CN115944545A - Triple-helix collagen dermal hydrogel and preparation method and application thereof - Google Patents
Triple-helix collagen dermal hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a triple-helix collagen dermal hydrogel and a preparation method and application thereof, and relates to the field of beauty. The preparation method comprises (1) the pretreatment of the pigskin; (2) performing deimmunization treatment on the pigskin; (3) filter aid adsorption; (4) separating and purifying; (5) The preparation method comprises the steps of hydrogel preparation and (6) degerming and filtering, wherein pigskin is used as a collagen source, the obtained active collagen has a complete triple-spiral structure consistent with human skin collagen through specific treatment, and the collagen hydrogel is obtained through polysaccharide modification and can be used for supplementing skin extracellular matrix collagen to achieve an anti-aging effect.
Description
Technical Field
The invention relates to the field of beauty, in particular to a triple-helix collagen dermal hydrogel and a preparation method and application thereof.
Background
The skin is the largest organ of the human body and the outer barrier of the human body, and the integrity of the skin plays an important role for human beings. With age, aging skin cells accumulate gradually and can transmit the aging phenotype to neighboring cells by paracrine means, which can induce or accelerate aging-related dysfunction of the skin's neighboring cells. Resulting in a decrease in skin thickness, regenerative capacity and barrier effect, characterized by thinning of the skin, fine lines.
Collagen is the most main component of connective tissue of the dermis layer of the skin, has good biological properties and functions, and is mainly shown in the following steps: 1. low antigenicity; 2. biodegradability: under the action of protease in vivo, the peptide chain of collagen is hydrolyzed and gradually broken to cause the destruction of triple helix structure, so that the collagen is hydrolyzed, 80% of collagen degradation products can be recycled as amino acid, and 20% of the collagen degradation products are metabolized to form urea; 3. good biocompatibility: the collagen has good interaction with host cells and tissues, shows the coordination of mutual influence and has good capacity of inducing the regeneration of autologous collagen.
However, since natural collagen has a large molecular weight and is a protein substance, the later processing is limited to a certain extent, and particularly, when the natural collagen is applied to cosmetics, besides good film-forming moisturizing performance, better technical inventions and applications are needed in the aspects of improving solubility and exerting anti-aging effect through self-assembly. In the disclosed patent, the treatment of lipid is difficult to remove fully, the obtained collagen solution is mostly in an opaque state, and in addition, the collagen obtained by the existing patent has poor water solubility and large molecular weight, so the application of the collagen in the field of skin care products is greatly limited.
The prior art of collagen focuses more on extraction and purification of collagen, and is mainly used for the form of an implant in the field of beauty, the technology of the collagen focuses on the completeness of a triple-helix structure, the more complete the collagen structure is, the larger the molecular weight is, the poorer the water solubility is, and even the collagen is completely insoluble in water, the smoothness and the dispersion uniformity of the injection are influenced in the aspect of practical application, and only film forming and moisture retention can be achieved on a smearing type skin care product, but the collagen cannot permeate and resist aging at all.
For example, chinese patent application 202111067335.1 discloses a process for extracting high purity collagen, which comprises an achilles tendon pretreatment step and a collagen extraction and purification step, wherein in the implementation process, the removal efficiency of achilles tendon fat in the pretreatment process is obviously improved by reasonably controlling the reagents in the pretreatment process, the concentrations of various reagents, the mixing mode and the addition sequence, the purity and the activity of the collagen are improved, and the obtained collagen has a complete triple helix structure, has better safety, can meet the requirements of cosmetics, medical instruments, tissue engineering and the like, but the collagen prepared by the application does not pay attention to the water solubility and the molecular weight.
For another example, chinese patent application 202111067165.7 discloses a collagen implant and a method for preparing the same, and the present invention also provides a method for preparing collagen fiber, including: (1) pretreatment of an animal achilles tendon; (2) carrying out enzymolysis digestion; (3) filter aid adsorption; (4) separating and purifying; (5) The collagen molecules obtained by the method have a fiber structure with clear shape after self-aggregation, each fiber is completely exposed and mutually staggered to form a fiber net, and meanwhile, a periodic transverse striation D zone of the collagen fiber can be clearly observed, so that a better fiber shape can be formed, and the molecular regularity is higher; the collagen fiber has uniform thickness and better elastic strength, but the collagen prepared by the application does not pay attention to the water solubility and the molecular weight.
Therefore, it is required to provide an active collagen which is safe, non-toxic, and has de-immunogenicity, has a complete triple-helical structure consistent with human skin collagen, and is modified to obtain a collagen hydrogel for supplementing skin extracellular matrix collagen to achieve an anti-aging effect.
Disclosure of Invention
In order to achieve the above objects, the present application provides a triple-helical collagen dermal hydrogel, a method for preparing the same, and applications thereof. The application adopts the pigskin as a collagen source, the obtained active collagen has a complete triple-helix structure consistent with the collagen of human skin through specific treatment, and the collagen hydrogel is obtained through polysaccharide modification, so that the collagen hydrogel can be used for supplementing skin extracellular matrix collagen, and the anti-aging effect is achieved.
