WO2022089287A1 - Preparation method and use method for biomimetic collagen solution for skin surface - Google Patents
Preparation method and use method for biomimetic collagen solution for skin surface Download PDFInfo
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- WO2022089287A1 WO2022089287A1 PCT/CN2021/125186 CN2021125186W WO2022089287A1 WO 2022089287 A1 WO2022089287 A1 WO 2022089287A1 CN 2021125186 W CN2021125186 W CN 2021125186W WO 2022089287 A1 WO2022089287 A1 WO 2022089287A1
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- WIPO (PCT)
- Prior art keywords
- solution
- collagen
- skin surface
- collagen solution
- biomimetic
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Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 195
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- 230000003592 biomimetic effect Effects 0.000 title claims abstract description 61
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Images
Classifications
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- 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
- 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
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
-
- 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
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
- A61K8/20—Halogens; Compounds thereof
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- 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
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
- A61K8/24—Phosphorous; Compounds thereof
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- 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
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
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- 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
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- 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
- A61Q19/08—Anti-ageing preparations
Definitions
- the invention relates to a biological material and a method for preparing and using the same, in particular to a method for preparing and using a biomimetic collagen solution on the skin surface.
- collagen In skin tissue, collagen is one of the components of the supporting structure of the dermis. Its mesh structure provides protection and elasticity for the skin, and a large amount of water, extracellular matrix and functional cells are distributed between the fibers. An important biochemical reaction site, providing moisture and nutrients to the epidermis.
- the diffusion coefficient of the molecule is inversely proportional to the square root or cube root of the molecular weight. The larger the molecular weight, the larger the molecular volume and the smaller the diffusion coefficient. Therefore, it is difficult for collagen molecules with relative molecular mass ranging from about 2kD to 300kD to diffuse into the dermis through the stratum corneum, and be absorbed by the skin tissue to exert its repair and regeneration effect.
- collagen molecules can only be absorbed through skin appendages such as hair follicles, sebaceous glands and sweat glands.
- skin appendages such as hair follicles, sebaceous glands and sweat glands.
- the total area of hair follicles, sweat glands and sebaceous glands is less than 1% of the total surface area of the skin, so the absorption of collagen molecules by hair follicles, sweat glands and sebaceous glands is also very small.
- the skin is an organ mainly for excretion, and the efficiency of passive diffusion is obviously not too high.
- the conventional method is to reduce the molecular weight of collagen molecules to use collagen peptides of small molecular weight.
- collagen peptides are made of collagen or gelatin after being degraded by protease, and their biological activity is poor; and due to their small molecular weight, collagen peptides are easily degraded in the body and easily digested and absorbed by skin tissue, so the skin The repair effect is limited.
- the effect of promoting collagen absorption can also be achieved by means of the functions of introduction instruments (conductivity, ultrasonic introduction, radio frequency introduction, microcrystal, microneedle introduction, etc.).
- introduction instruments conductivity, ultrasonic introduction, radio frequency introduction, microcrystal, microneedle introduction, etc.
- long-term disordered use of the instrument can cause certain damage to the skin.
- complexity of the operation is not conducive to large-scale promotion and use.
- the present invention develops a biomimetic collagen solution for use on the skin surface from a biomimetic perspective: (1) the collagen raw material selects type I, type III or type V collagen specific to skin tissue; (2) Add simulated body fluid components, adjust the pH value of the solution, and simulate the microenvironment of skin tissue; (3) Add linking molecules to promote the absorption of collagen molecules in skin tissue (especially the dermis).
- the developed low-concentration biomimetic collagen solution is applied on the skin surface by smearing, spraying or immersion.
- the invention provides an aqueous solution of biomimetic collagen and a preparation method and use method thereof.
- step (2) under low temperature conditions, the collagen solution obtained in step (1) is slowly added to simulate body fluid components in the stirring process to fully dissolve;
- the pH value of the collagen solution obtained in step (2) is adjusted to be greater than or equal to 4 but less than or equal to 10 by an alkaline solution or an acidic solution to form a homogeneous solution;
- the linking molecule solution is added to the collagen solution obtained in step (3) and the preparation is completed after stirring evenly. After the preparation is completed, the biomimetic collagen solution is stored under low temperature conditions.
- the range of low temperature conditions in step (2), step (3) and step (4) is greater than or equal to 0°C but less than or equal to 10°C; the solid collagen material is selected from type I, type III
- the pH value of the collagen solution is adjusted to be greater than or equal to 6 but less than or equal to 8 through an alkaline solution or an acidic solution.
- type I, type III or type V collagen is a type of collagen unique to skin tissue.
- the collagen solution is prepared as far as possible according to the content of different types of collagen in the skin tissue, but in order to facilitate the concentration of collagen molecules Flow, the collagen solution is finally a dilute solution, and the total concentration of all types of collagen is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%.
- the acidic solution in step (1) and step (3) is at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, tartaric acid, citric acid, oxalic acid, acetic acid and formic acid ;
- Described alkaline solution in described step (3) comprises triethylamine, tetramethylethylenediamine, pyridine, piperidine, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammoniacal liquor, phosphoric acid
- At least one of disodium hydrogen and sodium bicarbonate; the concentration of the acidic solution and the alkaline solution is greater than or equal to 0.01M but less than 10M.
- the acidic solution in the step (1) is added to fully dissolve the solid collagen material.
- the addition of the acidic solution is a trace addition, and does not significantly change the concentration of the collagen solution, so long as the collagen is dissolved to form a homogeneous solution.
- adding an alkaline solution or an acidic solution in the step (3) is to adjust the pH value of the collagen solution.
- the addition of the acidic solution or the alkaline solution is a micro-addition, which does not significantly change the concentration of the collagen solution, as long as the solution reaches a preset pH value and forms a homogeneous solution.
- the simulated body fluid components include Na + , K + , Mg 2+ , Ca 2+ , Cl - , HCO 3 - , HPO 4 2- , SO 4 2- , CO 3 2- , PO 4 One or a combination of two or more of 3- and H 2 PO 4 - .
- the purpose of adding simulated body fluid components is to adapt the ionic environment of the collagen solution to the microenvironment in the skin tissue.
- the addition of simulated body fluid components is a small amount, and does not significantly change the concentration of collagen solution therein.
- the final ion concentration of each ion in the biomimetic collagen solution for the skin surface was in the range of 0.1-500 mM, and the final total ion concentration was in the range of 0.1-1000 mM.
- the linking molecules are isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide esters (NHS esters), sulfonyl chlorides, aldehydes, epoxides, aryl halides , one or more mixtures of imide ester, carbodiimide, acid anhydride, fluorophenyl ester, proanthocyanidin and genipin.
- the linker molecules are added to enhance the absorption of collagen molecules into the skin tissue. The addition of the linker molecule solution did not significantly change the concentration of the collagen solution therein, and the final concentration of the linker molecules in the biomimetic collagen solution for skin surface was 0.0001 wt.% or more but 10 wt.% or less.
- the final concentration of the linking molecule in the biomimetic collagen solution for skin surface is greater than or equal to 0.0001 wt.% but less than or equal to 1 wt.%.
- the final concentration of the linking molecule in the biomimetic collagen solution for skin surface is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%.
- the final concentration of the linking molecules in the biomimetic collagen solution for skin surface is greater than or equal to 0.001 wt.% but less than or equal to 0.01 wt.%.
- biomimetic collagen solution for a skin surface
- the biomimetic collagen solution being the biomimetic collagen solution for a skin surface according to any one of the preceding embodiments. prepared by the method of preparation.
- the biomimetic collagen solution is applied to the skin surface by one or more combinations of self-smearing, spraying, and immersion. .
- the embodiments of the present invention are proposed solutions from the perspective of bionics for the problem that collagen molecules are difficult to absorb through the skin.
- the embodiments of the present invention are mainly optimized as follows: (1) According to the type of collagen molecules contained in the skin tissue, Select type I, type III or type V collagen, prepare collagen solution according to the content of different types of collagen in the skin tissue, and match the composition of the solution with the skin tissue as much as possible; (2) According to the composition and content of ions in the body fluid and the pH environment , adding simulated body fluid components to the solution, by regulating the ionic components and ionic strength, and further regulating the pH value, so that the collagen solution matches the microenvironment of the skin tissue; (3) Compared with the conventional method, the collagen molecule is directly combined with the skin tissue.
- the method for realizing the absorption of collagen molecules adds connecting molecule components in the collagen solution, and the connecting molecules can react with the collagen molecules in the collagen solution and the collagen components in the skin tissue at the same time, thereby combining the collagen molecules in the skin tissue,
- the high-efficiency reaction of linking molecules can improve the absorption rate of collagen molecules in skin tissue.
