on transdermal test of recombinant humanized collagen:
1. a test method of a chemical skin absorption in vitro test method GB/T27818-2011 is adopted. The skin to be tested is selected from 2-month-old white pig, abdominal skin is removed, fat and hair tissues are removed, and the skin is soaked in physiological saline (after superficial wound treatment, the skin is soaked in physiological saline) and stored at normal temperature.
2. And (3) testing conditions are as follows:
a diffusion cell: the volume is 15mL;
diffusion liquid: PBS buffer solution with pH7.0;
a receiving pool: the volume is 50mL;
receiving liquid: PBS buffer solution with pH7.0;
concentration of the test substance: 0.05%, 0.1%, 0.5% and 1% of recombinant humanized collagen solution, which is prepared by dissolving the recombinant humanized collagen in normal saline;
transdermal time points: 0.5h, 1h, 2h, 4h, 8h, 16h and 24h;
area of skin coating: 2.5cm 2 。
3. And (3) collecting the receiving liquid by the high performance liquid chromatography to a receiving pool at a corresponding time point, detecting the recombinant humanized collagen, diluting the receiving liquid with purified water according to a corresponding proportion, and filtering and injecting 20u mu L of sample. The results are shown in Table 1, calculated by area normalization. As can be seen from Table 1, no percutaneous absorption was observed in the recombinant humanized collagen (0.05%, 0.1%, 0.5% and 1% concentration) for seven periods of 0.5h, 1h, 2h, 4h, 8h, 16h and 24 h. The results of the superficial wound transdermal test are as follows: the wound transdermal absorption phenomenon is not found in the 24-hour time period of the recombinant humanized collagen with the concentration of 1%. Therefore, the molecular weight of the recombinant humanized collagen is 55kDa, and when the recombinant humanized collagen is used as the epidermal non-chronic wound dressing, the recombinant humanized collagen cannot penetrate through a skin structure and cannot be absorbed by a human body, thereby meeting the basic application requirements of II-class medical dressings.
TABLE 1 non-transdermal absorption test data for non-chronic wound application of recombinant humanized collagen
Example 2
Cell growth assay with different preservatives
6 different preservative formulas are selected to carry out cell growth influence test research on human epidermal cells and human fibroblasts. Wherein No. 1: 3% pentanediol; no. 2: 2% pentanediol +1% hexanediol; no. 3: 0.5% of BHC (available from Shanghai Dry chemical Co., ltd.); no. 4: 0.1% methylparaben; no. 5: 0.05% methylparaben +0.05% phenoxyethanol; no. 6: 0.02% methylparaben and 0.02% phenoxyethanol.
The results are shown in fig. 2 and 3, and it can be seen that preservative formulation No.5 has no potential cytotoxicity to human epidermis and human fibroblasts, and exhibits the best biocompatibility.
Example 3
The recombinant humanized collagen mucosa repair preparation comprises the following components in percentage by mass: 0.05% of recombinant humanized collagen, 1.0% of carbomer (thickening agent), 2% of glycerin (humectant), 0.05% of sodium hyaluronate, 0.05% of methylparaben, 0.05% of phenoxyethanol, 0.01% of pH regulator and the balance of purified water.
The preparation method comprises the following steps: putting 6L of purified water into a pot, heating to 70 ℃, putting 100g of carbomer, and stirring for 30r/min and 30min; dissolving 5g of methyl hydroxybenzoate in purified water at 80 deg.C, adding into a pot, stirring for 30r/min, and 10min; cooling to 40 deg.C; pre-dissolving 5g of phenoxyethanol in 200g of glycerol, putting the glycerol into a pot, and adding 5g of sodium hyaluronate and 5g of recombinant humanized collagen which are pre-dissolved respectively in advance by using purified water; adding 1g sodium hydroxide (pH regulator), stirring to desired volume, packaging, and sterilizing at 106 deg.C for 60min.
Example 4
The recombinant humanized collagen mucosa repair preparation consists of the following components in percentage by mass: 0.05% of recombinant humanized collagen, 1.0% of carbomer (thickening agent), 3% of glycerol (humectant), 0.1% of sodium hyaluronate, 0.05% of methylparaben, 0.05% of phenoxyethanol, 0.02% of pH regulator and the balance of purified water.