In order to achieve the technical effects of the invention, the invention is realized by the following technical scheme:
in one aspect, the present application provides a method for preparing a triple-helical collagen dermal hydrogel, comprising the steps of:
(1) Pretreatment of pigskin:
adding the animal pigskin into a trypsin solution containing EDTA for soaking and washing; then soaking and shaking the pigskin by using a descaling agent solution, and washing the pigskin to obtain pretreated pigskin;
(2) Deimmunization treatment of pigskin:
adding the pigskin pretreated in the step (1) into a neutral protease solution for soaking and washing; then soaking and shaking the mixture by using a descaling agent solution, and washing the mixture; repeating the step (1) and the step (2) for 1-3 times to obtain the pig skin without immunogenicity;
(3) Alpha-1, 3-galactose antigen treatment of pigskin:
soaking the pig skin without immunogenicity obtained in the step (2) in an alpha-1, 3-galactosidase solution, and washing to obtain a precipitate A;
(4) Filter aid and adsorption:
adding a filter aid solution into the precipitate A obtained in the step (3), stirring, filtering, and collecting supernatant for later use;
(5) Separation and purification:
concentrating the supernatant collected in the step (4), adsorbing collagen through a cation exchange medium, and then eluting and separating to finally obtain an eluent;
(6) Preparing hydrogel:
adding modified polysaccharide with the concentration of 10-40mg/mL into the eluent in the step (5), wherein the mass ratio of the eluent to the modified polysaccharide is (5) - (1-1);
(7) And (3) degerming and filtering:
and (4) sterilizing and filtering the hydrogel obtained in the step (6) by using a 0.22um filter membrane to obtain a sterile soluble collagen hydrogel solution.
The concentration of the EDTA in the trypsin solution containing EDTA in the step (1) is 0.01-0.04 wt%; the concentration of the trypsin is 0.25wt% -0.35wt%;
preferably, the concentration of EDTA in the EDTA-containing trypsin solution described in the above step (1) is 0.025wt%; the concentration of the trypsin was 0.3wt%.
The detergent solution in the step (1) and the step (2) is a phosphate buffer solution containing 0.05-0.15wt% of an ionic detergent, the ionic detergent is a mixture of sodium dodecyl sulfate and TritonX-200, and the mass ratio of the sodium dodecyl sulfate to the TritonX-200 is 4:6.
wherein the phosphate buffer solution is pH 6.0-8.0 sodium phosphate buffer solution, preferably pH7.2 sodium phosphate buffer solution.
The temperature for soaking by the trypsin solution containing EDTA in the step (1) is 30-37 ℃, preferably 37 ℃; the time is 12-24h, preferably 24h.
The temperature for soaking the water in the descaling agent solution in the step (1) is 24-32 ℃, and preferably 30 ℃; the time is 12-24h, preferably 20h.
The concentration of the neutral protease solution in the step (2) is 500-700U/L; preferably 700U/L.
The concentration of the alpha-1, 3-galactosidase solution in the step (3) is 10-20 mu g/mL; preferably 20. Mu.g/mL.
The temperature for soaking by the neutral protease solution in the step (2) is 28-35 ℃, and preferably 35 ℃; the time is 12-24h, preferably 24h.
The temperature for soaking the water in the descaling agent solution in the step (2) is 24-32 ℃, and preferably 30 ℃; the time is 12-24h, preferably 20h.
The temperature for soaking the alpha-1, 3-galactosidase solution in the step (3) is 24-37 ℃, and preferably 37 ℃; the time is 1-4h, preferably 3h.
The filter aid in the step (4) is selected from one or more of diatomite, perlite, cellulose, asbestos, magnesium oxide and activated carbon; preferably diatomaceous earth.
The mass ratio of the filter aid to the precipitate A is 0.5-3:1; preferably 1:1.
the cation exchange medium in the step (5) is one or more selected from DEAE (N, N-diethylaminoethyl) Sepharose Fast Flow, Q (N, N-diethylamino-2-hydroxypropyl), SP (sulfopropyl) -Sephadex dextran gel; preferably an SP (sulfopropyl) -Sephadex dextran gel.
The elution solution for the elution separation described in the above step (5) is 10mM Tris-HCl aqueous solution containing 0.5-0.9wt% of sodium salt.
The modified polysaccharide in the step (6) is one or more of methacrylic anhydride modified hyaluronic acid, oat beta-glucan, aldehyde modified hyaluronic acid and N-hydroxy thiosuccinimide;
preferably, the modified polysaccharide is prepared by mixing the following components in a mass ratio of 2:5:3 methacrylic anhydride modified hyaluronic acid, oat beta-glucan and aldehyde group modified hyaluronic acid.
The preparation method of the Methacrylic Anhydride (MA) modified hyaluronic acid comprises the following steps:
hyaluronic Acid (HA) powder 1g was weighed and dissolved in 100mL of deionized water, and the solution was stirred well to prepare a 1% HA solution. Subsequently, 064mL of MA was slowly added dropwise to the HA solution, and the reaction solution was adjusted to and maintained at pH 8.0 while dropwise addition was carried out for 4 hours while maintaining the pH 8.0 during the reaction. After the reaction is finished, repeatedly washing the reaction solution with ethanol to obtain flocculent precipitate, centrifuging, then re-dissolving the white precipitate obtained by centrifuging in deionized water, dialyzing for 5 days by using a dialysis bag (with the molecular weight cutoff of 8000-14000 kDa), and drying in a vacuum freeze dryer to obtain the modified spongy HAMA.
