A carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel and a preparation method thereof

Abstract

The invention discloses a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel and a preparation method thereof. Recombinant human type I collagen, recombinant human type III collagen and carboxylated chitosan are used as main materials. Recombinant human type I / III collagen microgel is first prepared by emulsion polymerization, and then added into carboxylated chitosan hydrogel to obtain a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel. The recombinant human type I / III collagen microgel comprises raw materials: methacrylic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, oil phase, surfactant, photoinitiator, recombinant human type I and type III collagen; the invention prepares a carboxylated chitosan hydrogel dressing containing recombinant human type I collagen and recombinant human type III collagen with an adjustable ratio according to the dynamic repair process of the skin, which is suitable for skin repair.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel dressings, and particularly relates to a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel and a preparation method thereof. Background Art

[0002] Natural collagen is a type of biopolymer material, the main component of animal connective tissue, and the most abundant and widely distributed structural protein in mammals. In addition, collagen is the main component of the extracellular matrix and can be used to prepare collagen hydrogels. Its unique physical and chemical properties, excellent biological activity, degradability, and ability to stimulate cell attachment and growth make it widely used in high-end medical fields and human tissue repair.

[0003] Compared with animal collagen, recombinant human collagen is produced through genetic engineering technology, avoiding the risk of virus and bacterial contamination that animal collagen may carry, so it has higher biosafety. The amino acid sequence of recombinant human collagen is highly consistent with the collagen sequence of the human body, and its immunogenicity is lower than that of animal collagen, reducing the chance of human body rejection. In addition, recombinant human collagen has good water solubility, is compatible with other water-soluble ingredients, and can be mixed with different active ingredients, moisturizers, antioxidants, etc., thus providing more diverse product application possibilities. Water-soluble recombinant collagen molecules are more likely to penetrate the surface of the skin, thereby better playing a moisturizing, nourishing and repairing role; water-soluble recombinant human collagen makes it easier for human tissues to absorb and utilize, which helps to improve the bioavailability of collagen, thereby more effectively replenishing collagen in the skin and tissues; water-soluble recombinant human collagen does not involve organic solvents in the preparation process, so it is easier to process and prepare various medical beauty products, such as facial masks, essences, injections, etc.

[0004] Type I and type III collagen are the two main structural collagens in human skin, and they play a dynamic repair role in the process of skin injury wound repair. At present, the preparation methods of collagen hydrogels mainly include physical cross-linking and chemical cross-linking. The commonly used technical means of physical cross-linking include dry heat cross-linking treatment, radiation, photooxidation and thermal dehydrogenation. Chemical methods change its properties through modifiers, mainly metal ions, glutaraldehyde, genipin, etc., but these modifiers are cytotoxic in the body and affect cell adsorption, proliferation and growth. For example, the toxicity of the commonly used cross-linking agent glutaraldehyde will slow down the rate of wound healing and cell infiltration, and the low toxicity of genipin will also affect gene expression in cells. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant human type I / III collagen microgel for use in skin wound repair.

[0006] Another object of the present invention is to prepare a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel, and mix the recombinant human type I / III collagen microgel with the carboxylated chitosan hydrogel to obtain a mixed hydrogel dressing. The dressing can adjust the ratio of recombinant human type I and type III collagen in the microgel (1:10-10:1) according to the process of skin wound repair to maintain the needs of dynamic skin repair.

[0007] The present invention provides a method for preparing recombinant human type I / III collagen microgels. Recombinant human type I / III collagen microgels with different particle sizes are prepared by adjusting different rotation speeds to achieve the effect of slowly and controllably releasing type I / III collagen.

[0008] The technical solution adopted by the present invention is:

[0009] A recombinant human type I / III collagen microgel is obtained by emulsification polymerization of the following raw materials: methacrylic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, an oil phase, a surfactant, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), recombinant human type I collagen and recombinant human type III collagen; the particle size is between 10 um and 100 um; the ratio of recombinant human type I collagen to recombinant human type III collagen is adjustable between 1:10 and 10:1.