- the storage time of the collagen solution after adding the linking molecule is greater than or equal to 0 min but less than or equal to 60 min, so as not to affect its use effect.
- the concentration of the collagen solution is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%.
- the choice of low-concentration collagen solution is conducive to the flow transmission of collagen molecules and the absorption of collagen molecules in skin tissue.
- the solution environment in the biomimetic collagen solution of the present invention is closer to the microenvironment of the skin tissue, and its biocompatibility is better than that of the conventional collagen solution, which is more conducive to the absorption of collagen molecules into the skin tissue (especially in the dermis);
- the role of molecules in skin tissue can delay the degradation of collagen molecules, exert its biological activity, and promote skin tissue repair and regeneration.
- FIG. 1 is a graph showing the effect of subcutaneous absorption of collagen molecules labeled by fluorescein isothiocyanate (FITC) fluorescent staining in a biomimetic collagen solution according to an embodiment of the present invention
- FIG. 1A is a sample of Example 1
- FIG. 1B is a sample of Example 2
- Figure 1C is a sample of Example 3
- Figure 1D is a sample of Example 4
- Figure 1E is a sample of Comparative Example 1
- Figure 1F is a sample of Comparative Example 2
- FIG. 2 is an effect diagram of live/dead cell staining in a biomimetic collagen solution according to an embodiment of the present invention
- FIG. 2A is the sample of Example 1
- FIG. 2B is the sample of Example 2
- FIG. 2C is the sample of Example 3
- FIG. 2D It is a sample of Example 4
- Figure 2E is a sample of Comparative Example 1
- Figure 2F is a sample of Comparative Example 2);
- FIG. 3 is an effect diagram of HE staining (hematoxylin-eosin staining) of biomimetic collagen solution implanted in rats according to an embodiment of the present invention
- FIG. 3A is the sample of Example 1
- FIG. 3B is the sample of Example 2
- Figure 3C is a sample of Example 3
- Figure 3D is a sample of Example 4
- Figure 3E is a sample of Comparative Example 1
- Figure 3F is a sample of Comparative Example 2
- a preparation method for a biomimetic collagen solution on a skin surface wherein, the steps are sequentially included:
- the pH value of the collagen solution obtained in step (2) was adjusted to 7.2 by 0.01M NaOH solution or 0.01M acetic acid solution to form a homogeneous solution;
- a method of using a biomimetic collagen solution for skin surface the biomimetic collagen solution is prepared according to the above preparation method, and the using method includes: 10 minutes (min) after the biomimetic collagen solution is prepared, evenly smearing it On the pre-repaired skin surface, the skin surface was washed after 2 hours (h).
- a preparation method for a biomimetic collagen solution on a skin surface wherein, the steps are sequentially included:
- step (3) at 2°C, adjust the pH value of the collagen solution obtained in step (2) to 10 by 0.2M KOH solution or 0.1M ammonia solution to form a homogeneous solution;
- a method of using a biomimetic collagen solution for skin surface the biomimetic collagen solution is prepared according to the above preparation method, and the using method includes: after the biomimetic collagen solution is prepared for 60 minutes, uniformly spraying it on pre-repaired skin surface, and the skin surface was washed after 1 h.
- a preparation method for a biomimetic collagen solution on a skin surface wherein, the steps are sequentially included:
- the pH value of the collagen solution obtained in step (2) is adjusted to 4 by 1M formic acid solution or 0.2M phosphoric acid solution to form a homogeneous solution;
- a method for using a biomimetic collagen solution for skin surface the biomimetic collagen solution is prepared according to the above preparation method, and the using method comprises: immersing the pre-repaired skin surface in the solution 0 min after the biomimetic collagen solution is prepared , the skin surface was washed after 3 h.
- a method for preparing a biomimetic collagen solution on a skin surface comprising the steps of:
- the pH value of the collagen solution obtained in step (2) is adjusted to 6 by 0.01M pyridine solution or 2M tartaric acid solution to form a homogeneous solution;
- a method for using a biomimetic collagen solution for skin surface the biomimetic collagen solution is prepared according to the above-mentioned preparation method, and the using method comprises: after preparing the biomimetic collagen solution for 30 minutes, spraying it evenly and then smearing it on The skin surface was pre-repaired, and the skin surface was washed after 2 hours.
- Comparative Example 1 a preparation method for a collagen solution on the skin surface and a method for using the same, comprising the following steps in turn:
- the collagen solution is applied to the skin surface by spraying.
- Comparative Example 2 a preparation method for a collagen solution on the skin surface and a method for using the same, wherein the following steps are included in sequence:
- the collagen solution is applied to the surface of the skin by smearing.
- Collagen solutions were prepared according to the preparation methods in Examples 1-4 and Comparative Examples 1-2, and 1 mL and 6 ⁇ 10 5 rat skin fibroblasts were taken respectively (Shanghai Cell Bank, Chinese Academy of Sciences)
- the cells were mixed to form collagen hydrogel-encapsulated cells at 37°C for three-dimensional culture experiments.
- the medium was high glucose DMEM (HyClone) medium containing 10% (v/v%) fetal bovine serum, 1% streptomycin and penicillin, and the medium was changed once a day.
- the temperature of the incubator (STERI 371, Thermo Electron Corporation) was set at 37°C and humidified air containing 5% (v/v%) CO 2 .
- the cells in the hydrogel were stained with live/dead cells, and the staining solution was 2mmol/L calcein-AM (calcein acetoxymethyl ester solution, Sigma) (used to stain live cells and emit green fluorescence) ) and 2 mmol/L EthD-1 solution (ethidium bromide dimer 1, Sigma) (used to stain dead cells, emit red fluorescence), observe the samples on A1 confocal microscope (Nikon), green excitation light The wavelength is 488 nm, and the wavelength of the red excitation light is 562 nm. Live/dead cell staining in collagen hydrogels is shown in Figure 2.
- the binding effect test shows that, compared with Comparative Example 1-2, the collagen solution in Comparative Example 1-2 is a strongly acidic solution, which is not conducive to the use of the collagen solution on the skin surface;
- the solution is neutral and can be adjusted according to different skins, which is more suitable for the microenvironment of the skin surface;
- Examples 1-4 add ionic components similar to those of body fluids, so that the ionic strength and permeability of the collagen solution are comparable to those of the skin.
- the microenvironment is closer, which is conducive to the transmission of collagen molecules; in Examples 1-4, connecting molecules are added, which can realize the combination of collagen molecules and collagen components in the dermis during the process of collagen molecules entering the dermis, which is beneficial to the collagen molecules in the solution.
- Layer enrichment and absorption; collagen solutions in Examples 1-4 are more favorable for cell growth and fusion with in vivo tissues.
Abstract
A biomimetic collagen solution for skin surface, and a preparation method and a use method for same. Type I, type III or type V collagen specific to skin tissue is selected, a simulated body fluid component is added to regulate ionic components and ionic strength of the solution, and the pH value of the solution is adjusted to simulate a microenvironment of the skin tissue; the addition of linker molecules enhances binding between collagen molecules in the solution and the skin tissue. The collagen solution has a concentration of 0.0001-0.1wt.%, and can be applied to skin surface by means of smearing, spray coating or immersion. The biomimetic collagen solution simulates some features of skin tissue, has good biocompatibility, facilitates absorption of collagen molecules in the skin tissue (particularly in the dermis), delays degradation of the collagen molecules, and promotes reparative regeneration of the skin tissue.
Description
本发明涉及一种生物材料及其制备方法和使用方法，具体涉及一种用于皮肤表面的仿生胶原溶液的制备方法和使用方法。The invention relates to a biological material and a method for preparing and using the same, in particular to a method for preparing and using a biomimetic collagen solution on the skin surface.
在皮肤组织中，胶原蛋白是真皮的支撑结构组成成份之一，其网状架构为皮肤提供了保护和弹性，在纤维之间则分布着大量的水分、细胞外基质和功能性细胞，是皮肤重要的生化反应场所，为表皮层提供水分和营养。然而，当胶原分子接触皮肤表面角质层时，由于角质层的屏障作用，分子的扩散系数与分子量的平方根或立方根成反比，分子量愈大，分子体积愈大，扩散系数愈小。因此，相对分子质量从约2kD至300kD不等的胶原分子很难通过角质层扩散进入真皮层，被皮肤组织吸收发挥其修复再生功效。In skin tissue, collagen is one of the components of the supporting structure of the dermis. Its mesh structure provides protection and elasticity for the skin, and a large amount of water, extracellular matrix and functional cells are distributed between the fibers. An important biochemical reaction site, providing moisture and nutrients to the epidermis. However, when collagen molecules contact the stratum corneum on the surface of the skin, due to the barrier effect of the stratum corneum, the diffusion coefficient of the molecule is inversely proportional to the square root or cube root of the molecular weight. The larger the molecular weight, the larger the molecular volume and the smaller the diffusion coefficient. Therefore, it is difficult for collagen molecules with relative molecular mass ranging from about 2kD to 300kD to diffuse into the dermis through the stratum corneum, and be absorbed by the skin tissue to exert its repair and regeneration effect.