The preparation method comprises the following steps: adding 7L of purified water into a pot, heating to 70 deg.C, adding 100g carbomer, stirring for 30r/min and 30min; dissolving 5g of methyl hydroxybenzoate in purified water at 80 deg.C, adding into a pot, stirring for 30r/min, and 10min; cooling and cooling to 35 ℃; taking 300g of glycerol to pre-dissolve 5g of phenoxyethanol, putting into a pot, and adding 10g of sodium hyaluronate and 5g of recombinant humanized collagen which are pre-dissolved respectively by using purified water in advance; adding 2g sodium hydroxide, stirring to desired volume, bottling, and sterilizing (106 deg.C, 60 min).
Example 6
The recombinant humanized collagen mucosa repair preparation comprises the following components in percentage by mass: 0.1% of recombinant humanized collagen, 1.0% of carbomer (thickening agent), 5% of glycerol (humectant), 0.1% of sodium hyaluronate, 0.05% of methylparaben, 0.05% of phenoxyethanol, 0.02% of pH regulator and the balance of purified water.
The preparation method comprises the following steps: adding 8L of purified water into a pot, heating to 70 deg.C, adding 100g carbomer, stirring for 30r/min and 30min; dissolving 5g of methyl hydroxybenzoate in purified water at 80 deg.C, adding into a pot, stirring for 30r/min, and 10min; cooling, and cooling to 40 ℃; pre-dissolving 500g of glycerol and 5g of phenoxyethanol, putting into a pot, and adding 10g of sodium hyaluronate and 10g of recombinant humanized collagen which are pre-dissolved respectively by purified water in advance; adding 2g sodium hydroxide, stirring to desired volume, bottling, and sterilizing (106 deg.C, 60 min).
Example 7
1. Cell proliferation assay
The cell proliferation test of epidermal cells and fibroblasts was performed on examples 1 to 3 and a control group of a commercially available mucosal repair product by using the MTT method, and the number of living cells was judged based on the measured absorbance value (OD value), and the larger the OD value, the stronger the cell activity.
The statistical method comprises the following steps: mean ± standard deviation (x ± s); cell viability% = OD value of sample group/OD value of blank group × 100%.
As shown in FIG. 4, in the 3 examples and the commercial control group, the cell viability value of example 3 was the highest, and the biological activities of examples 1-3 were higher than those of the commercial control group, wherein the biological activity of example 3 was the best and the cell stimulation effect was the least, and the sample of example 3 was subsequently selected to perform the mucosal stimulation test.
2. Rectal mucosa irritation test
Rectal irritation evaluation tests were carried out on 12 new zealand pure white rabbits (6 tests, 6 controls) according to the method requirements of GB/T16886.10-2017. The sample of example 3 above was leached with sesame oil using sodium chloride injection. Negative control solutions were prepared under the same conditions. A short (6 cm) tube or a blunt cannula is linked to a syringe with a volume greater than 1ml, and the syringe and catheter are filled to allow the animal to receive 1ml of test sample. A set of syringe and connecting catheter was prepared for each animal. The animal is placed in a fixture for fixation so as to contact the perineum. The catheter is wetted with a control solution or a suitable lubricant. The animal's tail was lifted to expose the perineum, and the wet-treated catheter was gently inserted into the rectum and 1ml of test sample or control solution was injected with a syringe. The above steps are repeated at daily intervals (24 + -2 h) for 5 consecutive days.
The results are shown in fig. 5, and the rectal mucosa of the animals in the negative control group (sodium chloride injection and sesame oil) is intact during the test process, and the phenomena of overflowing, erythema and edema do not occur. The rectal mucosa skin of the animals in the test group is intact, and rectal irritation reactions such as effusion, erythema and edema do not occur. No stimulation response is found in the animal rectum histoscope observation of the test group and the control group. The result shows that the recombinant humanized collagen mucosa repair preparation has no rectal mucosa irritation reaction in a rectal irritation test.