[0010] A method for preparing recombinant human type I / III collagen microgel comprises the following steps:

[0011] 1) Add methacrylic acid (MAA) to a recombinant human type I collagen solution and stir to prepare methacrylic anhydride recombinant human type I collagen (RHCⅠ-MA solution), then collect the supernatant of the RHCⅠ-MA solution by centrifugation, dilute, filter, dialyze, and freeze-dry to obtain a RHCⅠ-MA sponge-like solid sample; prepare a RHC Ⅲ-MA sponge-like solid sample by the same method;

[0012] 2) The freeze-dried RHCⅠ-MA sponge-like solid sample was dissolved in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution and reacted for 2-4 hours, and then the solution was filtered, dialyzed, and freeze-dried to obtain the RHCⅠ-MA-EDC sponge-like solid sample; the RHCⅢ-MA-EDC sponge-like solid sample was prepared by the same method;

[0013] 3) The RHCⅠ-MA-EDC sponge-like solid sample is dissolved in a photoinitiator with a concentration of 0.1%-1% (w / v) to obtain an RHCⅠ-MA-EDC solution. Similarly, the RHCⅢ-MA-EDC sponge-like solid sample is dissolved in a certain amount of photoinitiator solution with a concentration of 0.1%-1% (w / v) to obtain an RHCⅢ-MA-EDC solution. Subsequently, the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution are uniformly mixed at a volume ratio of 1:10-10:1;

[0014] 4) The mixed solution obtained in step (3) is added dropwise to the mixed solution of the oil phase and the surfactant at different rotation speeds and stirred for a certain period of time. After ultraviolet light irradiation for 3-10 min, the supernatant is removed by centrifugation, PBS is added and shaken evenly to remove the remaining oil phase, and finally PBS buffer is added to rinse it. The recombinant human type I / III collagen microgel with an adjustable type I / III collagen ratio is obtained by filtration and collection.

[0015] In step 1), the recombinant human type I collagen solution is prepared by dissolving recombinant human type I collagen powder in PBS buffer to obtain a recombinant human type I collagen solution with a concentration of 10%-20% (w / v).

[0016] In step 1), the amount of methacrylic acid solution added is 3%-12% (V / V) of the volume of the recombinant human type I collagen solution to prepare methacrylic anhydride recombinant human type I collagen solution (RHCⅠ-MA solution), and the preferred amount added is 6% (V / V).

[0017] In step 2), the EDC concentration is 0.1 M-1 M.

[0018] The photoinitiator in step 3) can be irgacure 2959, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), preferably LAP;

[0019] The oil phase in step 4) can be liquid paraffin, rapeseed oil, mineral oil, soybean oil, preferably rapeseed oil; the surfactant can be lecithin, Tween, span 80, preferably span 80.

[0020] In step 4), the volume ratio of the oil phase to the surfactant is 10:2.

[0021] In step 4), the ultraviolet light irradiation is performed using 405 nm ultraviolet light for 3-10 min.

[0022] In step 4), the different rotation speeds are 500-2000 rpm, more preferably 600-1600 rpm. Different rotation speeds can obtain microgels with different particle sizes. The rotation speed can be gradually increased from low speed to high speed to obtain microgels with different particle sizes, which can achieve the effect of slow and controllable release of type I / III collagen.

[0023] The EDC concentration for dissolving the freeze-dried samples of RHCⅠ-MA and RHCⅢ-MA is 0.1 M to 1 M, preferably 0.1 M;

[0024] The mixing volume ratio of the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution dissolved in the photoinitiator LAP is 1:10-10:1, preferably 4:1.

[0025] When the concentration of recombinant human type I / III collagen solution was 10% (w / v): at 600 rpm, the particle size of the microgel was mainly distributed between 50 um and 60 um; at 1000 rpm, the particle size of the microgel was mainly distributed between 45 um and 50 um; and at 1600 rpm, the particle size of the microgel was mainly distributed between 20 um and 25 um.

[0026] When the concentration is 15% (w / v): at 600 rpm, the particle size of the microgel is mainly distributed between 40 um and 50 um; at 1000 rpm, the particle size of the microgel is mainly distributed between 30 um and 40 um; at 1600 rpm, the particle size of the microgel is mainly distributed between 20 um and 30 um;

[0027] When the concentration was 20% (w / v): at 600 rpm, the particle size of the microgel was mainly distributed between 30 um and 40 um; at 1000 rpm, the particle size of the microgel was mainly distributed between 20 um and 25 um; at 1600 rpm, the particle size of the microgel was mainly distributed between 8 um and 10 um.

[0028] The particle size is inversely proportional to the rotation speed and concentration, and the sustained release rate is inversely proportional to the particle size. By controlling the rotation speed at 500~2000 rpm, microgels of different particle sizes (10 um-100 um) can be obtained, which can achieve the effect of slow and controlled release of type I / III collagen.

