CN117088963A - PEG modified long-acting recombinant collagen for scalp repair and preparation method thereof - Google Patents
PEG modified long-acting recombinant collagen for scalp repair and preparation method thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Dermatology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Birds (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hematology (AREA)
- Materials Engineering (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of biomedicine, in particular to IPC C07K19, and more particularly relates to PEG modified long-acting recombinant collagen for scalp repair and a preparation method thereof. The main steps in the invention are as follows: in a 2- (N-morpholinyl) ethanesulfonic acid (MES) buffer solution system, the carboxyl of the long-acting recombinant collagen and the amino of cystamine are connected by carbodiimide (EDC), so that disulfide bonds carried by cystamine are reduced by tris [ 2-carboxyethyl ] phosphine (TCEP), and the generated sulfhydryl reacts with PEG-maleimide (mPEG-MAL) to obtain the PEG modified long-acting recombinant collagen. The PEG modified long-acting recombinant collagen can obviously improve the transdermal effect of the long-acting recombinant collagen, can obviously promote proliferation of hDPC cells, promote healing of damaged skin and promote hair growth at damaged parts.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to IPC C07K19, and more particularly relates to PEG modified long-acting recombinant collagen for scalp repair and a preparation method thereof.
Background
Collagen (COL) is one of the most widely distributed proteins in the body, which is widely distributed in various tissues in the body, as a scaffold of extracellular matrix, and the composition of 70% of protein components of human skin is Collagen. Collagen plays an important role on human skin, and is closely related to skin youth, wound repair, cell support, regeneration and other actions. At present, collagen in the market is mainly divided into a natural extraction method and a recombinant method, wherein the natural extraction method mainly comprises a relatively mature process of a collagen hydrolysis method, and collagen is extracted from biological materials such as fish skin by utilizing methods such as enzyme hydrolysis and the like. However, the temperature and pH have a large impact on this process, and the denaturation temperature of the collagen naturally extracted from animal sources is typically 39.5 ℃. When the temperature exceeds the temperature, the performance is exponentially reduced, and collagen is immediately degraded into gelatin by temperature and acid-base change, so that the biological efficacy is lost. Since most of the natural extracts are prepared from animal sources, the natural extracts have high immunogenicity and are easy to sensitize. Compared with natural extraction, the recombinant collagen has low immunogenicity, high denaturation temperature and high purity.
COL is currently obtained mainly by two routes: firstly, extracting from tissue cells or body fluid of organisms; and secondly, constructing an expression vector by using a genetic engineering technology, and expressing recombinant COL by using a host. Because the natural COL has large molecular weight and low expression feasibility of the whole sequence, mainly the structural domains with different functions of COL are selected for expression or combined expression so as to obtain recombinant COL with different functions. However, even recombinant COL belongs to a macromolecular protein, and cannot be absorbed transdermally.
The prior patent CN202110358113.9 discloses a saccharomyces cerevisiae expressed long-acting recombinant type III collagen and application thereof in cosmetics, wherein the long-acting recombinant type III collagen has the advantages of solubility, no virus hidden trouble and low rejection reaction. But its percutaneous absorption effect and hair follicle growth promoting effect are yet to be improved.
Disclosure of Invention
In order to solve the problems in the prior art, the first aspect of the invention provides a preparation method of PEG modified long-acting recombinant collagen with high transdermal absorbability and hair restoration efficacy, which comprises the following steps:
s1: adding rhCOL protein stock solution into MES (2- (N-morpholinyl) ethanesulfonic acid) buffer solution, and then adding EDC (carbodiimide) solution and cystamine to react for 2.5-3.5 hours at the temperature of 4 ℃;
s2: after the reaction, adding TCEP (tri (2-carboxyethyl) phosphine) solution to perform reduction reaction, and reacting for 1.5-2.5 hours at 4 ℃;
s3: after the reduction reaction, adding mPEG2-MAL into a reaction system, and reacting for 18-22 hours at the temperature of 4 ℃, namely, carrying out fixed point modification on the C-terminal carboxyl of rhCOL to obtain a PEG modified long-acting recombinant collagen solution;
s4: and separating the modified product in the PEG modified long-acting recombinant collagen solution by adopting a chromatographic medium to obtain the PEG modified long-acting recombinant collagen for scalp repair.
Preferably, the rhCOL protein stock solution comprises rhCOL III protein stock solution or rhCOL XVII protein stock solution.