一般而言，胶原分子只能通过毛囊、皮脂腺和汗腺等皮肤附属器官吸收。然而，毛囊、汗腺和皮脂腺总面积小于皮肤总表面积的1％，所以毛囊、汗腺和皮脂腺对于胶原分子的吸收量也很小。而且，皮肤是一个以排泄为主的器官，靠被动扩散效率显然不会太高。In general, collagen molecules can only be absorbed through skin appendages such as hair follicles, sebaceous glands and sweat glands. However, the total area of hair follicles, sweat glands and sebaceous glands is less than 1% of the total surface area of the skin, so the absorption of collagen molecules by hair follicles, sweat glands and sebaceous glands is also very small. Moreover, the skin is an organ mainly for excretion, and the efficiency of passive diffusion is obviously not too high.
为了提高胶原在皮肤表面(尤其在真皮层)的吸收进而修复皮肤损伤(如皱纹)，常规的方法是降低胶原分子的分子量以使用小分子量的胶原蛋白肽。然而，胶原蛋白肽是由胶原或明胶经蛋白酶降解处理后制成的，其生物活性差；而且由于分子量较小，胶原蛋白肽在体内很容易降解，也很容易被皮肤组织消化吸收，因此皮肤修复效果有限。In order to improve the absorption of collagen on the skin surface (especially in the dermis layer) to repair skin damage (such as wrinkles), the conventional method is to reduce the molecular weight of collagen molecules to use collagen peptides of small molecular weight. However, collagen peptides are made of collagen or gelatin after being degraded by protease, and their biological activity is poor; and due to their small molecular weight, collagen peptides are easily degraded in the body and easily digested and absorbed by skin tissue, so the skin The repair effect is limited.
另外，也可以借助导入仪器(电导、超声波导入、射频导入以及微晶、微针导入等)的功能达到促进胶原吸收的效果。然而，仪器的长期无序使用会对皮肤会造成一定的伤害。而且操作的复杂性也不利于大规模的推广和使用。In addition, the effect of promoting collagen absorption can also be achieved by means of the functions of introduction instruments (conductivity, ultrasonic introduction, radio frequency introduction, microcrystal, microneedle introduction, etc.). However, long-term disordered use of the instrument can cause certain damage to the skin. And the complexity of the operation is not conducive to large-scale promotion and use.
发明内容SUMMARY OF THE INVENTION
针对以上胶原分子经皮肤吸收的问题的至少一个方面，本发明从仿生的角度开发用于皮肤表面的仿生胶原溶液：(1)胶原原材料选用皮肤组织特有的I型、III型或V型胶原；(2)添加模拟体液成份，调控溶液pH值，模拟皮肤组织的微环境；(3)添加连接分子，促进胶原分子在皮肤组织中(特别是真皮层)的吸收。所开发的低浓度仿生胶原溶液经由涂抹、喷涂或浸没的方式在皮肤表面使用。In view of at least one aspect of the problem of the above collagen molecules being absorbed through the skin, the present invention develops a biomimetic collagen solution for use on the skin surface from a biomimetic perspective: (1) the collagen raw material selects type I, type III or type V collagen specific to skin tissue; (2) Add simulated body fluid components, adjust the pH value of the solution, and simulate the microenvironment of skin tissue; (3) Add linking molecules to promote the absorption of collagen molecules in skin tissue (especially the dermis). The developed low-concentration biomimetic collagen solution is applied on the skin surface by smearing, spraying or immersion.
本发明提供一种仿生胶原水溶液及其制备方法和使用方法。The invention provides an aqueous solution of biomimetic collagen and a preparation method and use method thereof.
根据本发明的一个方面，提供一种用于皮肤表面的仿生胶原溶液的制备方法，其中，依次包括如下步骤：According to one aspect of the present invention, there is provided a method for preparing a biomimetic collagen solution on the skin surface, wherein, the following steps are included in sequence:
(1)将固体胶原材料置入水中，在搅拌过程中添加酸性溶液溶解所述固体胶原材料形成均质溶液，胶原溶液的浓度为大于等于0.0001wt.％但小于等于0.1wt.％；(1) Put the solid collagen material into water, add an acidic solution to dissolve the solid collagen material during stirring to form a homogeneous solution, and the concentration of the collagen solution is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%;
(2)低温条件下，将步骤(1)所得胶原溶液在搅拌过程中缓慢加入模拟体液成份，充分溶解；(2) under low temperature conditions, the collagen solution obtained in step (1) is slowly added to simulate body fluid components in the stirring process to fully dissolve;
(3)低温条件下，通过碱性溶液或酸性溶液调节步骤(2)所得胶原溶液pH值至大于等于4但小于等于10，形成均质溶液；(3) Under low temperature conditions, the pH value of the collagen solution obtained in step (2) is adjusted to be greater than or equal to 4 but less than or equal to 10 by an alkaline solution or an acidic solution to form a homogeneous solution;
(4)低温条件下，在步骤(3)所得胶原溶液中加入连接分子溶液搅拌均匀后制备完成，在制备完成后，在低温条件下保存所述仿生胶原溶液。(4) Under low temperature conditions, the linking molecule solution is added to the collagen solution obtained in step (3) and the preparation is completed after stirring evenly. After the preparation is completed, the biomimetic collagen solution is stored under low temperature conditions.
在本发明的实施例中，步骤(2)、步骤(3)和步骤(4)中的低温条件的范围为大于等于0℃但小于等于10℃；所述固体胶原材料选自I型、III型、V型胶原中的一种或两种以上的组合；在所述步骤(3)中，通过碱性溶液或酸性溶液调节胶原溶液pH值至大于等于6但小于等于8。In the embodiment of the present invention, the range of low temperature conditions in step (2), step (3) and step (4) is greater than or equal to 0°C but less than or equal to 10°C; the solid collagen material is selected from type I, type III In the step (3), the pH value of the collagen solution is adjusted to be greater than or equal to 6 but less than or equal to 8 through an alkaline solution or an acidic solution.
在本发明的实施例中，I型、III型或V型胶原是皮肤组织特有的胶原类型，在制备过程中尽可能按照皮肤组织中不同类型胶原的含量制备胶原溶液，但为了利于胶原分子的流动，胶原溶液最终为稀溶液，所有类型胶原的总浓度为大于等于0.0001wt.％但小于等于0.1wt.％。In the embodiment of the present invention, type I, type III or type V collagen is a type of collagen unique to skin tissue. In the preparation process, the collagen solution is prepared as far as possible according to the content of different types of collagen in the skin tissue, but in order to facilitate the concentration of collagen molecules Flow, the collagen solution is finally a dilute solution, and the total concentration of all types of collagen is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%.
在本发明的实施例中，在步骤(1)和步骤(3)中的所述酸性溶液同为包括盐酸、硝酸、硫酸、磷酸、酒石酸、柠檬酸、草酸、乙酸和甲酸中的至少一种；所述步骤(3)中的所述碱性溶液包括三乙胺、四甲基乙二胺、吡啶、哌啶、氢氧化钠、氢氧化钾、氢氧化钙、氢氧化镁、氨水、磷酸氢二钠和碳酸氢钠中的至少一种；所述酸性溶液、碱性溶液的浓度为大于等于0.01M但小于10M。In an embodiment of the present invention, the acidic solution in step (1) and step (3) is at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, tartaric acid, citric acid, oxalic acid, acetic acid and formic acid ; Described alkaline solution in described step (3) comprises triethylamine, tetramethylethylenediamine, pyridine, piperidine, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammoniacal liquor, phosphoric acid At least one of disodium hydrogen and sodium bicarbonate; the concentration of the acidic solution and the alkaline solution is greater than or equal to 0.01M but less than 10M.
在本发明的实施例中，所述步骤(1)中的酸性溶液是为了充分溶解固体胶原材料添加的。该酸性溶液的添加为微量添加，不显著改变其中胶原溶液浓度，以溶解胶原形成均质溶液为准。In the embodiment of the present invention, the acidic solution in the step (1) is added to fully dissolve the solid collagen material. The addition of the acidic solution is a trace addition, and does not significantly change the concentration of the collagen solution, so long as the collagen is dissolved to form a homogeneous solution.