[0029] A method for preparing a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel, comprising the steps of first preparing a recombinant human collagen microgel with an adjustable type I / III collagen ratio, then preparing a carboxylated chitosan hydrogel, and finally mixing the recombinant human type I / III collagen microgel with the carboxylated chitosan hydrogel to obtain a mixed hydrogel dressing that can be used for dynamic repair of skin wounds.

[0030] The following steps are included:

[0031] In the first step, a certain amount of methacrylic acid (MAA) is added to the recombinant human type I collagen solution, and the reaction is carried out under magnetic stirring for 3-5 hours to prepare a methacrylic anhydride recombinant human type I collagen solution (RHCⅠ-MA solution); the RHCⅠ-MA solution is centrifuged to collect the supernatant, diluted, filtered through a 0.22 um disposable syringe filter, and then dialyzed using a dialysis bag for 3-4 days, and freeze-dried to obtain an RHCⅠ-MA sponge solid sample; the RHCⅢ-MA sponge solid sample is prepared in the same way;

[0032] In the second step, a certain amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was weighed and dissolved in PBS buffer to obtain a 0.1 M-1 M EDC solution, which was filtered through a 0.22 um disposable syringe filter and set aside for later use. The freeze-dried RHCⅠ-MA sample was fully dissolved in the cross-linking agent EDC, and reacted for 2-4 h to obtain an RHCⅠ-MA-EDC solution, which was filtered through a 0.22 um disposable syringe filter again, dialyzed again using a dialysis bag for 3-4 days, and freeze-dried to obtain an RHCⅠ-MA-EDC sponge solid sample; the RHCⅢ-MA-EDC sponge solid sample was prepared in the same way;

[0033] The third step is to dissolve the freeze-dried RHCⅠ-MA-EDC sponge-like solid sample in a certain amount of a photoinitiator solution with a concentration of 0.1%-1% (w / v) to obtain a RHCⅠ-MA-EDC solution; prepare a RHCⅢ-MA-EDC solution by the same method, and then mix the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution evenly according to a certain volume ratio;

[0034] The fourth step is to slowly add the mixed solution obtained in the third step into the mixed solution of the oil phase and the surfactant through a syringe at different speeds and stir for a certain period of time. After curing by ultraviolet light for 3-10 minutes, the supernatant is removed by centrifugation, PBS is added and shaken evenly to remove the remaining oil phase, and PBS buffer is added 1-3 times to remove the oil phase. Finally, PBS buffer is added to rinse, and recombinant human type I / III collagen microgels with adjustable type I / III collagen ratios are obtained by filtration and collection;

[0035] (5) Step 5: weigh a certain amount of carboxylated chitosan powder, dissolve it in PBS phosphate buffer under magnetic stirring at room temperature to prepare 10%-20% (w / v) carboxylated chitosan hydrogel, and then add appropriate amount of xanthan gum to adjust the viscosity of the carboxylated chitosan hydrogel;

[0036] (6) Finally, the recombinant human type I / III collagen microgel obtained in step (4) and the carboxylated chitosan hydrogel obtained in step (5) are evenly mixed, and bubbles are removed by negative pressure to obtain a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel.

[0037] In step 5), 0.1%-3% (w / v) xanthan gum is added to the carboxylated chitosan hydrogel and stirred to dissolve to adjust the viscosity of the hydrogel dressing, preferably 2-3% (w / v).

[0038] In step 6), the recombinant human type I / III collagen microgel and the carboxylated chitosan hydrogel are uniformly mixed in a volume ratio of 1:5-5:1, preferably 2:1.

[0039] The carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel prepared by the above method has a particle size of the recombinant human type I / III microgel in the dressing between 10 um and 100 um, preferably between 50 um and 75 um.

[0040] In the present invention, recombinant human type I and recombinant human type III collagen can be obtained from microbial expression systems (such as Escherichia coli, Streptococcus, Saccharomyces cerevisiae, Pichia pastoris, etc.), mammalian expression systems (such as transgenic mice, CHO cells, human embryonic kidney cells, etc.), plant expression systems (Arabidopsis, tobacco, soybean, corn, etc.) and insect expression systems (Spodoptera frugiperda cells, Trichoplusia cells, Bombyx mori cells, Helicoverpa armigera cells, etc.).

[0041] The recombinant human collagen microgel of the present invention has different particle sizes, that is, different specific surface areas, thereby achieving the effect of slowly and controllably releasing type I / III collagen.

[0042] Application of carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel in the preparation of skin injury repair materials or materials for promoting skin-related cell proliferation.