Claims (10)
1. The recombinant humanized collagen mucosa repair preparation is characterized by comprising the following components in percentage by mass: 0.05-1% of recombinant humanized collagen, 0.6-2% of thickening agent, 2-5% of humectant, 0.05-1% of sodium hyaluronate, 0.02-0.05% of methyl hydroxybenzoate, 0.02-0.05% of phenoxyethanol, 0.01-0.2% of pH regulator and the balance of water, wherein the recombinant humanized collagen is produced by fermentation of Pichia pastoris with the preservation number of CGMCC No. 5021.
2. The recombinant humanized collagen mucosal repair preparation according to claim 1, consisting of the following components in mass percent: 0.05-0.1 percent of recombinant humanized collagen, 1 percent of thickening agent, 2-5 percent of humectant, 0.05-0.1 percent of sodium hyaluronate, 0.02-0.05 percent of methyl hydroxybenzoate, 0.02-0.05 percent of phenoxyethanol, 0.01-0.02 percent of pH regulator and the balance of water.
3. The recombinant humanized collagen mucosal repair formulation according to claim 1, wherein the thickening agent is carbomer.
4. The recombinant humanized collagen mucosal repair preparation of claim 1, wherein the humectant is glycerol.
5. The mucosal repair preparation of claim 1, wherein the pH regulator is sodium hydroxide.
6. The recombinant humanized collagen mucosal repair preparation according to claim 1, consisting of the following components in mass percent: 0.1% of recombinant humanized collagen, 1% of carbomer, 5% of glycerol, 0.1% of sodium hyaluronate, 0.05% of methylparaben, 0.05% of phenoxyethanol, 0.02% of pH regulator and the balance of water.
7. The preparation method of the recombinant humanized collagen mucosal repair preparation according to any one of claims 1 to 6, characterized by comprising the following specific steps:
step 1: heating purified water to 70-80 ℃ according to the proportion, adding carbomer, stirring until the mixture is uniformly mixed, and cooling to below 35-40 ℃;
and 2, step: pre-dissolving methylparaben in purified water at the temperature of 80 ℃ according to the proportion, then adding the dissolved methylparaben into the solution obtained in the step (1), and cooling the solution to the temperature of below 35-40 ℃;
and 3, step 3: pre-dissolving phenoxyethanol with glycerol according to a ratio, uniformly stirring, and then adding into the solution in the step (2);
and 4, step 4: pre-dissolving recombinant humanized collagen and sodium hyaluronate with purified water according to a ratio, and then adding the re-dissolved recombinant humanized collagen and sodium hyaluronate into the solution of 3;
and 5: and (4) adding a pH regulator into the solution obtained in the step (4) according to the proportion, uniformly stirring, discharging after constant volume of purified water, filling and sterilizing to obtain the recombinant humanized collagen mucosa repair preparation.
8. The method according to claim 7, wherein the purified water is added in an amount of 60 to 80% based on the total amount of water in step 1.
9. The method according to claim 7, wherein the stirring speed is 20 to 40r/min in the steps 1 to 5.
10. The method according to claim 7, wherein the sterilization temperature is 105 ℃ and the sterilization time is 60min in step 5.
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| CN117860959A (en) * | 2024-01-15 | 2024-04-12 | 钱望(浙江)生物科技有限责任公司 | Repair-promoting recombinant humanized collagen gel and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106491518A (en) * | 2016-11-25 | 2017-03-15 | 陕西巨子生物技术有限公司 | A kind of Human-like Collagen gynecological cell migration gel |
| CN115212295A (en) * | 2021-04-20 | 2022-10-21 | 江苏江山聚源生物技术有限公司 | Application of recombinant humanized collagen in preparation of burn and scald healing promotion material |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106491518A (en) * | 2016-11-25 | 2017-03-15 | 陕西巨子生物技术有限公司 | A kind of Human-like Collagen gynecological cell migration gel |
| CN115212295A (en) * | 2021-04-20 | 2022-10-21 | 江苏江山聚源生物技术有限公司 | Application of recombinant humanized collagen in preparation of burn and scald healing promotion material |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117860959A (en) * | 2024-01-15 | 2024-04-12 | 钱望(浙江)生物科技有限责任公司 | Repair-promoting recombinant humanized collagen gel and preparation method thereof |
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