Preferably, the MES buffer has a concentration of 0.05mo1/L and a pH of 5.5.
Preferably, the concentration of the EDC solution is 1.5-2.5 times of that of the rhCOL protein stock solution; further preferably, it is 2 times.
Preferably, the molar ratio of EDC to cystamine in the EDC solution is (0.8-1.2): 1, a step of; further preferred is 1:1.
preferably, the concentration of the TCEP solution is 40-60 mM.
Preferably, the molar ratio of TCEP to cystamine in the TCEP solution is (0.8 to 1.2): 1, a step of; further preferred is 1:1.
preferably, the mPEG2-MAL has a molecular weight of 10kD.
Preferably, the mole ratio of the mPEG2-MAL to the rhCOL protein (based on the protein content) is (3-5): 1, a step of; further preferably, it is 4:1.
preferably, the specific steps in S4 are as follows: separating a modified product in the PEG modified long-acting recombinant collagen solution by adopting a DEAE Sepharose FF chromatographic medium, diluting the PEG modified long-acting recombinant collagen solution by 3-7 times by using a ppH 6-7 and 8-12 mmol/L phosphate buffer solution, and then loading the diluted PEG modified long-acting recombinant collagen solution, wherein the balancing solution is a pH 6-7 and 8-12 mmol/L phosphate buffer solution.
Preferably, the specific step in S4 further includes: setting DEAE Sepharose FF chromatographic medium separation parameters: and (3) performing linear gradient elution on 0.1-0.3mol/L NaCl for 5CV, wherein the flow rate is 5.0mL/min, the detection wavelength is 280nm, collecting elution peaks, and obtaining the PEG modified long-acting recombinant collagen after ultrafiltration, desalination and concentration.
In the invention, the conditions of a specific modification method, PEG modifier, reaction pH and the like are selected, so that the C-terminal of the III type collagen and the XVII type collagen are subjected to site-directed modification to obtain the PEG modified long-acting recombinant collagen which is a uniform product, has few isomers and excellent activity retention, and the immunogenicity is greatly reduced. Due to the presence of multiple alpha-NH's in the long-acting recombinant collagens (rhCOL III and rhCOL XVII) 2 And epsilon-NH 2 The random modification of the group and the amino group can lead to obvious reduction of the activity of the medicine, so that the C-terminal of the collagen needs to be subjected to site-directed modification through specific modification methods, PEG modifier, reaction pH and other conditions. The inventor connects carboxyl of long-acting recombinant type III collagen with amino of cystamine by carbodiimide (EDC) in a 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution (pH 5.0) system through a series of creative labor and accidental factors, and controls rhCOL: cystamine: the ratio of carbodiimide was 1:2:2. making disulfide bond carried by cystamine pass through tri [ 2-carboxyethyl group]After phosphine (TCEP) is reduced, the generated sulfhydryl reacts with PEG-maleimide (mPEG-MAL) to obtain the rhCOL with the C terminal carboxyl selectively modified, namely, PEG modified long-acting recombinant collagen. The inventor speculates that the PEG has the advantages of no toxicity, no immunogenicity, no antigenicity, good water solubility and the like, and long-acting recombinant III typeAfter the XVII type collagen is modified by PEG, the lubricity, the moisturizing property, the dispersibility and the adhesiveness of the long-acting recombinant III and XVII type collagen can be improved, and the physical and chemical properties such as the conformation, the electrostatic combination and the hydrophobicity of the long-acting recombinant III and XVII type collagen are changed, so that the pharmacokinetics, the pharmacodynamics and the immunology of protein medicines are improved, and the treatment effect of the PEG modified long-acting recombinant III and XVII type collagen is further enhanced.
In the invention, PEG site-directed modification technology is adopted to modify the C-terminal carboxyl of the long-acting recombinant collagen, so that the transdermal effect of the long-acting recombinant collagen is obviously improved, and an immunohistochemical test proves that the modified protein can act on hair follicles through skin. Collagen belongs to biological macromolecules, and has the defects of incapability of percutaneous absorption, incapability of entering hair follicles and the like. The existing PEG new drugs approved by FDA are mostly randomly modified products, the randomly modified protein takes epsilon-NH 2 or alpha-NH 2 of lysine as a modification target, and the modification causes a plurality of lysines in the protein to be modified due to the fact that the number of lysines in the protein is large, and the obtained product is a mixture of polyethylene glycol modified isomers. The inventor creatively carries out PEG fixed-point modification on saccharomyces cerevisiae expression long-acting recombinant collagen to obtain the PEG modified long-acting recombinant collagen with reduced administration times, high curative effect, improved tolerance and reduced severity and incidence of adverse events. Meanwhile, the solubility and stability of long-acting recombinant III and XVII type collagen can be increased by adopting PEG fixed point modification technology to modify long-acting recombinant collagen, and the utilization rate of long-acting recombinant III type collagen is improved.