在本发明的实施例中，所述步骤(3)中添加碱性溶液或酸性溶液是为了调节胶原溶液的pH值。该酸性溶液或碱性溶液的添加为微量添加，不显著改变其中胶原溶液浓度，以溶液达到预设pH值且形成均质溶液为准。In the embodiment of the present invention, adding an alkaline solution or an acidic solution in the step (3) is to adjust the pH value of the collagen solution. The addition of the acidic solution or the alkaline solution is a micro-addition, which does not significantly change the concentration of the collagen solution, as long as the solution reaches a preset pH value and forms a homogeneous solution.
在本发明的实施例中，模拟体液成份包括Na
+、K
+、Mg
2+、Ca
2+、Cl
-、HCO
3
-、HPO
4
2-、SO
4
2-、CO
3
2-、PO
4
3-、H
2PO
4
-中的一种或两种以上的组合。添加模拟体液成份是为了使胶原溶液的离子环境与皮肤组织内的微环境相适应。模拟体液成份的添加为微量添加，不显著改变其中胶原溶液浓度。在用于皮肤表面的仿生胶原溶液中各离子的最终离子浓度范围为0.1-500mM，最终总离子浓度范围为0.1-1000mM。
In the embodiment of the present invention, the simulated body fluid components include Na + , K + , Mg 2+ , Ca 2+ , Cl - , HCO 3 - , HPO 4 2- , SO 4 2- , CO 3 2- , PO 4 One or a combination of two or more of 3- and H 2 PO 4 - . The purpose of adding simulated body fluid components is to adapt the ionic environment of the collagen solution to the microenvironment in the skin tissue. The addition of simulated body fluid components is a small amount, and does not significantly change the concentration of collagen solution therein. The final ion concentration of each ion in the biomimetic collagen solution for the skin surface was in the range of 0.1-500 mM, and the final total ion concentration was in the range of 0.1-1000 mM.
在本发明的实施例中，连接分子为异硫氰酸酯、异氰酸酯、酰基叠氮化物、N-羟基琥珀酰亚胺酯(NHS酯)、磺酰氯、醛、环氧化物、芳基卤化物、酰亚胺酯、碳二亚胺、酸酐、氟苯基酯、原花青素、京尼平中的一种或两种以上的混合物。添加连接分子是为了增强胶原分子在皮肤组织中的吸收。连接分子溶液的添加不显著改变其中胶原溶液浓度，所述连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.0001wt.％但小于等于10wt.％。In embodiments of the present invention, the linking molecules are isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide esters (NHS esters), sulfonyl chlorides, aldehydes, epoxides, aryl halides , one or more mixtures of imide ester, carbodiimide, acid anhydride, fluorophenyl ester, proanthocyanidin and genipin. The linker molecules are added to enhance the absorption of collagen molecules into the skin tissue. The addition of the linker molecule solution did not significantly change the concentration of the collagen solution therein, and the final concentration of the linker molecules in the biomimetic collagen solution for skin surface was 0.0001 wt.% or more but 10 wt.% or less.
在本发明的实施例中，连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.0001wt.％但小于等于1wt.％。In the embodiment of the present invention, the final concentration of the linking molecule in the biomimetic collagen solution for skin surface is greater than or equal to 0.0001 wt.% but less than or equal to 1 wt.%.
在本发明的实施例中，连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.0001wt.％但小于等于0.1wt.％。In an embodiment of the present invention, the final concentration of the linking molecule in the biomimetic collagen solution for skin surface is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%.
在本发明的实施例中，连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.001wt.％但小于等于0.01wt.％。In the embodiment of the present invention, the final concentration of the linking molecules in the biomimetic collagen solution for skin surface is greater than or equal to 0.001 wt.% but less than or equal to 0.01 wt.%.
根据本发明的另一方面，提供了一种用于皮肤表面的仿生胶原溶液的使用方法，所述仿生胶原溶液是根据前述实施例中任一项所述的用于皮肤表面的仿生胶原溶液的制备方法所制备得到的。According to another aspect of the present invention, there is provided a method of using a biomimetic collagen solution for a skin surface, the biomimetic collagen solution being the biomimetic collagen solution for a skin surface according to any one of the preceding embodiments. prepared by the method of preparation.
在本发明的实施例中，在制备完成用于皮肤表面的仿生胶原溶液后一小时内，采用自涂抹、喷涂、浸没中的一种或两种以上的组合方式将仿生胶原溶液应用于皮肤表面。In the embodiment of the present invention, within one hour after the preparation of the biomimetic collagen solution for the skin surface, the biomimetic collagen solution is applied to the skin surface by one or more combinations of self-smearing, spraying, and immersion. .
本发明的实施例是针对胶原分子经皮肤吸收困难的问题从仿生角度提出的解决方案。为了提高胶原溶液的生物相容性，增强胶原分子在皮肤组织中(特别在真皮层)的吸收，本发明的实施例主要进行如下优化：(1)按照皮肤组织中所含胶原分子的类型，选用I型、III型或V型胶原，按照皮肤组织中不同类型胶原的含量制备胶原溶液，在溶液组成上尽可能与皮肤组织相匹配；(2)按照体液中离子的成份和含量以及pH环境，在溶液中添加模拟体液成份，通过调控离子成份和离子强度，并进一步调控pH值，使胶原溶液与皮肤组织的微环境相匹配；(3)相较于常规通过胶原分子直接与皮肤组织结合实现胶原分子吸收的方法， 本发明在胶原溶液中添加了连接分子成份，连接分子可以与胶原溶液中的胶原分子和皮肤组织中的胶原成份同时发生反应，从而将胶原分子结合在皮肤组织中，连接分子的高效反应可以提高胶原分子在皮肤组织中的吸收率。The embodiments of the present invention are proposed solutions from the perspective of bionics for the problem that collagen molecules are difficult to absorb through the skin. In order to improve the biocompatibility of the collagen solution and enhance the absorption of collagen molecules in the skin tissue (especially in the dermis), the embodiments of the present invention are mainly optimized as follows: (1) According to the type of collagen molecules contained in the skin tissue, Select type I, type III or type V collagen, prepare collagen solution according to the content of different types of collagen in the skin tissue, and match the composition of the solution with the skin tissue as much as possible; (2) According to the composition and content of ions in the body fluid and the pH environment , adding simulated body fluid components to the solution, by regulating the ionic components and ionic strength, and further regulating the pH value, so that the collagen solution matches the microenvironment of the skin tissue; (3) Compared with the conventional method, the collagen molecule is directly combined with the skin tissue. The method for realizing the absorption of collagen molecules, the present invention adds connecting molecule components in the collagen solution, and the connecting molecules can react with the collagen molecules in the collagen solution and the collagen components in the skin tissue at the same time, thereby combining the collagen molecules in the skin tissue, The high-efficiency reaction of linking molecules can improve the absorption rate of collagen molecules in skin tissue.
在本发明的实施例中，由于添加了连接分子，在胶原溶液未使用在皮肤表面前，有可能会发生胶原分子与连接分子发生反应的情况，从而影响溶液中胶原分子的流动性，进而影响其在皮肤组织中的吸收。因此，在本发明的实施例的制备方法中，添加连接分子后胶原溶液的保存时间为大于等于0min但小于等于60min，以不影响其使用效果。In the embodiment of the present invention, due to the addition of linking molecules, before the collagen solution is used on the skin surface, the reaction between the collagen molecules and the linking molecules may occur, thereby affecting the fluidity of the collagen molecules in the solution, thereby affecting the Its absorption in skin tissue. Therefore, in the preparation method of the embodiment of the present invention, the storage time of the collagen solution after adding the linking molecule is greater than or equal to 0 min but less than or equal to 60 min, so as not to affect its use effect.
在本发明的实施例中，胶原溶液的浓度为大于等于0.0001wt.％但小于等于0.1wt.％。选用低浓度胶原溶液有利于胶原分子的流动传输，利于胶原分子在皮肤组织中的吸收。In the embodiment of the present invention, the concentration of the collagen solution is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%. The choice of low-concentration collagen solution is conducive to the flow transmission of collagen molecules and the absorption of collagen molecules in skin tissue.
本发明的仿生胶原溶液中的溶液环境与皮肤组织的微环境更接近，其生物相容性比常规的胶原溶液更好，更利于胶原分子进入皮肤组织中(特别在真皮层)吸收；由于连接分子在皮肤组织中的作用，可以延缓胶原分子的降解性，发挥其生物活性，促进皮肤组织修复再生。The solution environment in the biomimetic collagen solution of the present invention is closer to the microenvironment of the skin tissue, and its biocompatibility is better than that of the conventional collagen solution, which is more conducive to the absorption of collagen molecules into the skin tissue (especially in the dermis); The role of molecules in skin tissue can delay the degradation of collagen molecules, exert its biological activity, and promote skin tissue repair and regeneration.