[0043] For example, it is used in non-chronic wound repair, such as absorbing wound exudate or draining water from the wound, and covering non-chronic wounds such as postoperative suture wounds. It is suitable for the care of adults with non-chronic wounds (such as superficial wounds, postoperative suture wounds, mechanical trauma, small wounds, abrasions, cut wounds, puncture sites of puncture instruments, first-degree or shallow second-degree burn wounds, laser / photon / acid peel / micro-plastic surgery wounds), providing a microenvironment for wound healing, etc.

[0044] The ratio of type I to type III collagen is adjustable from 1:10 to 10:1.

[0045] A skin wound dressing prepared by mixing recombinant human collagen microgel with adjustable ratio of type I and type III collagen with carboxylated chitosan hydrogel can better meet the collagen demand of skin damage in the repair stage, and has a good effect in promoting wound healing, reducing scar formation, improving anti-infection ability and promoting the proliferation of skin-related cells.

[0046] In the present invention, type I and type III collagen are selected as the raw materials of hydrogel dressings for two main reasons. First, type I and type III collagen are the two main structural collagens in human skin, and their proportions vary greatly at different ages. For example, type I collagen accounts for about 80% in adult skin, and most of the rest is type III collagen, while the proportion of type III collagen in infant skin is higher. Second, the roles of type I and type III collagen are different in the process of skin injury wound repair. In the early stage of wound formation, inflammatory mediators promote the synthesis of type III collagen, so type III collagen helps to provide structural support for the wound in the early stage; when the wound healing enters the repair period, type III collagen decreases, and type I collagen begins to gradually replace type III collagen as the main structural protein. At this stage, the synthesis of type I collagen will increase significantly, which helps to enhance the structural stability and strength of the wound. Type I collagen is dominant at the end of wound healing, and it can provide more lasting support and protection. Therefore, choosing collagen with a controllable ratio of type I to type III to prepare hydrogel is more in line with the physiological structure of the skin and the needs of each stage of wound healing.

[0047] Methacrylic acid is used to react with recombinant human type I / III collagen solution to prepare photosensitive recombinant human type I / III collagen, and recombinant human type I / III collagen microgel is prepared by emulsion polymerization and photocrosslinking. The crosslinking agent EDC is added to promote the reaction between the carboxyl and amino groups on the collagen to form a stable peptide bond and enhance the stability of the microgel. The present invention prepares recombinant human type I / III collagen microgels with adjustable type I / III collagen ratio and adjustable microgel size through an emulsion polymerization method and by adjusting the rotation speed. The microgels prepared by this method have different specific surface areas, and can achieve the effect of slowly and controllably releasing type I / III collagen, which is more in line with the dynamic process of skin repair.

[0048] EDC can activate the carboxyl groups on the collagen molecules, prompting the activated carboxyl groups to react quickly with the amino groups on the collagen molecules to form neutral amide bonds, thereby achieving the purpose of cross-linking, thereby enhancing the stability of the collagen molecules. During the above reaction process, EDC was not grafted onto the collagen molecules. At the same time, the water-soluble isourea byproducts produced during the reaction process can also be completely removed by simple operations such as washing, soaking, and dialysis, so that the cross-linked collagen still has good biocompatibility. The gelation of polypeptides and collagen can be achieved by photocrosslinking. First, by treating collagen with methacrylic anhydride, the collagen is given the property of being photocurable. After being evenly mixed with the photoinitiator, the gelation reaction can be rapidly carried out under light of a specific wavelength. No toxic substances are produced during this photocrosslinking process. Therefore, the collagen hydrogel prepared by photocrosslinking has good biocompatibility and can provide a good attachment and proliferation environment for cells.

[0049] The photoinitiator LAP is a highly water-soluble, visible light-sensitive, biocompatible type I photoinitiator, which is often used for photopolymerization of hydrogel materials. It has a wide absorption wavelength range for ultraviolet light and low requirements for light sources; when it is exposed to ultraviolet light and absorbs light energy, it is excited to an excited state and decomposes to produce active free radicals, which can quickly initiate polymerization or cross-linking reactions. Therefore, using LAP as a photoinitiator has the advantage of rapid curing, which can shorten the curing time and improve efficiency. Rapeseed oil has a low melting point and viscosity. As a natural plant oil, it is friendly to the human body and has good biocompatibility. Therefore, rapeseed oil is used as the oil phase. When preparing recombinant human type I / III collagen microgels, adding the surfactant span 80 to rapeseed oil can prevent coagulation or deposition between recombinant human type I / III collagen microgels and maintain the stability of the microgels. Span 80 has good solubility and dispersion properties, can form effective emulsions in water and oil phases, and can emulsify oil and water phases very well, so that recombinant human type I / III collagen microgels are evenly dispersed in the oil phase system, greatly improving the stability and uniformity of recombinant human type I / III collagen microgels.