The preparation method of the aldehyde hyaluronic acid comprises the following steps:
hyaluronic Acid (HA) and sodium periodate, having a molecular weight of 60KDa, were dissolved in deionized water, respectively. And slowly dripping the sodium periodate solution into the HA solution, reacting for 3 hours at room temperature in a dark place, putting into a dialysis bag (with molecular weight cut-off of 8000-14000 kDa) for dialysis for 5 days after the reaction is finished, and putting into a vacuum freeze dryer for drying after the reaction is finished to obtain the spongy aldehyde hyaluronic acid.
Wherein the hyaluronic acid is moistuHA produced by Dongying Foster bioengineering limited company, and molecular weight of hyaluronic acid is 20KDa-2000KDa; oat beta-glucan (1%) was produced by Guangzhou beta health Biotech limited, lot number BT2212101005B
In another aspect, the present invention also provides a triple-helical collagen dermal hydrogel prepared by the above method.
In still another aspect, the present invention also provides the use of the triple-helical collagen dermal hydrogel in the preparation of cosmetics.
The cosmetic is selected from one or more of essence water, lotion, essence cream and facial cream.
Compared with the prior art, the invention has the beneficial effects that:
(1) The collagen hydrogel prepared by the application preserves a complete triple-helix structure, is equal to or larger than 9% in hydroxyproline content of collagen through detection, is consistent with human skin collagen, can be used for supplementing skin extracellular matrix collagen, and achieves an anti-aging effect;
(2) The collagen hydrogel prepared by the method has good water solubility, the prepared hydrogel has good water absorption and swelling properties, and a large amount of water is absorbed by hydrophilic macromolecules;
(3) The collagen hydrogel prepared by the method has small molecular weight and can be better absorbed;
(4) The collagen hydrogel prepared by the method has good light transmission and transparency.
Drawings
FIG. 1 is a gel-like collagen hydrogel prepared in example 3 after stirring at 37 ℃ at room temperature;
FIG. 2 is a porous three-dimensional structural view of a collagen hydrogel prepared in example 3;
fig. 3 is a fluorescent microscope observation result of the permeation behavior of the collagen hydrogel prepared in example 3.
Detailed Description
The features mentioned above, or those mentioned in the embodiments, can be combined in any way. All the features disclosed in this specification may be combined in any suitable manner and each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The following examples are conducted under conditions specified, usually according to conventional conditions or according to conditions recommended by the manufacturer. All percentages and fractions are by weight unless otherwise specified.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are exemplary only.
The trypsin and the neutral protease used in the following examples are both produced by Shandong Kete enzyme preparation GmbH, and the activities thereof are 4K units and 10W units, respectively; the alpha-1, 3-galactosidase used was produced by Xiasang (Beijing) Biotechnology development, inc., and its activity was 500 units; the hyaluronic acid is moistuHA produced by Dongying Foster bioengineering limited company, and the molecular weight of the hyaluronic acid is 20KDa-2000KDa; the oat beta-glucan (1%) is produced by Guangzhou beta health biotechnology limited company, and the batch number is BT2212101005B.
The Methacrylic Anhydride (MA) modified hyaluronic acid described in basic example 1 was prepared as follows:
hyaluronic Acid (HA) powder 1g was weighed and dissolved in 100mL of deionized water, and the solution was stirred well to prepare a 1% HA solution. Subsequently, 064mL of MA was slowly added dropwise to the HA solution, and the reaction solution was adjusted and maintained at pH 8.0 while dropwise addition was carried out for 4 hours, while maintaining the reaction solution at pH 8.0 during the reaction. After the reaction is finished, repeatedly washing the reaction solution with ethanol to obtain flocculent precipitate, centrifuging, then re-dissolving the white precipitate obtained by centrifuging in deionized water, dialyzing for 5 days by using a dialysis bag (with the molecular weight cutoff of 8000-14000 kDa), and drying in a vacuum freeze dryer to obtain the modified spongy HAMA.
The preparation method of the aldehyde-modified hyaluronic acid described in basic embodiment 2 is as follows:
hyaluronic Acid (HA) and sodium periodate, having a molecular weight of 60KDa, were dissolved in deionized water, respectively. And slowly dripping the sodium periodate solution into the HA solution, reacting for 3 hours at room temperature in a dark place, putting into a dialysis bag (with molecular weight cut-off of 8000-14000 kDa) for dialysis for 5 days after the reaction is finished, and putting into a vacuum freeze dryer for drying after the reaction is finished to obtain the spongy aldehyde hyaluronic acid.