图1为根据本发明的实施例的仿生胶原溶液中的、异硫氰酸荧光素(FITC)荧光染色标记的胶原分子在大鼠皮下的吸收情况效果图(图1A是实施例1样品，图1B是实施例2样品，图1C是实施例3样品，图1D是实施例4样品，图1E是对比例1样品，图1F是对比例2样品)；1 is a graph showing the effect of subcutaneous absorption of collagen molecules labeled by fluorescein isothiocyanate (FITC) fluorescent staining in a biomimetic collagen solution according to an embodiment of the present invention (FIG. 1A is a sample of Example 1, FIG. 1B is a sample of Example 2, Figure 1C is a sample of Example 3, Figure 1D is a sample of Example 4, Figure 1E is a sample of Comparative Example 1, and Figure 1F is a sample of Comparative Example 2);
图2为根据本发明的实施例的仿生胶原溶液中的活/死细胞染色情况效果图(图2A是实施例1样品，图2B是实施例2样品，图2C是实施例3样品，图2D是实施例4样品，图2E是对比例1样品，图2F是对比例2样品)；2 is an effect diagram of live/dead cell staining in a biomimetic collagen solution according to an embodiment of the present invention ( FIG. 2A is the sample of Example 1, FIG. 2B is the sample of Example 2, FIG. 2C is the sample of Example 3, and FIG. 2D It is a sample of Example 4, Figure 2E is a sample of Comparative Example 1, and Figure 2F is a sample of Comparative Example 2);
图3为根据本发明的实施例的仿生胶原溶液植入大鼠体内的HE染色(苏木精—伊红染色)情况效果图(图3A是实施例1样品，图3B是实施例2样品，图3C是实施例3样品，图3D是实施例4样品，图3E是对比例1样品，图3F是对比例2样品)。3 is an effect diagram of HE staining (hematoxylin-eosin staining) of biomimetic collagen solution implanted in rats according to an embodiment of the present invention (FIG. 3A is the sample of Example 1, FIG. 3B is the sample of Example 2, Figure 3C is a sample of Example 3, Figure 3D is a sample of Example 4, Figure 3E is a sample of Comparative Example 1, and Figure 3F is a sample of Comparative Example 2).
下面通过实施例，并结合附图，对本发明的技术方案作进一步具体的说明。在说明书中，相同或相似的附图标号指示相同或相似的部件。下述参照附图对本发明实施方式的说明旨在对本发明的总体发明构思进行解释，而不应当理解为对本发明的一种限制。The technical solutions of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. In the specification, the same or similar reference numerals refer to the same or similar parts. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the general inventive concept of the present invention, and should not be construed as a limitation of the present invention.
实施例1Example 1
一种用于皮肤表面的仿生胶原溶液的制备方法，其中，依次包括如下步骤：A preparation method for a biomimetic collagen solution on a skin surface, wherein, the steps are sequentially included:
(1)将0.1g III型胶原材料置入5000g去离子水中，在搅拌过程中添加0.1M乙酸溶液溶解胶原材料形成均质溶液；(1) Put 0.1g type III collagen material into 5000g deionized water, add 0.1M acetic acid solution during stirring to dissolve the collagen material to form a homogeneous solution;
(2)在2℃下，将步骤(1)所得胶原溶液在搅拌过程中缓慢加入1g NaH
2PO
4和0.5g KHCO
3，充分溶解；
(2) at 2°C, slowly add 1 g of NaH 2 PO 4 and 0.5 g of KHCO 3 to the collagen solution obtained in step (1) during stirring to fully dissolve;
(3)在5℃下，通过0.01M NaOH溶液或0.01M乙酸溶液调节步骤(2)所得胶原溶液pH值至7.2，形成均质溶液；(3) at 5°C, the pH value of the collagen solution obtained in step (2) was adjusted to 7.2 by 0.01M NaOH solution or 0.01M acetic acid solution to form a homogeneous solution;
(4)在0℃下，在步骤(3)所得胶原溶液中加入0.01wt.％原花青素溶液，搅拌均匀。(4) At 0° C., add 0.01 wt.% procyanidin solution to the collagen solution obtained in step (3), and stir evenly.
一种用于皮肤表面的仿生胶原溶液的使用方法，所述仿生胶原溶液是根据上述制备方法制备得到的，所述使用方法包括：在仿生胶原溶液配制10分钟(min)后，将其均匀涂抹于预修复皮肤表面，在2小时(h)后清洗皮肤表面。A method of using a biomimetic collagen solution for skin surface, the biomimetic collagen solution is prepared according to the above preparation method, and the using method includes: 10 minutes (min) after the biomimetic collagen solution is prepared, evenly smearing it On the pre-repaired skin surface, the skin surface was washed after 2 hours (h).
实施例2Example 2
一种用于皮肤表面的仿生胶原溶液的制备方法，其中，依次包括如下步骤：A preparation method for a biomimetic collagen solution on a skin surface, wherein, the steps are sequentially included:
(1)将1g I型胶原材料和0.1g III型胶原材料置入10000g去离子水中，在搅拌过程中添加0.03M盐酸溶液溶解胶原材料形成均质溶液；(1) Put 1g of type I collagen material and 0.1g of type III collagen material into 10000g of deionized water, and add 0.03M hydrochloric acid solution during stirring to dissolve the collagen material to form a homogeneous solution;
(2)在10℃下，将步骤(1)所得胶原溶液在搅拌过程中缓慢加入2g NaCl和5g Na
2CO
3，充分溶解；
(2) at 10°C, slowly add 2g NaCl and 5g Na 2 CO 3 to the collagen solution obtained in step (1) during stirring to fully dissolve;
(3)在2℃下，通过0.2M KOH溶液或0.1M氨水溶液调节步骤(2)所得胶原溶液pH值至10，形成均质溶液；(3) at 2°C, adjust the pH value of the collagen solution obtained in step (2) to 10 by 0.2M KOH solution or 0.1M ammonia solution to form a homogeneous solution;
(4)在2℃下，在步骤(3)所得胶原溶液中加入2wt.％碳二亚胺溶液，搅拌均匀。(4) At 2° C., add 2wt.% carbodiimide solution to the collagen solution obtained in step (3), and stir uniformly.
一种用于皮肤表面的仿生胶原溶液的使用方法，所述仿生胶原溶液是根据上述制备方法制备得到的，所述使用方法包括：在仿生胶原溶液配制60min后，将其均匀喷涂于预修复皮肤表面，在1h后清洗皮肤表面。A method of using a biomimetic collagen solution for skin surface, the biomimetic collagen solution is prepared according to the above preparation method, and the using method includes: after the biomimetic collagen solution is prepared for 60 minutes, uniformly spraying it on pre-repaired skin surface, and the skin surface was washed after 1 h.
实施例3Example 3
一种用于皮肤表面的仿生胶原溶液的制备方法，其中，依次包括如下步骤：A preparation method for a biomimetic collagen solution on a skin surface, wherein, the steps are sequentially included:
(1)将1g V型胶原材料置入1000000g去离子水中，搅拌过程中添加10M草酸溶液溶解胶原材料形成均质溶液；(1) Put 1g of V-type collagen material into 1,000,000g of deionized water, and add 10M oxalic acid solution to dissolve the collagen material during stirring to form a homogeneous solution;
(2)在0℃下，将步骤(1)所得胶原溶液在搅拌过程中缓慢加入2g Na
3PO
4和4g NaHCO
3，充分溶解；
(2) at 0°C, slowly add 2g Na 3 PO 4 and 4g NaHCO 3 to the collagen solution obtained in step (1) during stirring to fully dissolve;
(3)在5℃下，通过1M甲酸溶液或0.2M磷酸溶液调节步骤(2)所得胶原溶液pH值至4，形成均质溶液；(3) at 5°C, the pH value of the collagen solution obtained in step (2) is adjusted to 4 by 1M formic acid solution or 0.2M phosphoric acid solution to form a homogeneous solution;
(4)在8℃下，在步骤(3)所得胶原溶液中加入10wt.％NHS酯溶液和1wt.％京尼平溶液，搅拌均匀。(4) At 8° C., add 10 wt.% NHS ester solution and 1 wt.% genipin solution to the collagen solution obtained in step (3), and stir uniformly.