[0050] Carboxylated chitosan is a derivative of chitosan. Through chemical modification, carboxyl (-COOH) groups are introduced into the molecular structure of chitosan, which can enhance the water solubility, biocompatibility and biodegradability of chitosan. Carboxylated chitosan has good water solubility, so it is easier to prepare hydrogels than chitosan. The use of carboxylated chitosan eliminates the use of organic acids. Second, the antibacterial properties of carboxylated chitosan are improved, which can more effectively inhibit the growth of bacteria. Third, the biocompatibility of carboxylated chitosan is better than that of chitosan.

[0051] Xanthan gum is a highly water-absorbent polysaccharide that can form a stable gel in water. Xanthan gum has excellent water absorption properties and can absorb the surrounding exudate, maintain a moist environment on the wound or skin surface, and help promote wound healing. The role of xanthan gum in hydrogel dressings is mainly reflected in gel formation, water absorption, permeation control and adhesion. The adhesion properties of xanthan gum can help the dressing fit the skin surface, reduce displacement, and improve the stability and comfort of the dressing. Therefore, adding xanthan gum to carboxylated chitosan hydrogel can optimize the practicality of the hydrogel dressing.

[0052] The present invention prepares recombinant human type I / III collagen microgels with different particle sizes by adjusting different rotation speeds, so that the composite skin gel dressing can achieve the effect of slowly and controllably releasing type I / III collagen during use. The structure of the hydrogel dressing without microspheres is relatively simple and does not have the characteristics of slowly and controllably releasing collagen. The present invention adds a microsphere structure to the carboxylated chitosan hydrogel, and can load various types of active ingredients on the microspheres, so that the skin dressing can slowly and controllably release active molecules, thereby giving the skin dressing better potential. Beneficial Effects

[0053] The present invention aims at the dynamic change process of the content of type I collagen and type III collagen in the process of skin wound repair and the characteristics of different content of type I and type III collagen in human skin of different age groups, prepares a recombinant human collagen microgel with adjustable ratio of type I and type III collagen, and mixes it with carboxylated chitosan hydrogel to prepare a mixed hydrogel dressing. The mixed hydrogel dressing can not only adjust the ratio of type I collagen and type III collagen in time according to the wound recovery process, but also provide a good moisturizing, antibacterial and anti-inflammatory microenvironment for the skin by adding carboxylated chitosan and coordinating with recombinant human type I / III collagen, and has good moisturizing and anti-infection effects on skin wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The present invention is a flowchart of the method for preparing recombinant human type I / III collagen microgel.

[0055] Figure 2 The diagram shows the recombinant human type I / III collagen microgels prepared at three different rotation speeds in the present invention and the sustained release rate of the recombinant human collagen in different microgels.

[0056] Figure 3 FTIR images of RHC Ⅰ before and after grafting with MAA.

[0057] Figure 4 FTIR images of RHC Ⅲ before and after grafting with MAA.

[0058] Figure 5 H NMR spectrum of RHC Ⅰ-MA.

[0059] Figure 6 H NMR spectrum of RHC Ⅲ-MA.

[0060] Figure 7 The present invention is a flow chart of mixing recombinant human type I / III collagen microgel and carboxylated chitosan hydrogel.

[0061] Figure 8 This is a real picture of the carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel of the present invention.

[0062] Fig. 9 The live / dead fluorescence staining micrographs of human skin fibroblasts cultured in the carboxylated chitosan hydrogel dressing environment containing recombinant human type I / III collagen microgel of the present invention and in the complete culture medium environment for 24 hours.

[0063] Fig.10 The live / dead fluorescence staining micrographs of human umbilical vein endothelial cells cultured in the carboxylated chitosan hydrogel dressing environment containing recombinant human type I / III collagen microgel of the present invention and in the complete culture medium environment for 24 hours.

[0064] Fig.11 The wound surfaces of SD rats on days 0, 7, and 10. The unit scale is 1 mm and the total length is 20 mm. DETAILED DESCRIPTION

[0065] The present invention will be further described below in conjunction with specific embodiments and drawings.