Example 1 preparation of triple helix collagen dermal hydrogel
The method comprises the following steps:
(1) Pretreatment of pigskin:
adding animal Corii Sus Domestica into 0.25wt% trypsin solution containing 0.01wt% of EDTA, soaking at 30 deg.C for 24 hr, and washing; then soaking the pigskin for 24 hours at 24 ℃ by using phosphate buffer solution (pH 6.0) containing 0.05wt% of ionic detergent, shaking and washing to obtain pretreated pigskin;
(2) Deimmunization treatment of pigskin:
adding the pigskin pretreated in the step (1) into a neutral protease solution with the concentration of 500U/L, soaking for 24h at 28 ℃, and washing; then soaking the mixture for 24 hours at 24 ℃ by using phosphate buffer solution (pH 6.0) containing 0.05wt% of ionic detergent, shaking and washing; repeating the step (1) and the step (2) for 1-3 times to obtain the pig skin without immunogenicity;
(3) Alpha-1, 3-galactose antigen treatment of pigskin:
soaking the deimmunized pigskin obtained in the step (2) in 10 microgram/mL alpha-1, 3-galactosidase solution at 24 ℃ for 4 hours, and washing to obtain a precipitate A;
(4) Filter aid and adsorption:
adding a diatomite solution into the precipitate A obtained in the step (3), stirring, filtering, and collecting a supernatant for later use;
(5) Separation and purification:
concentrating the supernatant collected in the step (4), adsorbing collagen by SP (sulfopropyl) -Sephadex Sephadex gel, eluting and separating by using 10mM Tris-HCl aqueous solution containing 0.5wt% sodium salt, and finally obtaining eluent;
(6) Preparing hydrogel:
adding modified polysaccharide with the concentration of 10mg/mL into the eluent in the step (5), wherein the mass ratio of the eluent to the modified polysaccharide is 1;
(7) And (3) degerming and filtering:
and (5) sterilizing and filtering the hydrogel obtained in the step (6) by using a 0.22um filter membrane to obtain a sterile soluble collagen hydrogel solution.
The ionic descaling agent in the step (1) and the step (2) is prepared from the following components in a mass ratio of 4:6, a mixture of sodium dodecyl sulfate and TritonX-200;
the mass ratio of the filter aid to the precipitate A in the step (4) is 0.5:1;
the modified polysaccharide in the step (6) is prepared by the following steps of (1) by mass: 5:3 methacrylic anhydride modified hyaluronic acid, oat beta-glucan and aldehyde group modified hyaluronic acid.
Example 2 preparation method of triple helix collagen dermal hydrogel
The method comprises the following steps:
(1) Pretreatment of pigskin:
adding animal Corii Sus Domestica into 0.35wt% trypsin solution containing 0.04wt% of EDTA, soaking at 37 deg.C for 12 hr, and washing; then soaking the pigskin for 12 hours at 32 ℃ by using a phosphate buffer solution (pH 8.0) containing 0.15wt% of an ionic detergent, oscillating and washing to obtain pretreated pigskin;
(2) Deimmunization treatment of pigskin:
adding the pigskin pretreated in the step (1) into a neutral protease solution with the concentration of 600U/L, soaking for 12h at 35 ℃, and washing; then soaking the mixture for 12 hours at 32 ℃ by using a phosphate buffer solution (pH 8.0) containing 0.15wt% of an ionic detergent, shaking and washing the mixture; repeating the step (1) and the step (2) for 1-3 times to obtain the pig skin without immunogenicity;
(3) Alpha-1, 3-galactose antigen treatment of pigskin:
soaking the deimmunized pigskin obtained in the step (2) in 15 mu g/mL alpha-1, 3-galactosidase solution at 37 ℃ for 1h, and washing to obtain a precipitate A;
(4) Filter aid and adsorption:
adding a diatomite solution into the precipitate A obtained in the step (3), stirring, filtering, and collecting a supernatant for later use;
(5) Separation and purification:
concentrating the supernatant collected in the step (4), adsorbing collagen by SP (sulfopropyl) -Sephadex Sephadex gel, eluting and separating by using 10mM Tris-HCl aqueous solution containing 0.9wt% sodium salt, and finally obtaining eluent;
(6) Preparing hydrogel:
adding modified polysaccharide with the concentration of 40mg/mL into the eluent in the step (5), wherein the mass ratio of the eluent to the modified polysaccharide is 5;
(7) And (3) degerming and filtering:
and (4) sterilizing and filtering the hydrogel obtained in the step (6) by using a 0.22um filter membrane to obtain a sterile soluble collagen hydrogel solution.
The ionic descaling agent in the step (1) and the step (2) is prepared from the following components in a mass ratio of 4:6, a mixture of sodium dodecyl sulfate and TritonX-200;
the mass ratio of the filter aid to the precipitate A in the step (4) is 3:1;
the modified polysaccharide in the step (6) is prepared by the following steps of (1) by mass: 5:3 methacrylic anhydride modified hyaluronic acid, oat beta-glucan and aldehyde group modified hyaluronic acid.