The second aspect of the invention provides the PEG modified long-acting recombinant collagen for scalp repair, which is prepared by the preparation method of the PEG modified long-acting recombinant collagen for scalp repair.
The third aspect of the invention provides application of PEG modified long-acting recombinant collagen for scalp repair, which is applied to preparation of scalp field products, wherein the scalp field products comprise scalp skin care products, scalp medical devices and scalp medicines.
Preferably, the scalp care product comprises shampoo, hair conditioner and hair mask.
Preferably, the scalp medical device comprises a wound dressing.
Advantageous effects
1. In the invention, PEG site-directed modification technology is adopted to modify the C-terminal carboxyl of the long-acting recombinant collagen, so that the transdermal effect of the long-acting recombinant collagen is obviously improved, and an immunohistochemical test proves that the modified protein can act on hair follicles through skin.
2. In the invention, the conditions of a specific modification method, a PEG modifier, reaction pH and the like are selected, so that the C end of the collagen is subjected to site-directed modification, and the PEG modified long-acting recombinant collagen is obtained as a uniform product, has fewer isomers and excellent activity retention, and has greatly reduced immunogenicity.
3. The PEG modified long-acting recombinant III and XVII type collagen obtained in the invention has obvious effects of promoting proliferation of hDPC cells, promoting healing of damaged skin and promoting hair to recover and grow at damaged parts in-vitro tests and animal tests.
4. The long-acting recombinant III and XVII type collagen in the PEG modified long-acting recombinant collagen belongs to biological proteins, has the advantages of easy decomposition and no residue, and has the great advantage of no side effect compared with common chemical cosmetics and hormone medicines.
5. The PEG modification method is simple to operate, the recombinant collagen is obtained by expressing a saccharomyces cerevisiae secretion expression system, and the method has the advantages of simple production process, low cost, uniform product and no immunogenicity, and is suitable for commercial production.
Drawings
FIG. 1 is a graph showing the purity identification result of PEG site-directed modification long-acting recombinant type III collagen in example 1; wherein M: a Marker;1: PEG-rhCOL III after purification.
FIG. 2 shows the cellular status of hDPC observed under a 72h well-mirror in Performance test 1; observing the state of hDPC (100X) cells in the PEG modified collagen high-dose group under a sample hole microscope; on the right are the cell status of the negative control under the porosimetry (100×).
FIG. 3 is a bar graph showing the number of proliferating cells in each of the normal collagen group, the PEG-modified collagen high dose group, the PEG-modified collagen medium dose group, and the PEG-modified collagen low dose group of Performance test 1 after 7d culture.
FIG. 4 is a schematic diagram of the operation of a murine peel-Franz cell diffusion cell in Performance test 2.
FIG. 5 is a graph of the cumulative permeation Q of PEG modified long-acting recombinant type III collagen versus time for performance test 2.
FIG. 6 is a graph showing the results of immunohistochemistry (100X) performed on the isolated mouse skin of test 24h in performance test 2.
Wherein A: PEG-rhCOL III 0.3mg/mL group; b: PEG-rhCOL III 0.1mg/mL group; c: rhCOL III 0.3mg/mL group; d: blank control (normal saline).
Fig. 7 is a graph showing the results of the model establishment of the skin damage of the mice after the 8% sodium sulfide treatment in performance test 3, wherein a: visual observation of the treated back of the mice; b: CBS dermoscope polarized light observation; c: CBS dermoscope ordinary light observation.
FIG. 8 shows skin repair and hair change (CBS polarized light, 50X) for each time period of the experimental and control groups in Performance test 3; the first layer is the skin repair and hair change conditions of the test groups at 1d, 3d, 7d and 10 d; the second layer is the skin repair and hair change conditions of the control groups at 1d, 3d, 7d and 10 d.