[0066] Recombinant human type I collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.;

[0067] Recombinant human type III collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.;

[0068] Carboxylated chitosan was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0069] The reagents used in the following examples are all conventional reagents in the art and can be purchased from commercial sources. Example 1

[0070] The purpose of this example is to prepare recombinant human type I / III collagen microgels at a speed of 600 rpm. The preparation process is as follows: Figure 1 As shown:

[0071] Weigh 1 g of recombinant human type I collagen powder and dissolve it in 10 mL PBS buffer to prepare a 10% (w / v) recombinant human type I collagen solution. Weigh 1 g of recombinant human type III collagen powder and dissolve it in 10 mL PBS buffer to prepare a 10% (w / v) recombinant human type III collagen solution. Then, add 300 ul of methacrylic acid to each of the recombinant human type I collagen solution and the recombinant human type III collagen solution, react for 3 h under magnetic stirring to prepare RHCⅠ-MA solution and RHCⅢ-MA solution. Then, centrifuge at 6000 rpm for 4 min to collect the supernatant, dilute it 2 times, filter it through a 0.22 um disposable syringe filter, dialyze it for 3-4 days using a dialysis bag, and freeze-dry it to obtain two types of sponge-like solid samples, RHCⅠ-MA and RHCⅢ-MA.

[0072] Weigh a certain amount of EDC and dissolve it in PBS buffer to obtain a 0.1 M EDC solution, filter it with a 0.22 um disposable syringe filter and set it aside. Dissolve the freeze-dried RHCⅠ-MA sample in the EDC solution and react for 3 h to obtain the RHCⅠ-MA-EDC solution. Similarly, dissolve the freeze-dried RHCⅢ-MA sample in the EDC solution and react for 3 h to obtain the RHCⅢ-MA-EDC solution. After filtering the two sample solutions again with a 0.22 um disposable syringe filter, dialyze them with a dialysis bag for 3-4 days, and freeze-dry them to obtain two types of sponge-like solids, RHCⅠ-MA-EDC and RHCⅢ-MA-EDC.

[0073] (3) Take 0.5 g of the RHCⅠ-MA-EDC freeze-dried sample and dissolve it in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 10% (w / v) RHCⅠ-MA-EDC solution, take 0.5 g of the RHCⅢ-MA-EDC freeze-dried sample and dissolve it in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 10% (w / v) RHCⅢ-MA-EDC solution, and then mix the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution in a volume ratio of 1:4;

[0074] (4) The uniformly mixed photosensitive recombinant human type I / III collagen solution was then slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span 80 through a syringe at a rotation speed of 600 rpm and stirred for 5 min. It was then cured by ultraviolet light for 5 min under stirring. Finally, the particle size was observed under a microscope. The particle size of the microgel at 600 rpm was measured by ImageJ software to be in the range of 50 um-60 um. Example 2

[0075] The purpose of this example is to prepare recombinant human type I / III collagen microgels at a speed of 1000 rpm.

[0076] (1) According to steps (1) to (3) in Example 1, a recombinant human type I / III collagen solution was prepared in a volume ratio of 1:4 between RHCⅠ-MA-EDC solution and RHCⅢ-MA-EDC solution;

[0077] (2) The uniformly mixed recombinant human type I / III collagen solution was then slowly added to a mixed solution of 10 mL rapeseed oil and 2 mL span 80 through a syringe at a rotation speed of 1000 rpm and stirred for 5 min. The mixture was then cured under ultraviolet light for 5 min under stirring. Finally, the particle size was observed under a microscope. The particle size of the microgel at 1000 rpm was measured to be in the range of 45 um to 50 um using ImageJ software. Example 3

[0078] The purpose of the embodiment is to prepare recombinant human type I / III collagen microgel at a speed of 1600 rpm

[0079] (1) According to steps (1) to (3) in Example 1, a recombinant human type I / III collagen solution was prepared in a volume ratio of 1:4 between RHCⅠ-MA-EDC solution and RHCⅢ-MA-EDC solution;

[0080] (2) The uniformly mixed recombinant human type I / III collagen solution was then slowly added to a mixed solution of 10 mL rapeseed oil and 2 mL span 80 through a syringe at a rotation speed of 1600 rpm and stirred for 5 min. The solution was then cured under ultraviolet light for 5 min under stirring. Finally, the particle size was observed under a microscope. The particle size of the microgel at 1600 rpm was measured to be in the range of 20 um-25 um using ImageJ software.