Example 3 preparation of triple helix collagen dermal hydrogel
The method comprises the following steps:
(1) Pretreatment of pigskin:
adding animal Corii Sus Domestica into 0.3wt% trypsin solution containing 0.025wt% EDTA, soaking at 37 deg.C for 24 hr, and washing; then soaking the pigskin in a phosphate buffer solution (pH 7.2) containing 0.10wt% of an ionic detergent at 30 ℃ for 20 hours, shaking and washing to obtain pretreated pigskin;
(2) Deimmunization treatment of pigskin:
adding the pigskin pretreated in the step (1) into a neutral protease solution with the concentration of 700U/L, soaking for 24h at 35 ℃, and washing; then soaking the mixture for 20 hours at 30 ℃ by using a phosphate buffer solution (pH 7.2) containing 0.10wt% of an ionic detergent, shaking and washing; repeating the step (1) and the step (2) for 1-3 times to obtain the pig skin without immunogenicity;
(3) Alpha-1, 3-galactose antigen treatment of pigskin:
soaking the deimmunized pigskin obtained in the step (2) in 20 microgram/mL alpha-1, 3-galactosidase solution at 37 ℃ for 3 hours, and washing to obtain a precipitate A;
(4) Filter aid and adsorption:
adding a diatomite solution into the precipitate A obtained in the step (3), stirring, filtering, and collecting a supernatant for later use;
(5) Separation and purification:
concentrating the supernatant collected in the step (4), adsorbing collagen by SP (sulfopropyl) -Sephadex Sephadex gel, eluting and separating by using 10mM Tris-HCl aqueous solution containing 0.8wt% sodium salt, and finally obtaining eluent;
(6) Preparing hydrogel:
adding modified polysaccharide with the concentration of 25mg/mL into the eluent in the step (5), wherein the mass ratio of the eluent to the modified polysaccharide is 3.5;
(7) And (3) degerming and filtering:
and (5) sterilizing and filtering the hydrogel obtained in the step (6) by using a 0.22um filter membrane to obtain a sterile soluble collagen hydrogel solution.
The ionic descaling agent in the step (1) and the step (2) is prepared from the following components in a mass ratio of 4:6, a mixture of sodium dodecyl sulfate and TritonX-200;
the mass ratio of the filter aid to the precipitate A in the step (4) is 1:1;
the modified polysaccharide in the step (6) is prepared by the following steps of (1) by mass: 5:3 methacrylic anhydride modified hyaluronic acid, oat beta-glucan and aldehyde group modified hyaluronic acid.
Comparative example 1
The difference from example 2 is that: no scale remover is added in the step (1) and the step (2), namely:
(1) Pretreatment of pigskin:
adding animal Corii Sus Domestica into 0.3wt% trypsin solution containing 0.025wt% EDTA, soaking at 37 deg.C for 24 hr, and washing; then soaking the pigskin in phosphate buffer solution (pH 7.2) at 30 ℃ for 20h, shaking and washing to obtain pretreated pigskin;
(2) Deimmunization treatment of pigskin:
adding the pigskin pretreated in the step (1) into a neutral protease solution with the concentration of 700U/L, soaking for 24h at 35 ℃, and washing; then soaking the mixture for 20 hours at 30 ℃ by using a phosphate buffer solution (pH 7.2), shaking and washing the mixture; and (3) repeating the step (1) and the step (2) for 1-3 times to obtain the deimmunized pigskin.
Other operations and steps are the same as in example 3.
Comparative example 2
The difference from example 2 is that: the scale remover added in the step (1) and the step (2) is sodium dodecyl sulfate only, namely:
(1) Pretreatment of pigskin:
adding animal Corii Sus Domestica into 0.3wt% trypsin solution containing EDTA 0.025wt%, soaking at 37 deg.C for 24 hr, and washing; then soaking the pigskin for 20 hours at 30 ℃ by using phosphate buffer solution (pH 7.2) containing 0.10wt% of sodium dodecyl sulfate, shaking and washing to obtain pretreated pigskin;
(2) Deimmunization treatment of pigskin:
adding the pigskin pretreated in the step (1) into a neutral protease solution with the concentration of 700U/L, soaking for 24h at 35 ℃, and washing; then soaking the mixture for 20 hours at 30 ℃ by using a phosphate buffer solution (pH 7.2) containing 0.10wt% of sodium dodecyl sulfate, shaking and washing; and (3) repeating the step (1) and the step (2) for 1-3 times to obtain the deimmunized pigskin.
Comparative example 3
The difference from example 3 is that: the modified polysaccharide in the step (6) is hyaluronic acid and oat beta-glucan in a mass ratio of 1.
Comparative example 4
The difference from example 3 is that: the modified polysaccharide in the step (6) is prepared from the following components in a mass ratio of 4:5:1 methacrylic anhydride-modified hyaluronic acid, oat beta-glucan and aldehyde-group-modified hyaluronic acid.
Comparative example 5
The difference from example 3 is that: the modified polysaccharide in the step (6) is prepared from the following components in a mass ratio of 1:5:4 methacrylic anhydride modified hyaluronic acid, oat beta-glucan and aldehyde group modified hyaluronic acid.
Effect verification:
1. detection result of three-spiral structure characteristics of collagen hydrogel
1.1 protein content
The total protein content is determined by a physicochemical method according to the Kjeldahl method of the pharmacopoeia of the people's republic of China for protein content determination.
The test method comprises the following steps:
methyl red ethanol solution (1 g/L): 0.1g of methyl red was weighed, dissolved in 95% ethanol and diluted to 100mL with 95% ethanol.
Bromocresol green ethanol solution (1 g/L): 0.1g of bromocresol green was weighed out and dissolved in 95% ethanol and diluted to 100mL with 95% ethanol.