Detailed Description
Example 1
The first aspect of the present embodiment provides a method for preparing a PEG-modified long-acting recombinant collagen with high transdermal absorbability and hair restoration efficacy, comprising the steps of:
s1: 20mL of rhCOL III protein stock solution (5 mg/mL) was added to 80mL of 0.05mo1/L MES buffer solution at pH5.5, and then the same volume of 2-fold concentration EDC solution of the protein stock solution and cystamine were added to react at 4℃for 3 hours (molar ratio of rhCOL III, EDC, cystamine is 1:2:2), wherein the molar ratio of EDC and cystamine in the EDC solution is 1:1, a step of;
s2: after the reaction, a 50mM TCEP solution was added to carry out the reduction reaction, the molar ratio of TCEP to cystamine in the TCEP solution being 1: reacting for 2 hours at 1,4 ℃;
s3: after the reduction reaction, mPEG2-MAL having a molecular weight of 10kD was added to the reaction system, and the molar ratio of mPEG2-MAL to rhCOL III protein (based on the protein content) was 4:1, reacting for 20 hours at 4 ℃, namely, carrying out fixed point modification on the C-terminal carboxyl of rhCOL III to obtain PEG modified long-acting recombinant type III collagen solution;
s4: separating a modified product in the PEG modified long-acting recombinant type III collagen solution by adopting a DEAE Sepharose FF chromatographic medium, diluting the PEG modified long-acting recombinant type III collagen solution by 5 times with a phosphate buffer solution with the pH value of 6.5 and 10mmol/L, loading the diluted solution, balancing the solution with the phosphate buffer solution with the pH value of 6.5 and 10mmol/L, and setting DEAE Sepharose FF chromatographic medium separation parameters: and (3) performing linear gradient elution on 0.1-0.3mol/L NaCl for 5CV, wherein the flow rate is 5.0mL/min, the detection wavelength is 280nm, collecting elution peaks, and obtaining the PEG modified long-acting recombinant type III collagen after ultrafiltration, desalination and concentration.
The purity of the sample was checked by SDS-PAGE, and the detection results are shown in FIG. 1. The molecular weight of the PEG modifier used in the modification reaction is 10kD, the molecular weight of rhCOL III is 45kD, so that the newly-appearing band with the molecular weight of about 55kD can be deduced to be the PEGylated long-acting recombinant type III collagen, and a PEG chain is connected to a long-acting recombinant type III collagen molecule, which indicates that mPEG is only modified on the C-terminal carboxyl of fibronectin.
The DEAE Sepharose FF (anion exchange chromatography packing) was purchased from GE Healthcare.
The MES buffer, EDC solution, cystamine, PEGylation modifier (mPEG 2-MAL) related reagents were all purchased from sigma.
The chromatographic medium separation equipment is an AKTA purifier.
The rhCOL III protein stock solution is prepared according to patent number 202110358113.9 and batch number 20230220.
The second aspect of the present embodiment provides a PEG-modified long-acting recombinant type III collagen with high transdermal absorbability and hair restoration efficacy.
In a third aspect, the present embodiment provides an application of PEG-modified long-acting recombinant type III collagen, which is applied to the preparation of an article in the scalp field, where the article in the scalp field is a medical device for scalp.
The medical device for scalp comprises a wound dressing.
Example 2
The embodiment of example 2 is the same as that of example 1 except that the rhCOL protein stock solution used is rhCOL XVII protein stock solution. The rhCOL XVII protein stock solution is prepared according to the preparation method in the example 3 of the patent number 2023105645238.
Performance test 1: PEG-rhCOL III biological Activity assay
According to the requirements in YY/T1849-2022 recombinant collagen, the biological activity of the collagen prepared in example 1 is detected, and the in vitro biological activity detection is mainly carried out by using cell proliferation promotion.
1. Principle of cell proliferation promoting experiment
Dermal papilla mesenchymal cells (dermal papillary cell) are located at the bottom of hair follicles and have a unique role in inducing hair formation. The growth of hair depends on the follicular cells of the hair root, which takes at least three months from overgrowth to telogen, during which dermal papilla mesenchymal cells located at the bottom of the hair follicle are the core of the association and control of the entire follicular cell population, a part that helps to synthesize new hair and transport nutrients, and a key to helping the division of hair matrix cells into new hair. If DP cells are subjected to molecular fracture, the internal environment of the hair follicle is deteriorated, malnutrition of the hair root occurs, the division of hair mother cells is inhibited, and finally the whole hair follicle cells are always in the resting stage, so that the conditions of alopecia, hair breakage, hair quality deterioration and difficult growth of new hair are easy to occur at the moment, and even the death of the hair root cells can be caused.