[0081] (3) Weigh 1 g of microspheres at 600 rpm, 1000 rpm, and 1600 rpm, respectively, and add them to 10 mL of the same release medium (PBS solution). Sampling is performed at the same time intervals to determine the concentration of collagen in the medium.

[0082] The results of Example 1, Example 2 and Example 3 are as follows Figure 2 It shows that the particle size is inversely proportional to the rotation speed. At the same time, the sustained release rate is inversely proportional to the particle size.

[0083] like Figure 3 , Figure 4 As shown in the figure, with the addition of MAA, the absorption peak is significantly enhanced, which can be attributed to the anhydride reaction between MAA and the amide group on the RHC repeating unit to form an ester bond. In the RHC spectrum, after reacting with MAA molecules, the 1650 cm -1 and 1520cm -1 The two peaks at 2910 cm -1 The CH stretching vibration peak at 0 shifted to a higher wavelength after the addition of MAA. These results confirmed that MAA interacted with RHCⅠ and RHCⅢ and that the grafting rate was positively correlated with the MAA concentration.

[0084] like Figure 5 , Figure 6 As shown in the figure, by comparison, it can be found that new peaks appear in RHC after grafting. These two new peaks come from the acrylic acid protons (2H) of MAA, and the peak enhancement at 1.84 ppm can be attributed to the methyl protons (3H) grafted by methacrylic anhydride. These results further indicate that RHC reacts with MAA. The integral operation further proves that the grafting rate increases with the increase of methacrylic anhydride concentration. Example 4

[0085] The purpose of this embodiment is to prepare a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel. The preparation process is as follows: Figure 1 , Figure 7 As shown, specifically:

[0086] Weigh 2 g of recombinant human type I collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type I collagen solution; weigh 2 g of recombinant human type III collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type III collagen solution, then add 300 ul of methacrylic acid to each of the recombinant human type I collagen solution and the recombinant human type III collagen solution, react for 3 h under magnetic stirring to prepare RHCⅠ-MA solution and RHCⅢ-MA solution, then centrifuge at 6000 rpm for 4 min to collect the supernatant, dilute 2 times, pass through a 0.22 um disposable syringe filter, dialyze using a dialysis bag for 3-4 days, and freeze-dry to obtain two types of sponge-like solid samples, RHCⅠ-MA and RHCⅢ-MA;

[0087] The freeze-dried RHCⅠ-MA sample was dissolved in a 0.1 M EDC solution and reacted for 3 h to obtain an RHCⅠ-MA-EDC solution. The freeze-dried RHCⅢ-MA sample was dissolved in a 0.1 M EDC solution and reacted for 3 h to obtain an RHCⅢ-MA-EDC solution. The two sample solutions were filtered again through a 0.22 um disposable syringe filter, and then dialyzed again using a dialysis bag for 3-4 days, and freeze-dried to obtain two types of sponge-like solid samples, RHCⅠ-MA-EDC and RHCⅢ-MA-EDC.

[0088] 0.5 g of RHCⅠ-MA-EDC freeze-dried sample was dissolved in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 10% (w / v) RHCⅠ-MA-EDC solution; 0.5 g of RHCⅢ-MA-EDC freeze-dried sample was dissolved in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 10% (w / v) RHCⅢ-MA-EDC solution, and then the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution were mixed evenly at a volume ratio of 5:5;

[0089] Then, the mixed recombinant human type I / III collagen solution was slowly added dropwise to a mixed solution of 10 mL rapeseed oil and 2 mL span 80 through a syringe at a speed of 1600 rpm, stirred for 5 min, and then cured by ultraviolet light for 5 min, centrifuged to remove the supernatant, added PBS and shaken to evenly remove the remaining oil phase, and PBS buffer was added 3 times to remove the oil phase, and finally PBS buffer was added for washing, and recombinant human type I / III collagen microgels with a type I / III collagen ratio of 5:5 were collected by filtration;

[0090] 1 g of carboxylated chitosan powder was weighed and dissolved in PBS phosphate buffer at room temperature to prepare 10% (w / v) carboxylated chitosan hydrogel; then 0.3 g of xanthan gum was added to adjust the viscosity of the carboxylated chitosan hydrogel;

[0091] The recombinant human type I / III collagen microgel with a ratio of type I to type III collagen of 5:5 prepared at a rotation speed of 1600 rpm was mixed evenly with 10% (w / v) carboxylated chitosan hydrogel, and the carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel was obtained after removing bubbles under negative pressure (such as Figure 8 Store in a refrigerator at 4°C. Example 5

[0092] The purpose of this example is to prepare a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel.