一种用于皮肤表面的仿生胶原溶液的使用方法，所述仿生胶原溶液是根据上述制备方法制备得到的，所述使用方法包括：在仿生胶原溶液配制0min后，将预修复皮肤表面浸没在溶液中，在3h后清洗皮肤表面。A method for using a biomimetic collagen solution for skin surface, the biomimetic collagen solution is prepared according to the above preparation method, and the using method comprises: immersing the pre-repaired skin surface in the solution 0 min after the biomimetic collagen solution is prepared , the skin surface was washed after 3 h.
实施例4Example 4
一种用于皮肤表面的仿生胶原溶液的制备方法，依次包括如下步骤：A method for preparing a biomimetic collagen solution on a skin surface, comprising the steps of:
(1)将2g I型、0.5g III型和0.1g V型胶原材料置入200000g去离子水中，搅拌过程中添加1M硝酸溶液溶解胶原形成均质溶液；(1) Put 2g type I, 0.5g type III and 0.1g type V collagen materials into 200000g deionized water, add 1M nitric acid solution to dissolve the collagen during stirring to form a homogeneous solution;
(2)在3℃下，将步骤(1)所得胶原溶液在搅拌过程中缓慢加入10g K
2SO
4和20g Na
2HPO
4，充分溶解；
(2) at 3° C., slowly add 10 g K 2 SO 4 and 20 g Na 2 HPO 4 to the collagen solution obtained in step (1) during stirring to fully dissolve;
(3)在7℃下，通过0.01M吡啶溶液或2M酒石酸溶液调节步骤(2)所得胶原溶液pH值至6，形成均质溶液；(3) at 7°C, the pH value of the collagen solution obtained in step (2) is adjusted to 6 by 0.01M pyridine solution or 2M tartaric acid solution to form a homogeneous solution;
(4)在0℃下，在步骤(3)所得胶原溶液中加入0.001wt.％酰基叠氮化物溶液，搅拌均匀。(4) At 0° C., add 0.001 wt.% acyl azide solution to the collagen solution obtained in step (3), and stir uniformly.
一种用于皮肤表面的仿生胶原溶液的使用方法，所述仿生胶原溶液是根据上述制备方法制备得到的，所述使用方法包括：在仿生胶原溶液配制30min后，将其先均匀喷涂再涂抹于预修复皮肤表面，在2h后清洗皮肤表面。A method for using a biomimetic collagen solution for skin surface, the biomimetic collagen solution is prepared according to the above-mentioned preparation method, and the using method comprises: after preparing the biomimetic collagen solution for 30 minutes, spraying it evenly and then smearing it on The skin surface was pre-repaired, and the skin surface was washed after 2 hours.
实施例5Example 5
用于皮肤表面的仿生胶原溶液效果比较试验Comparative test of the effect of biomimetic collagen solution on skin surface
一、对比胶原水溶液的制备1. Preparation of Collagen Aqueous Solution
对比例1：一种用于皮肤表面的胶原溶液的制备方法及其使用方法，依次包括如下步骤：Comparative Example 1: a preparation method for a collagen solution on the skin surface and a method for using the same, comprising the following steps in turn:
将2g I型胶原材料置入100g去离子水中，在搅拌过程中添加0.5M乙酸溶液溶解胶原材料形成均质溶液；Put 2g of type I collagen material into 100g of deionized water, and add 0.5M acetic acid solution during stirring to dissolve the collagen material to form a homogeneous solution;
将胶原溶液通过喷涂的方式应用于皮肤表面。The collagen solution is applied to the skin surface by spraying.
对比例2：一种用于皮肤表面的胶原溶液的制备方法及其使用方法，其中，依次包括如下步骤：Comparative Example 2: a preparation method for a collagen solution on the skin surface and a method for using the same, wherein the following steps are included in sequence:
将1g I型胶原材料置入10000g去离子水中，在搅拌过程中添加1M盐酸溶液溶解胶原材料形成均质溶液；Put 1g of type I collagen material into 10000g of deionized water, and add 1M hydrochloric acid solution during stirring to dissolve the collagen material to form a homogeneous solution;
将胶原溶液通过涂抹的方式应用于皮肤表面。The collagen solution is applied to the surface of the skin by smearing.
二、效果试验对比Second, the effect test comparison
(1)胶原分子在皮肤组织中吸收情况对比：实施例1-4以及对比例1-2中的胶原原材料经FITC荧光染料标记后分别制备对应的胶原溶液，经过对应的方式应用于大鼠皮肤表面，24h后处死实验大鼠，剪取对应皮肤切片，在A1激光共聚焦显微镜上观察(Nikon)，绿色激发光波长为488nm，比较皮肤中胶原分子的吸收情况，结果见图1。由图1可知，由于实施例中的胶原溶液含有模拟体液成份及pH环境，能够更好的与皮肤微环境相适应，而且连接分子在胶原分子进入皮肤组织的传输过程中可以促进其与周围组织的结合，在皮肤切片中吸收了大量的胶原分子。由于测试胶原分子已标记有标记分子FITC(例如，绿色荧光)，对应于实施例1-4中的样品的图1A-1D发现有大量荧光信号(例如，绿色荧光信号)。然而，对比例中胶原溶液不能模拟皮肤微环境，也没有连接分子，胶原分子进入皮肤组织的传输效率很低，仅能吸收极少的胶原分子。所以，对应于对比例1-2中的样品的图1E-1F只发现很少的荧光信号(例如，绿色荧光信号)。因此，在本发明的实施例中的模拟体液成份的添加、pH值的调控以及连接分子的添加可以促进胶原分子在皮肤组织中的吸收。(1) Comparison of the absorption of collagen molecules in skin tissue: The collagen raw materials in Examples 1-4 and Comparative Examples 1-2 were labeled with FITC fluorescent dye to prepare corresponding collagen solutions, which were applied to rat skin in corresponding ways. On the surface, the experimental rats were sacrificed 24 hours later, and the corresponding skin sections were cut and observed on an A1 laser confocal microscope (Nikon). The wavelength of the green excitation light was 488 nm, and the absorption of collagen molecules in the skin was compared. As can be seen from Figure 1, since the collagen solution in the embodiment contains simulated body fluid components and pH environment, it can better adapt to the skin microenvironment, and the linking molecules can promote collagen molecules and surrounding tissues during the transmission process of collagen molecules into the skin tissue. , absorbed a large amount of collagen molecules in the skin slices. Since the tested collagen molecules have been labeled with the marker molecule FITC (eg, green fluorescence), a substantial amount of fluorescent signal (eg, green fluorescence signal) is found in Figures 1A-1D corresponding to the samples in Examples 1-4. However, the collagen solution in the comparative example cannot simulate the skin microenvironment and has no connecting molecules. The transmission efficiency of collagen molecules into the skin tissue is very low, and only a few collagen molecules can be absorbed. Therefore, only little fluorescent signal (eg, green fluorescent signal) was found in FIGS. 1E-1F corresponding to the samples in Comparative Examples 1-2. Therefore, the addition of simulated body fluid components, the regulation of pH value, and the addition of linking molecules in the embodiments of the present invention can promote the absorption of collagen molecules in skin tissue.