The growth activity promotion effect of the PEG modified long-acting recombinant type III collagen (PEG-rhCOL III) on human DP cells is explored through in vitro cell test analysis. The method detects the proliferation promoting effect of PEG-rhCOL III on cells through a proliferation promoting test of hDPC.
2. Experimental method
2.1 Experimental materials
Test article: PEG modified Long-acting recombinant collagen III (PEG-rhCOL III), prepared in example 1, lot number 20230223.
Control: rhCOL III, prepared according to patent No. 202110358113.9, lot No. 20230220.
Cell lines: human DP cells (hdcp), passage 5, frozen lot number 20221215, purchased from ATCC in the united states.
Other reagents (DMEM medium, PBS, 0.25% trypsin) and other items (96 well cell culture plates, TIP heads and micropipettes) were routine in the laboratory, sterilized or filter sterilized prior to testing, and sterile tested.
2.2 detection method
(1) The hDPC cell strain is cultured with DMEM culture solution at 37deg.C and 5% carbon dioxide, and the cell concentration is controlled to 1.0X10 per 1mL 5 -5.0×10 5 Individual cells, 24-36 hours after passage, were used for biological activity assays.
(2) The culture medium in the flask was discarded, and the cells were digested and collected using DMEM medium to prepare a culture medium containing 5.0X10 s/1 mL 4 ～8.0×10 4 The cell suspension of each cell is inoculated in a 96-well plate, shaking is carried out continuously in the inoculation process, the inoculation number of each well is kept the same, 100 mu L of each well is cultured at 37 ℃ under the condition of 5% carbon dioxide.
(3) After 24 hours of incubation, different doses of test solution and control were added, 100 μl per well, and incubated at 37deg.C with 5% carbon dioxide for 7d. In addition to the sample groups, negative controls (no test or control added) were also set, with 2 duplicate wells per group.
(4) MTT colorimetric method
mu.L of MTT solution was added to each well and incubated at 37℃for 5 hours with 5% carbon dioxide, and the above procedure was performed under aseptic conditions. After the liquid in the culture plate was discarded, 100. Mu.L of DMSO was added to each well, and after the mixture was dissolved and mixed well, absorbance was measured on an ELISA apparatus using 630nm as a reference wavelength and 570nm as a test wavelength, and the measurement result was recorded.
(5) Data processing
The test data are processed by a computer program or a four-parameter regression calculation method.
3. Result calculation
3.1 Cell status
The cell state is shown in FIGS. 1 and 2. Only a few cells survived the negative control well cells, and the negative control experiment was established.
3.2 proliferation promoting Rate
The results of the cell proliferation activity are shown in Table 1 and FIG. 3.
TABLE 1
As can be seen from the results in Table 1 and FIG. 3, the PEG-rhCOL III dose groups were higher than the proliferation effect of normal long-acting recombinant type III collagen hDPC cells.
Conclusion 4
The experimental result of the in vitro cytology efficacy model shows that the PEG modified long-acting recombinant type III collagen (PEG-rhCOL III) has the effect of promoting the proliferation of dermal papilla mesenchymal cells, thereby promoting the hair growth, and the proliferation promoting effect is better than that of the common long-acting recombinant type III collagen.
Performance test 2: percutaneous absorption test
Principle of test
The protein solution was subjected to transdermal absorption in a vertical Franz diffusion cell placed in a drug transdermal diffusion tester and incubated in a constant temperature circulating water bath at 37 ℃. Evaluation of in vitro transdermal Properties of PEG-modified Long-acting recombinant type III collagen and general Long-acting recombinant type III collagen
Two test methods
1. Test materials:
1.1 ex vivo skin: isolated skin was prepared by selecting 6-8 week old BALB/c mice.
1.2 main test reagents: PEG modified long-acting recombinant type III collagen (PEG-rhCOL III protein) prepared in example 1, lot number 20230223; a long-acting recombinant type iii collagen control, prepared according to patent No. 202110358113.9; physiological saline; type iii collagen mab (available from abcam, uk).