Weighing a proper amount of sample in a digestion tube, adding 0.4g of copper sulfate, 6g of potassium sulfate and 20mL of sulfuric acid into the digestion tube for digestion, continuing digestion for 1h after the temperature of the digestion tube reaches 420 ℃, taking out and cooling, adding 20mL of water, cooling, distilling for 7min on an automatic Kjeldahl apparatus, adding 1-2 drops of indicator mixed solution (1 part of methyl red ethanol solution and 5 parts of bromocresol green ethanol solution are prepared) and 10mL of boric acid solution (20 g/L) into a receiving bottle, receiving the distillate to 200mL, titrating with hydrochloric acid standard solution (0.100 mol/L), and simultaneously making a reagent blank.
1.2 hydroxyproline content
Detection by HPLC method
Test method
1.2.1 pretreatment method
Weighing a proper amount of uniformly mixed sample, putting the sample into a 50mL hydrolysis tube, adding 20mL 1mol/L hydrochloric acid solution, putting the mixture into an electrothermal blowing dry box, and hydrolyzing for 22h at 110 ℃. After being taken out and cooled, the solution is transferred to a 25mL colorimetric tube for constant volume.
Accurately taking 50 mu L of sample into a 15mL centrifuge tube, putting the centrifuge tube into a vacuum drying oven, drying the centrifuge tube for 2h at 60 ℃ (drying all solvents), filling nitrogen into the centrifuge tube, and accurately adding 50 mu L of derivative reagent: ethanol, phenyl isothiocyanate, water, triethylamine =7, 1 (prepared in situ, nitrogen is filled during preparation) at normal temperature for derivatization for 30min, a mobile phase A4.5mL is added, mixed uniformly, filtered through a 0.45 μm microporous filter membrane, and the mixture is subjected to computer measurement.
1.2.2 instrumental methods
(1) And (3) chromatographic column: c18 SHISEIDO (4.6 mm 250mm 5 μm);
(2) Sample introduction amount: 10 mu L of the solution;
(3) Column temperature: at 40 ℃;
(4) Wavelength: 254nm;
(5) Mobile phase: a:0.1mol/L anhydrous sodium acetate + acetonitrile =97+3, mixed well and adjusted to pH 6.5 (31.815 g sodium acetate +3880mL water +120mL acetonitrile); b: acetonitrile + water =80+20;
(6) The mobile phase gradients are shown in table 1 below.
TABLE 1
1.2.3 calculation formula
Hydroxyproline containingThe amount is calculated according to the formula:
in the formula:
w is the content of the target object in the sample, and the unit is mu g/kg;
c is the concentration of the target in the sample measuring solution, unit mug/L;
C 0 -the concentration of target in the blank in μ g/L;
v is constant volume, unit mL;
n-dilution factor;
m is the sample size in g.
1.3 Hydroxyllysine content
Detection by LC-MS method
Test method
1.3.1 pretreatment method
Taking out the biological material sample stored at ultralow temperature, and grinding (30Hz, 1min) to be powder by using a grinder (MM 400, retsch);
taking a proper amount of sample, carrying out ultrasonic extraction by pure water, adding 70% methanol to dilute and precipitate protein, centrifuging, taking supernate, filtering the supernate with a 0.22 mu m filter membrane, and placing the supernate in a sample injection bottle for LC-MS/MS analysis.
1.3.2 instrumental methods
(1) A chromatographic column: UPLC ACQUITY BEH Amide column (2.1mm 100 mm,1.7 μm);
(2) Mobile phase: phase A0.15% formic acid/water (10 mmol/L ammonium formate); phase B85% acetonitrile/Water (10 mmol/L ammonium formate)
(3) Elution gradient: 0minA/B (0 100V/V), 6.1minA/B (5.9;
(4) Flow rate: 0.4mL/min; column temperature: at 40 ℃; sample introduction amount: 2 μ L.
(5) The mass spectrum conditions mainly comprise: electrospray ion source (ESI), positive ion mode, scan range 50-400m/z, detection mode PRM mode, spray voltage 3.50kv, capillary temperature (capillary temp.) 320 ℃, each ion pair was scan detected according to optimized Declustering Potential (DP) and Collision Energy (CE).
1.3.3 calculation formula
The content of free amino acid is calculated according to the formula:
in the formula:
w is the content of the target object in the sample, and the unit is mg/Kg;
c is the concentration of the target object in the sample measuring solution, and the unit is mg/L;
C 0 -the concentration of target in the blank in mg/L;
v is constant volume, unit mL;
n-dilution factor;
m is the sample size in g.
Specific detection results are shown in Table 2 below
TABLE 2
Note: "-" indicates that no calculation was made for this item
According to the detection results in the table 2 above, it can be seen that the collagen dermal hydrogel prepared in the examples 1 to 3 of the present application has a high content of hydroxyproline, which indicates that the triple-helical structure in the collagen dermal hydrogel is relatively complete, whereas the use content and the type of the detergent in the purification operation are changed in the comparative examples 1 to 2, which may affect the purity of collagen to a certain extent, and thus affect the integrity of the triple-helical structure in the final collagen dermal hydrogel; comparative examples 3 to 5 change the kind and ratio of the modified polysaccharide greatly affects the integrity of the triple helix structure in the collagen dermal hydrogel of the final product, so that the content of the hydroxyproline in the collagen dermal hydrogel is significantly lower than that in examples 1 to 3.