(2)三维细胞培养情况比较：根据实施例1-4以及对比例1-2中的制备方法制备胶原溶液，分别取1mL与6×10
5大鼠皮肤成纤维细胞(中国科学院上海细胞库)混合，在37℃条件下形成胶原水凝胶包裹细胞进行三维培养实验。培养基为高糖DMEM(HyClone)培养基，含10％(v/v％)的胎牛血清，1％的链霉素和青霉素，培养基每天更换一次。培养箱(STERI 371,Thermo Electron Corporation)温度设定为37℃，含有5％(v/v％)CO
2的湿空气。培养10d后，水凝胶中的细胞经活/死细胞染色处理，染色液为2mmol/L的calcein-AM(钙黄绿素乙酰氧基甲酯溶液，Sigma)(用于染活细胞，发射绿色荧光)和2mmol/L的EthD-1溶液(溴乙啡锭二聚体1，Sigma)(用于染死细胞，发射红色荧光)，在A1激光共聚焦显微镜(Nikon)上观察样品，绿色激发光波长为488nm，红色激发光波长为562nm。胶原水凝胶中活/死细胞染色情况见图2。由图2可知，由于实施例1-4中的胶原溶液与皮肤组织微环境更接近， 其形成的胶原水凝胶更适宜皮肤细胞的生长，在实施例样品图2A-2D中代表活细胞的荧光信号(例如绿色荧光信号)更强，这说明细胞在根据本发明的实施例的制备方法制备的仿生胶原溶液中可以正常生长，材料的生物相容性好。然而，在对比例1-2中，胶原水凝胶不适宜皮肤细胞生长，在对比例样品图2E-2F中代表死细胞的荧光信号(例如红色荧光信号)更强，这说明细胞无法在根据对比例1-2中的制备方法制备的胶原溶液中正常生长，材料的生物相容性很差。因此，在本公开的实施例中，模拟体液成份的添加和pH值的调控更利于细胞生长，胶原溶液也表现出更好的生物相容性。
(2) Comparison of three-dimensional cell culture conditions: Collagen solutions were prepared according to the preparation methods in Examples 1-4 and Comparative Examples 1-2, and 1 mL and 6×10 5 rat skin fibroblasts were taken respectively (Shanghai Cell Bank, Chinese Academy of Sciences) The cells were mixed to form collagen hydrogel-encapsulated cells at 37°C for three-dimensional culture experiments. The medium was high glucose DMEM (HyClone) medium containing 10% (v/v%) fetal bovine serum, 1% streptomycin and penicillin, and the medium was changed once a day. The temperature of the incubator (STERI 371, Thermo Electron Corporation) was set at 37°C and humidified air containing 5% (v/v%) CO 2 . After culturing for 10 days, the cells in the hydrogel were stained with live/dead cells, and the staining solution was 2mmol/L calcein-AM (calcein acetoxymethyl ester solution, Sigma) (used to stain live cells and emit green fluorescence) ) and 2 mmol/L EthD-1 solution (ethidium bromide dimer 1, Sigma) (used to stain dead cells, emit red fluorescence), observe the samples on A1 confocal microscope (Nikon), green excitation light The wavelength is 488 nm, and the wavelength of the red excitation light is 562 nm. Live/dead cell staining in collagen hydrogels is shown in Figure 2. It can be seen from Figure 2 that since the collagen solution in Examples 1-4 is closer to the skin tissue microenvironment, the collagen hydrogel formed by it is more suitable for the growth of skin cells. The fluorescence signal (eg green fluorescence signal) is stronger, which indicates that cells can grow normally in the biomimetic collagen solution prepared according to the preparation method of the embodiment of the present invention, and the biocompatibility of the material is good. However, in Comparative Examples 1-2, the collagen hydrogels were not suitable for skin cell growth, and the fluorescent signals (such as red fluorescent signals) representing dead cells were stronger in the Comparative Examples Figures 2E-2F, which indicated that the cells could not grow according to The collagen solution prepared by the preparation method in Comparative Examples 1-2 grows normally, and the biocompatibility of the material is poor. Therefore, in the embodiments of the present disclosure, the addition of simulated body fluid components and the regulation of pH value are more conducive to cell growth, and the collagen solution also exhibits better biocompatibility.
(3)组织相容性比较：在大鼠肌肉分别植入根据实施例1-4以及对比例1-2的制备方法制备的胶原溶液，经1个月后处死实验大鼠，取出植入材料，并使用4wt.％的多聚甲醛固定液固定，进行HE染色。(3) Comparison of histocompatibility: The collagen solutions prepared according to the preparation methods of Examples 1-4 and Comparative Examples 1-2 were respectively implanted in the muscles of rats, and the experimental rats were sacrificed after 1 month, and the implanted materials were taken out. , and fixed with 4 wt.% paraformaldehyde fixative for HE staining.
具体步骤如下：Specific steps are as follows:
(1)将样品放在石蜡包埋机里进行包埋，然后用切片机进行切片，将切好的片子放入60℃的水浴锅里，用载玻片小心插入靠近切片的水里，将漂浮的石蜡切片转移到载玻片上，若有水泡，则使用针头挑出。(1) Put the sample in a paraffin embedding machine for embedding, then slice it with a microtome, put the sliced slices into a 60°C water bath, carefully insert a glass slide into the water close to the slice, and place the slices into the water close to the slices. Floating paraffin sections were transferred to glass slides, and blisters were picked out with a needle.
(2)使用二甲苯脱水5min并进行两次，使用100％酒精、95％酒精、85％酒精、70％酒精、50％酒精依次冲洗，再使用自来水冲洗，然后使用苏木精染色5min，再使用自来水冲洗使颜色变蓝。(2) Use xylene to dehydrate for 5 minutes and perform twice, rinse with 100% alcohol, 95% alcohol, 85% alcohol, 70% alcohol, and 50% alcohol in turn, then rinse with tap water, then stain with hematoxylin for 5 minutes, and then use Rinse with tap water to turn the color blue.
(3)放入1％盐酸乙醇溶液中褪色2-10秒(s)，颜色变红并较浅，再不适用自来水冲洗恢复蓝色。(3) Put it into 1% hydrochloric acid ethanol solution to fade for 2-10 seconds (s), the color becomes red and lighter, and it can be washed with tap water to restore the blue color.
(4)再分别放入50％酒精、70％酒精、80％酒精中并分别持续5min，再使用0.5％伊红酒精溶液对比染色1-3min。(4) Put them in 50% alcohol, 70% alcohol, and 80% alcohol respectively for 5 minutes, and then use 0.5% eosin alcohol solution for contrast staining for 1-3 minutes.
(5)将切片放入95％酒精中洗去多余红色，然后放入100％酒精中3-5min，再使用吸水纸吸取多余酒精，并将切片放入二甲苯Ⅰ、Ⅱ各3-5min。(5) Put the slices into 95% alcohol to wash off the excess redness, then put them into 100% alcohol for 3-5 minutes, then use absorbent paper to absorb the excess alcohol, and put the slices into xylene I and II for 3-5 minutes each.
(6)用中性树胶封存并进行固定。(6) Seal and fix with neutral gum.
不同样品的HE染色结果见图3。由图3可知，由于实施例1-4中(图3A-3D)的胶原溶液与大鼠体内组织的微环境更接近，所以材料植入体内后能很快与周围组织融合，只发现少量炎症细胞，而对比例1-2中(图3E-3F)则发现大量炎症细胞，材料的组织相容性很差。因此，在本公开的实施例中，模拟体液成份的添加和pH值的调控可以明显提高胶原溶液在体内的组织相容性。The HE staining results of different samples are shown in Figure 3. As can be seen from Figure 3, since the collagen solution in Examples 1-4 (Figures 3A-3D) is closer to the microenvironment of the tissue in the rat body, the material can quickly fuse with the surrounding tissue after being implanted in the body, and only a small amount of inflammation is found. However, in Comparative Examples 1-2 (FIG. 3E-3F), a large number of inflammatory cells were found, and the histocompatibility of the material was poor. Therefore, in the embodiments of the present disclosure, the addition of simulated body fluid components and the regulation of pH value can significantly improve the histocompatibility of the collagen solution in vivo.
结合效果试验可知，实施例1-4与对比例1-2相比，对比例1-2中胶原溶液为强酸性溶 液，不利于胶原溶液在皮肤表面的使用；实施例1-4中的胶原溶液偏中性，而且可根据不同的皮肤进行调控，更适宜皮肤表面的微环境；实施例1-4添加了与体液相似的离子成份，使胶原溶液的离子强度、渗透性等性能与皮肤的微环境更接近，利于胶原分子的传输；实施例1-4添加了连接分子，可以在胶原分子进入真皮层的过程中实现胶原分子与真皮层中胶原成份的结合，利于溶液中胶原分子在真皮层的富集和吸收；实施例1-4中的胶原溶液更利于细胞生长以及与体内组织的融合。The binding effect test shows that, compared with Comparative Example 1-2, the collagen solution in Comparative Example 1-2 is a strongly acidic solution, which is not conducive to the use of the collagen solution on the skin surface; The solution is neutral and can be adjusted according to different skins, which is more suitable for the microenvironment of the skin surface; Examples 1-4 add ionic components similar to those of body fluids, so that the ionic strength and permeability of the collagen solution are comparable to those of the skin. The microenvironment is closer, which is conducive to the transmission of collagen molecules; in Examples 1-4, connecting molecules are added, which can realize the combination of collagen molecules and collagen components in the dermis during the process of collagen molecules entering the dermis, which is beneficial to the collagen molecules in the solution. Layer enrichment and absorption; collagen solutions in Examples 1-4 are more favorable for cell growth and fusion with in vivo tissues.
以上所述的实施例仅仅是对本发明的优选实施方式进行描述，在不脱离本发明设计精神的前提下，本领域技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。The above-mentioned embodiments are only to describe the preferred embodiments of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by those skilled in the art to the technical solutions of the present invention shall fall within the scope of the present invention. within the scope of protection determined by the claims of the present invention.