1.3 laboratory apparatus: the Honghua ZTY intelligent transdermal tester has a maximum capacity of 20mL in a receiving tank,effective transdermal area 1.36cm 2 。
2 test procedure
2.1 ex vivo skin preparation
Selecting BALB/c mice with age of 6-8 weeks, performing cervical dislocation after the mice are adaptively raised, rapidly scraping off hair on the abdominal skin, peeling off the hairless abdominal skin, removing subcutaneous fat, repeatedly washing with normal saline, wiping, wrapping with aluminum foil paper, and storing in a safe box at-20 ℃ in a refrigerator for standby (not more than 1 month). The frozen products are taken out every other day before the experiment (0-5 ℃), and repeatedly rinsed with normal saline until the products are clarified before use. And simultaneously taking part of skin, and fixing and preparing for detection by using a fixing liquid.
2.2 in vitro transdermal test
2.2.1 murine cortex mounting
The transdermal absorption test was performed in a vertical Franz diffusion cell placed in a drug transdermal diffusion tester. The treated ex vivo skin was immobilized intermediate the supply and receiving reservoirs with the stratum corneum portion facing the supply reservoir and the dermis layer facing the receiving chamber. The temperature of the water bath system is regulated to 37 ℃, the stirring speed is 100r/min, physiological saline with the pre-temperature of 37 ℃ is added into the receiving chamber, and bubbles are discharged. To reduce the interference, the inner surface of the rat skin is contacted with the receiving liquid under the condition of no administration, and then 5mL of solutions with different concentrations are injected into the supply chamber to be closely attached to the rat skin.
2.2.2 sampling
1mL of the receiving solution was withdrawn as a sample solution by syringe at 1, 2, 4, 6, 8, 12 and 24 hours after the start of the test, respectively. At the same time, the receiving chamber was filled with an equal amount of physiological saline solution. Finally, the collected sample liquid in each time period is detected.
2.2.3 test groups
A blank group (normal saline), a rhCOL III group and a PEG-rhCOL III group are arranged as a III group, wherein two groups of protein groups are arranged as two subgroups, and the two groups of protein groups are diluted to the concentration of 0.1mg/L and 0.3mg/mL by using the normal saline, and 5 groups of samples are respectively added into a supply tank for simultaneous test.
3 results detection
3.1 protein content detection
The protein content in the receiving liquid is measured by adopting a coomassie brilliant blue method through receiving liquid samples taken at each time point, and the receiving liquid samples are prepared according to a formulaThe cumulative permeation quantity (Q) is calculated.
Q is the cumulative permeation quantity (mg cm) at the nth sampling 2 ) V is the volume of the receiving pool (20 mL), V i Volume for the ith sample (1 mL), C i Drug concentration in the receiving solution measured for the ith sample, C n The concentration of the sampling point at the nth time; s is the transdermal area.
3.2 preparation and observation of immunohistochemical sections
Immunohistochemical sections were prepared by fixing the transdermal portions (including the hair follicle sites and the non-hair follicle sites) of the murine skin after 24h of the 5-group experiments. The prepared immunohistochemical sections are placed under a microscope to observe the existence position of collagen in each group of skin tissues.
3.3 flow for preparation of immunohistochemical sections
(1) And (5) placing the slices into a drying oven to bake the slices for 30min at 66 ℃.
(2) 3 passes of xylene were sequentially passed, each for 5min.
(3) Sequentially passing through 3 times (100% -95% -80%) of ethanol for 3min each time.
(4) The slices were placed in a beaker, slowly rinsed with running water, and the ethanol was washed away until the slices were clean and transparent.
(5) Antigen high pressure repair: 2000mL of citrate repair liquid with pH of 6.0 is prepared in the pressure cooker, the electromagnetic oven is heated to boiling, the cut slices are put on the cover of the pressure cooker, the air injection is carried out for 2 minutes, then the heating is stopped, and the pressure cooker cover is slowly flushed by flowing water until the pressure cooker cover is cooled.
(6) Blocking endogenous peroxidases: slice is put into 3%H 2 O 2 Incubate at room temperature, wash 3 times with distilled water, draw a hydrophobic ring, wash 3 times with PBS-T.
(7) Removing excessive liquid on the slice, dripping primary antibody (III type collagen monoclonal antibody), capping, and incubating in a 37 ℃ incubator for 60min. Sections were removed and washed 3 times with PBS-T.