Similarly, according to the porous three-dimensional structure diagram of hydrogel collagen in fig. 2, it is apparent that the collagen dermal hydrogel prepared in example 3 of the present application has a better triple-helical structure.
2. Collagen hydrogel molecular weight detection
Test method
2.1 pretreatment method
Accurately weighing a proper amount of sample, dissolving the sample by using a mobile phase to prepare a solution with the concentration of about 1 to 3mg/mL, and testing the solution on a machine after the solution passes through a 0.22 mu m microporous filter membrane.
2.2 instrumental methods
(1) A chromatographic column: PL aquagel-OH Mixed-H,8 μm,7.5x300mm (molecular weight range 200-10000000);
(2) A detector: a differential detector, a dual-angle laser scattering detector, a viscosity detector;
(3) Flow rate: 1.0mL/min, column temperature: 45 ℃; sample injection amount: 50 mu L of the solution;
(4) Mobile phase: 0.1mol/L sodium nitrate (0.01% sodium azide), isocratic elution.
2.3 calculation of
The molecular weights Mp, mn, mw, mz, etc. of the samples were calculated by GPC software fitting from the viscosity, peak time, etc. of the samples.
The results of the measurements are shown in Table 3 below.
TABLE 3
Note: "-" indicates that the item was not detected
According to the detection results in table 3 above, it can be seen that the molecular weights Mp, mn, mw, and Mz of P2 in the low molecular region of the collagen hydrogels prepared in examples 1-3 of the present application are all small, which indicates that the collagen hydrogels prepared in examples 1-3 of the present application have small molecular weights and can be better absorbed; comparative examples 1-5 gave collagen hydrogels with molecular weight distributions that were not as good as those of examples 1-3.
3. Collagen hydrogel water absorption detection
The test method comprises the following steps:
3.1 treatment of the samples
Placing the collagen hydrogel in a vacuum drier with P 2 O 5 Drying at 40 ℃ to constant weight as a drying agent for later use. Hygroscopicity test the dried samples were used directly.
3.2 hygroscopicity test
Accurately weighing two parts of 0.5g samples; the samples were respectively added into weighing bottles with the diameter of 3cm, the weighing bottles were respectively placed in two driers, one drier was filled with a saturated solution of ferric sulfate (relative humidity RH = 81%), the other drier was filled with a saturated solution of sodium carbonate (relative humidity RH = 13%), the standing time was 48h, and the mass (Wo) before the sample was placed and the mass (Wv) after the sample was placed were respectively weighed. Moisture absorption% = [ (Wv-Wo)/Wo ]. Times.100% was calculated according to the following formula
The results of the measurements are shown in Table 4 below.
TABLE 4
As can be seen from the test data in table 4 above, the collagen hydrogels prepared in examples 1-3 of the present application have better water absorption, the water absorption of the collagen hydrogels prepared in examples 1-3 is slightly higher than that of hyaluronic acid when the relative humidity is RH =81%, and the water absorption of the collagen hydrogels prepared in examples 1-3 is significantly higher than that of hyaluronic acid when the relative humidity is RH = 13%; and the collagen hydrogels prepared in examples 1 to 3 had significantly higher water absorption than those of comparative examples 1 to 5 when the relative humidity was RH =81% or RH = 13%.
4. Collagen hydrogel transdermal assay
The test method comprises the following steps:
4.1 test grouping
4.2 test procedure
(1) Firstly, fixing the pigskin between a supply chamber and a receiving chamber of a Franz cell diffusion cell, wherein the skin cuticle of the pigskin faces the supply chamber, and one side of the dermis faces the receiving chamber;
(2) Adding 7.0mL of receiving liquid into the receiving chamber, tightening and fixing the pigskin, adding 1mL of receiving liquid (PBS) into the receiving chamber through a sampler, and exhausting air to ensure that the dermal layer of the skin is in close contact with the receiving liquid;
(3) Loading: the effective penetration area S of the sample into the skin surface in the supply chamber is about 3.14cm 2 . The sample is added to the surface of the pigskin and spread evenly from the center of the skin to the edge in a radial pattern. 3 replicates and parallel (three replicates taken of skin from a specific site of an independent donor) per sample;
(4) And (3) infiltration: starting an electromagnetic stirrer to stir at the speed of 300rpm, keeping a constant-temperature water bath at (32 +/-1) DEG C, and ensuring that a water bath interlayer has no bubbles;
(5) Collecting skin samples at 1h, 6h and 24h respectively, sucking PBS to clean the surface of the skin for 5 times, wiping residual liquid on the surface with a cotton swab, cutting the skin with a blade in a circular way, immersing in 4% paraformaldehyde solution for fixing (the fixing time is more than or equal to 24 h), and freezing and slicing for later use.