Claims (10)
- 一种用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，依次包括如下步骤：A method for preparing a biomimetic collagen solution for skin surface, comprising the steps of:(1)将固体胶原材料置入水中，在搅拌过程中添加酸性溶液溶解所述固体胶原材料形成均质溶液，胶原溶液的浓度为大于等于0.0001wt.％但小于等于0.1wt.％；(1) Put the solid collagen material into water, add an acidic solution to dissolve the solid collagen material during stirring to form a homogeneous solution, and the concentration of the collagen solution is greater than or equal to 0.0001 wt.% but less than or equal to 0.1 wt.%;(2)低温条件下，将步骤(1)所得胶原溶液在搅拌过程中缓慢加入模拟体液成份，充分溶解；(2) under low temperature conditions, the collagen solution obtained in step (1) is slowly added to simulate body fluid components in the stirring process to fully dissolve;(3)低温条件下，通过碱性溶液或酸性溶液调节步骤(2)所得胶原溶液pH值至大于等于4但小于等于10，形成均质溶液；(3) Under low temperature conditions, the pH value of the collagen solution obtained in step (2) is adjusted to be greater than or equal to 4 but less than or equal to 10 by an alkaline solution or an acidic solution to form a homogeneous solution;(4)低温条件下，在步骤(3)所得胶原溶液中加入连接分子溶液搅拌均匀后制备完成，在制备完成后，在低温条件下保存所述仿生胶原溶液。(4) Under low temperature conditions, the linking molecule solution is added to the collagen solution obtained in step (3) and the preparation is completed after stirring evenly. After the preparation is completed, the biomimetic collagen solution is stored under low temperature conditions.
- 如权利要求1所述的用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，步骤(2)、步骤(3)和步骤(4)中的低温条件的范围为大于等于0℃但小于等于10℃；所述固体胶原材料选自I型、III型、V型胶原中的一种或两种以上的组合；在所述步骤(3)中，通过碱性溶液或酸性溶液调节胶原溶液pH值至大于等于6但小于等于8。The method for preparing a biomimetic collagen solution for skin surface according to claim 1, wherein the range of low temperature conditions in step (2), step (3) and step (4) is greater than or equal to 0°C but less than is equal to 10°C; the solid collagen material is selected from one or a combination of two or more of collagen types I, III and V; in the step (3), the collagen solution is adjusted by an alkaline solution or an acidic solution pH value to 6 or more but less than or equal to 8.
- 如权利要求2所述的用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，模拟体液成份包括Na +、K +、Mg 2+、Ca 2+、Cl -、HCO 3 -、HPO 4 2-、SO 4 2-、CO 3 2-、PO 4 3-、H 2PO 4 -中的一种或两种以上的组合。 The method for preparing a biomimetic collagen solution for skin surface according to claim 2, wherein the simulated body fluid components include Na + , K + , Mg 2+ , Ca 2+ , Cl - , HCO 3 - , HPO 4 One or a combination of two or more of 2- , SO 4 2- , CO 3 2- , PO 4 3- , H 2 PO 4 - .
- 如权利要求3所述的用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，在步骤(1)和步骤(3)中的所述酸性溶液同为包括盐酸、硝酸、硫酸、磷酸、酒石酸、柠檬酸、草酸、乙酸和甲酸中的至少一种；所述步骤(3)中的所述碱性溶液包括三乙胺、四甲基乙二胺、吡啶、哌啶、氢氧化钠、氢氧化钾、氢氧化钙、氢氧化镁、氨水、磷酸氢二钠和碳酸氢钠中的至少一种；所述酸性溶液、碱性溶液的浓度为大于等于0.01M但小于10M。The method for preparing a biomimetic collagen solution for skin surface as claimed in claim 3, wherein the acidic solutions in step (1) and step (3) are both including hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, At least one of tartaric acid, citric acid, oxalic acid, acetic acid and formic acid; the alkaline solution in the step (3) includes triethylamine, tetramethylethylenediamine, pyridine, piperidine, sodium hydroxide, At least one of potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, disodium hydrogen phosphate and sodium bicarbonate; the concentration of the acidic solution and the alkaline solution is greater than or equal to 0.01M but less than 10M.
- 如权利要求4所述的用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，连接分子为异硫氰酸酯、异氰酸酯、酰基叠氮化物、N-羟基琥珀酰亚胺酯(NHS酯)、磺酰氯、醛、环氧化物、芳基卤化物、酰亚胺酯、碳二亚胺、酸酐、氟苯基酯、原花青素、京尼平中的一种或两种以上的混合物，所述连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.0001wt.％但小于等于10wt.％。The method for preparing a biomimetic collagen solution for skin surface according to claim 4, wherein the linking molecule is isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester (NHS ester) ), one or more mixtures of sulfonyl chloride, aldehyde, epoxide, aryl halide, imide ester, carbodiimide, acid anhydride, fluorophenyl ester, proanthocyanidin, genipin, etc. The final concentration of the linking molecules in the biomimetic collagen solution for skin surface is greater than or equal to 0.0001 wt.% but less than or equal to 10 wt.%.
- 如权利要求5所述的用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.0001wt.％但小于等于1 wt.％。The method for preparing a biomimetic collagen solution for skin surface according to claim 5, wherein the final concentration of linking molecules in the biomimetic collagen solution for skin surface is greater than or equal to 0.0001 wt.% but less than or equal to 1 wt .%.
- 如权利要求6所述的用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.0001wt.％但小于等于0.1wt.％。The method for preparing a biomimetic collagen solution for skin surface according to claim 6, wherein the final concentration of linking molecules in the biomimetic collagen solution for skin surface is greater than or equal to 0.0001wt.% but less than or equal to 0.1wt .%.
- 如权利要求7所述的用于皮肤表面的仿生胶原溶液的制备方法，其特征在于，连接分子在用于皮肤表面的仿生胶原溶液中的最终浓度为大于等于0.001wt.％但小于等于0.01wt.％。The method for preparing a biomimetic collagen solution for skin surface according to claim 7, wherein the final concentration of linking molecules in the biomimetic collagen solution for skin surface is greater than or equal to 0.001wt.% but less than or equal to 0.01wt .%.
- 一种用于皮肤表面的仿生胶原溶液的使用方法，所述仿生胶原溶液是根据权利要求1至8中任一项所述的用于皮肤表面的仿生胶原溶液的制备方法所制备得到的。A method for using a biomimetic collagen solution for skin surface, wherein the biomimetic collagen solution is prepared according to the method for preparing a biomimetic collagen solution for skin surface according to any one of claims 1 to 8.
- 如权利要求9所述的用于皮肤表面的仿生胶原溶液的使用方法，其特征在于，在制备完成用于皮肤表面的仿生胶原溶液后一小时内，采用自涂抹、喷涂、浸没中的一种或两种以上的组合方式将仿生胶原溶液应用于皮肤表面。The method for using the biomimetic collagen solution for the skin surface as claimed in claim 9, wherein within one hour after the preparation of the biomimetic collagen solution for the skin surface, one of self-smearing, spraying, and immersion is used. Or a combination of two or more to apply the biomimetic collagen solution to the skin surface.
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| CN112402289A (en) * | 2020-10-26 | 2021-02-26 | 杜明春 | Bionic collagen solution for skin surface and preparation method and use method thereof |
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| CN103691002A (en) * | 2013-12-13 | 2014-04-02 | 天津大学 | Bacterial cellulose/collagen/hydroxyapatite composite material, and preparation and application thereof |
| CN109675085B (en) * | 2019-02-26 | 2021-09-28 | 百澳瑞派（天津）生物科技有限公司 | Composite collagen dressing for burn wound repair and preparation method thereof |
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| CN104399120A (en) * | 2014-10-31 | 2015-03-11 | 陕西艾尔肤组织工程有限公司 | Preparation method of collagen membrane and collagen membrane thereof |
| CN105031740A (en) * | 2015-07-01 | 2015-11-11 | 世科志扬（北京）医疗科技有限公司 | Waterproof and breathable bionic artificial skin and preparation method thereof |
| CN105641740A (en) * | 2016-01-05 | 2016-06-08 | 重庆惠森驰恒医疗器械有限公司 | Biological nutrition dressing for thoroughly treating skin injury and preparation method thereof |
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| WO2018072679A1 (en) * | 2016-10-17 | 2018-04-26 | 张自强 | Biomimetic biomineralized artificial bone repair material and preparation method therefor and use thereof |
| CN106421931A (en) * | 2016-11-10 | 2017-02-22 | 广东泰宝医疗科技股份有限公司 | Skin repair material with biological activity and method for preparing skin repair material |
| CN112354013A (en) * | 2020-10-26 | 2021-02-12 | 杜明春 | Bionic collagen aqueous solution and preparation method and use method thereof |
| CN112402289A (en) * | 2020-10-26 | 2021-02-26 | 杜明春 | Bionic collagen solution for skin surface and preparation method and use method thereof |
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| KR20230018468A (en) | 2023-02-07 |
| CN112402289A (en) | 2021-02-26 |
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