(8) And (5) removing redundant liquid on the slices, dripping secondary antibody, capping, and placing in a 37 ℃ incubator for incubation for 30min. Sections were removed and washed 3 times with PBS-T.
(9) Removing excessive liquid on the slice, dripping DAB color-developing agent, controlling the color-developing time under a microscope, stopping the color development positively, and washing with distilled water.
(10) Lining with hematoxylin for 2-5min, and washing with water; the 1% hydrochloric acid alcohol is differentiated for a few seconds, and washed clean.
(11) The lithium carbonate solution was blued for 30s and washed clean with water.
(12) Conventional dehydration, xylene is transparent.
(13) And (5) sealing the neutral resin, and observing the result by a microscope. Brown was a positive reaction.
4 experimental results
4.1 sample protein concentration for each time period
Protein concentration detection is carried out on samples collected in each time period by adopting a Coomassie brilliant blue method, and the protein concentration detection is carried out according to a formula
The cumulative permeation per unit area (Q) at each time point was calculated, and the results of spotting the concentration of the long-acting recombinant type III collagen at each time point and the cumulative permeation per unit area Q are shown in Table 2.
The results of the concentration and the cumulative permeation quantity Q per unit area of the PEG modified long-acting recombinant III type collagen are shown in Table 3. Wherein, the unit of sample concentration is mug/mL; q value unit [ mu ] g/cm 2 。
TABLE 2
TABLE 3 Table 3
PEG modified long-acting recombinant type III collagen percutaneous permeation cumulative permeation quantity Q and time relation curve: the cumulative permeation per unit area versus time t linear curve is shown in figure 5 using Excel 2019 software plotted as Q versus time t.
Statistical analysis of the data was performed using SPSS 26.0. The t test between the test 3 group and the test 1 group and the test 2 group shows that P is less than 0.01 and has obvious difference; the difference is significant in p=0.015 < 0.05 between the test 3 and the test 4 groups.
From the results, the PEG modified long-acting recombinant type III collagen has transdermal performance, and the common long-acting recombinant type III collagen cannot be transdermal.
4.2 immunohistochemical results
Immunohistochemical detection was performed on the isolated mouse skin tested for 24h, and the results showed that: PEG-rhCOL III protein histone is transdermal through hair follicle (red arrow in figures 6A and 6B), presents obvious brown positive reaction, wherein 0.3mg/mL is compared with 0.1mg/mL group, scattered positive reaction exists at each part of epidermis and dermis layers, more protein penetrates into skin, and dose dependence is presented; whereas the rhCOL III proteome has substantially no protein penetration, no positive reaction at hair follicle (FIG. 6C, red arrow); the control hair follicles of fig. 6D also had no positive response.
Conclusion 5
According to the test result, the PEG modified long-acting recombinant III type collagen has obvious transdermal absorption effect.
Performance test 3: skin damage repair and hair growth test
1 test purpose:
in the test, the effect of the experimental sample on repairing skin damage and promoting hair growth is measured by smearing high-concentration sodium sulfide on the back skin of a mouse once to cause hair to fall off and damage the epidermis layer of the skin, simulating the condition of hair loss after scalp damage caused by chemical agents, and evaluating the repair time of skin damage and the hair regeneration condition of a hair removal area by using a dermatoscope for observation.
2 test materials and methods:
2.1 test animals: healthy adult black female C57 mice were selected for 10 animals, kept in a clean-grade environment, given full feed and free water feeding, fed water daily, and examined for animal physiological activity.
2.2 test reagents: PEG-rhCOL III protein prepared in example 1, batch number 20230223; a long-acting recombinant type III collagen rhCOL III control, prepared according to patent number 202110358113.9; 8% sodium sulfide;
2.3 test instruments and equipment: CBS dermoscope; a sterile cotton swab; alcohol cotton ball; iodophors, and the like.
2.4 test methods
2.4.1 test group: 10 adult mice were randomly aliquoted into 2 groups of 5 animals each, and the test group conditions are shown in Table 4.
TABLE 4 Table 4
2.4.2 establishing a mouse skin loss model: after the mice were anesthetized, the hair on the back was shaved, and the back was applied with 8% sodium sulfide for dehairing treatment, and after 5min, the sodium sulfide residue was washed off with purified water, and the back skin area was dehaired and the epidermis layer was damaged artificially.