4.3 test results
As can be seen from the fluorescence microscope observation results in FIG. 3, the permeation behavior of the sample collagen (0.1 mg/mL) occurs after 6h and 24h, and the sample permeation amount increases with time, and each time point is significantly increased compared with the previous time point.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. A preparation method of triple-helix collagen dermal hydrogel is characterized by comprising the following steps: the method comprises the following steps:
(1) Pretreatment of pigskin:
adding the animal pigskin into a trypsin solution containing EDTA for soaking and washing; then soaking and shaking the pigskin by using a descaling agent solution, and washing the pigskin to obtain pretreated pigskin;
(2) Deimmunization treatment of pigskin:
adding the pigskin pretreated in the step (1) into a neutral protease solution for soaking and washing; then soaking and shaking the mixture by using a descaling agent solution, and washing the mixture; repeating the step (1) and the step (2) for 1-3 times to obtain the pig skin without immunogenicity;
(3) Alpha-1, 3-galactose antigen treatment of pigskin:
soaking the deimmunized pigskin obtained in the step (2) with an alpha-1, 3-galactosidase solution, and washing to obtain a precipitate A;
(4) Filter aid and adsorption:
adding a filter aid solution into the precipitate A obtained in the step (3), stirring, filtering, and collecting supernatant for later use;
(5) Separation and purification:
concentrating the supernatant collected in the step (4), adsorbing collagen through a cation exchange medium, and then eluting and separating to finally obtain an eluent;
(6) Preparing hydrogel:
adding modified polysaccharide with the concentration of 10-40mg/mL into the eluent in the step (5), wherein the mass ratio of the eluent to the modified polysaccharide is 5;
the modified polysaccharide is one or more of methacrylic anhydride modified hyaluronic acid, oat beta-glucan, aldehyde modified hyaluronic acid and N-hydroxy thiosuccinimide;
(7) And (3) degerming and filtering:
and (4) sterilizing and filtering the hydrogel obtained in the step (6) by using a 0.22um filter membrane to obtain a sterile soluble collagen hydrogel solution.
2. The method of claim 1, wherein: the concentration of the EDTA in the trypsin solution containing the EDTA in the step (1) is 0.01-0.04 wt%; the concentration of the trypsin is 0.25wt% -0.35wt%.
3. The production method according to claim 1, characterized in that: the detergent solution in the step (1) and the step (2) is phosphate buffer solution containing 0.05-0.15wt% of ionic detergent.
4. The production method according to claim 3, characterized in that: the ionic descaling agent is a mixture of sodium dodecyl sulfate and TritonX-200, and the mass ratio of the sodium dodecyl sulfate to the TritonX-200 is (4): 6; the phosphate buffer solution is a pH 6.0-8.0 sodium phosphate buffer solution.
5. The method of claim 1, wherein: the concentration of the neutral protease solution in the step (2) is 500-700U/L.
6. The method of claim 1, wherein: the concentration of the alpha-1, 3-galactosidase solution in the step (3) is 10-20 mu g/mL.
7. The production method according to claim 1, characterized in that: the elution solution of the elution separation described in the step (5) is 10mM Tris-HCl aqueous solution containing 0.5-0.9wt% of sodium salt.
8. The method of claim 1, wherein: the modified polysaccharide in the step (6) is prepared by the following steps of (1) by mass: 5:3 methacrylic anhydride modified hyaluronic acid, oat beta-glucan and aldehyde group modified hyaluronic acid.
9. The triple-helical collagen dermal hydrogel prepared according to the preparation method of any one of claims 1 to 8.
10. Use of the triple-helical collagen dermal hydrogel prepared by the preparation method according to any one of claims 1 to 8 for the preparation of cosmetics.
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| US20090148534A1 (en) * | 2004-09-07 | 2009-06-11 | Chugai Seiyaku Kabushiki Kaisha | Process for producing water-soluble hyaluronic acid modification |
| CN106563173A (en) * | 2015-10-13 | 2017-04-19 | 温州华贞医疗科技有限公司 | Acellular biological dermal material, and preparation method and application thereof |
| CN113476657A (en) * | 2021-06-21 | 2021-10-08 | 四川大学 | Collagen-based hydrogel for cartilage tissue engineering and preparation method thereof |
| CN113768815A (en) * | 2021-09-13 | 2021-12-10 | 浙江崇山生物制品有限公司 | Collagen implant and preparation method thereof |
| CN114317661A (en) * | 2022-02-15 | 2022-04-12 | 无锡贝迪生物工程股份有限公司 | Method for preparing multiple active collagen |
| CN114540452A (en) * | 2022-04-26 | 2022-05-27 | 天新福（北京）医疗器材股份有限公司 | Collagen, inactivation and extraction method thereof and facial filler containing collagen |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090148534A1 (en) * | 2004-09-07 | 2009-06-11 | Chugai Seiyaku Kabushiki Kaisha | Process for producing water-soluble hyaluronic acid modification |
| CN106563173A (en) * | 2015-10-13 | 2017-04-19 | 温州华贞医疗科技有限公司 | Acellular biological dermal material, and preparation method and application thereof |
| CN113476657A (en) * | 2021-06-21 | 2021-10-08 | 四川大学 | Collagen-based hydrogel for cartilage tissue engineering and preparation method thereof |
| CN113768815A (en) * | 2021-09-13 | 2021-12-10 | 浙江崇山生物制品有限公司 | Collagen implant and preparation method thereof |
| CN114317661A (en) * | 2022-02-15 | 2022-04-12 | 无锡贝迪生物工程股份有限公司 | Method for preparing multiple active collagen |
| CN114540452A (en) * | 2022-04-26 | 2022-05-27 | 天新福（北京）医疗器材股份有限公司 | Collagen, inactivation and extraction method thereof and facial filler containing collagen |
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