2.4.3 treatment regimen: the different protein solutions were diluted to 0.3mg/mL with physiological saline, and the respective diluted solutions were applied to the dehairing areas of two groups of mice, once a day, for 7 consecutive days, respectively.
2.4.4 skin and hair observations: the skin damage repair state, hair growth state and skin color change of the mice are observed every day, and the skin change and hair occurrence time are counted until all the hair appears.
3. Test results
3.1 establishment of mouse skin damage model
Mice were treated according to the test protocol and after treatment were observed subcutaneously using CBS dermoscope with normal light skin surface and polarized light, respectively. From the observation result of the polarized light in fig. 7B, it is easier to observe whether the skin of the mouse is recovered subcutaneously or whether there is capillary congestion or the like.
3.2 skin and hair observations
After the skin damage model is established, the skin repair condition change and the hair occurrence time are counted, and typical changes of the experimental group and the control group are compared with each other, and the typical changes are shown in fig. 8.
As can be seen from the results of FIG. 2, PEG-rhCOL III promoted healing time of damaged skin 3-4 d earlier than rhCOL III. Hair growth has begun at the 7d broken skin after application, and the 10d skin has been substantially completely restored to normal hair growth.
Conclusion 4: the PEG-rhCOL III protein solution prepared in example 1 can promote healing of damaged skin and promote hair growth recovery at damaged parts.
Claims (10)
1. The preparation method of the PEG modified long-acting recombinant collagen for scalp repair is characterized by comprising the following steps of:
s1: adding the rhCOL protein stock solution into MES buffer solution, then adding EDC solution and cystamine to react for 2.5-3.5 hours at the temperature of 4 ℃;
s2: after the reaction, adding TCEP solution to perform reduction reaction, and reacting for 1.5-2.5 hours at 4 ℃;
s3: after the reduction reaction, adding mPEG2-MAL into a reaction system, and reacting for 18-22 hours at the temperature of 4 ℃, namely, carrying out fixed point modification on the C-terminal carboxyl of rhCOL to obtain a PEG modified long-acting recombinant collagen solution;
s4: and separating the modified product in the PEG modified long-acting recombinant collagen solution by adopting a chromatographic medium to obtain the PEG modified long-acting recombinant collagen for scalp repair.
2. The method for preparing a PEG-modified long-acting recombinant collagen for scalp repair according to claim 1, wherein the rhCOL protein stock solution comprises rhCOL III protein stock solution or rhCOL x vii protein stock solution.
3. The method for preparing PEG-modified long-acting recombinant collagen for scalp repair according to claim 1, wherein the molar ratio of EDC and cystamine in the EDC solution is (0.8-1.2): 1.
4. the method for preparing a PEG-modified long-acting recombinant collagen for scalp repair according to claim 1, wherein the concentration of the TCEP solution is 40 to 60mM.
5. The method for preparing the PEG-modified long-acting recombinant collagen for scalp repair according to claim 1, wherein the molar ratio of TCEP to cystamine in the TCEP solution is (0.8-1.2): 1.
6. the method for preparing a PEG-modified long-acting recombinant collagen for scalp repair according to claim 1, wherein the molecular weight of mPEG2-MAL is 10kD.
7. The method for preparing a PEG-modified long-acting recombinant collagen for scalp repair according to claim 1, wherein the molar ratio of mPEG2-MAL to rhCOL protein is (3-5): 1.
8. the method for preparing the PEG-modified long-acting recombinant collagen for scalp repair according to claim 1, wherein the specific steps in S4 are as follows: separating a modified product in the PEG modified long-acting recombinant collagen solution by adopting a DEAE Sepharose FF chromatographic medium, diluting the PEG modified long-acting recombinant collagen solution by 3-7 times by using a phosphate buffer solution with pH of 6-7 and 8-12 mmol/L, and then loading the diluted PEG modified long-acting recombinant collagen solution, wherein the balancing solution is the phosphate buffer solution with pH of 6-7 and 8-12 mmol/L.
9. A PEG-modified long-acting recombinant collagen for scalp repair prepared by the method for preparing a PEG-modified long-acting recombinant collagen for scalp repair according to any one of claims 1 to 8.
10. The use of PEG-modified long-acting recombinant collagen for scalp repair according to claim 9, characterized by being applied to the preparation of scalp field products including skin care products for scalp, medical devices for scalp, medicines for scalp.
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