[0093] Weigh 2 g of recombinant human type I collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type I collagen solution. Weigh 2 g of recombinant human type III collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type III collagen solution. Then, add 300 uL of methacrylic acid to each of the recombinant human type I collagen solution and the recombinant human type III collagen solution, react for 3 h under magnetic stirring to prepare RHCⅠ-MA solution and RHCⅢ-MA solution. Then, centrifuge at 6000 rpm for 4 min to collect the supernatant, dilute it 2 times, pass it through a 0.22 um disposable syringe filter, dialyze it using a dialysis bag for 3-4 days, and freeze-dry it to obtain two types of sponge-like solid samples, RHCⅠ-MA and RHCⅢ-MA.

[0094] The freeze-dried RHCⅠ-MA sample was dissolved in a 0.2 M EDC solution and reacted for 3 h to obtain an RHCⅠ-MA-EDC solution. The freeze-dried RHCⅢ-MA sample was dissolved in a 0.2 M EDC solution and reacted for 3 h to obtain an RHCⅢ-MA-EDC solution. The two sample solutions were filtered again through a 0.22 um disposable syringe filter, and then dialyzed again using a dialysis bag for 3-4 days, and freeze-dried to obtain two types of sponge-like solid samples, RHCⅠ-MA-EDC and RHCⅢ-MA-EDC.

[0095] 1 g of RHCⅠ-MA-EDC freeze-dried sample was dissolved in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 20% (w / v) RHCⅠ-MA-EDC solution, and 1 g of RHCⅢ-MA-EDC freeze-dried sample was dissolved in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 20% (w / v) RHCⅢ-MA-EDC solution, and then the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution were mixed evenly at a volume ratio of 8:2;

[0096] Then, the mixed recombinant human type I / III collagen solution was slowly added dropwise to a mixed solution of 10 mL rapeseed oil and 2 mL span 80 through a syringe at a speed of 1600 rpm, stirred for 5 min, and then cured by ultraviolet light for 5 min, centrifuged to remove the supernatant, added PBS and shaken to evenly remove the remaining oil phase, and PBS buffer was added 3 times to remove the oil phase, and finally PBS buffer was added for washing, and recombinant human type I / III collagen microgels with a type I / III collagen ratio of 8:2 were collected by filtration;

[0097] 2 g of carboxylated chitosan powder was weighed and dissolved in PBS phosphate buffer at room temperature to prepare 20% (w / v) carboxylated chitosan hydrogel, and then 0.3 g of xanthan gum was added to adjust the viscosity of the carboxylated chitosan hydrogel;

[0098] The recombinant human type I / III collagen microgel with a type I to type III collagen ratio of 8:2 prepared at 1600 rpm was mixed evenly with 20% (w / v) carboxylated chitosan hydrogel, and the bubbles were removed by negative pressure, and the microgel was stored in a refrigerator at 4°C. Example 6

[0099] (1) Weigh 2 g of recombinant human type I collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type I collagen solution. Weigh 2 g of recombinant human type III collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type III collagen solution. Then, add 600 ul of methacrylic acid to each of the recombinant human type I collagen solution and the recombinant human type III collagen solution, react for 3 h under magnetic stirring, and prepare RHCⅠ-MA solution and RHCⅢ-MA solution. Then, centrifuge at 6000 rpm for 4 min to collect the supernatant, dilute it 2 times, filter it through a 0.22 um disposable syringe filter, dialyze it using a dialysis bag for 3-4 days, and freeze-dry it to obtain two types of sponge-like solid samples, RHCⅠ-MA and RHCⅢ-MA.

[0100] (2) The freeze-dried RHCⅠ-MA sample was dissolved in a 0.3 M EDC solution and reacted for 3 h to obtain an RHCⅠ-MA-EDC solution. The freeze-dried RHCⅢ-MA sample was dissolved in a 0.3 M EDC solution and reacted for 3 h to obtain an RHCⅢ-MA-EDC solution. The two sample solutions were filtered again through a 0.22 um disposable syringe filter, and then dialyzed again using a dialysis bag for 3-4 days. The two types of sponge-like solid samples, RHCⅠ-MA-EDC and RHCⅢ-MA-EDC, were freeze-dried respectively;

[0101] (3) Take 1 g of the RHCⅠ-MA-EDC freeze-dried sample and dissolve it in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 20% (w / v) RHCⅠ-MA-EDC solution, take 1 g of the RHCⅢ-MA-EDC freeze-dried sample and dissolve it in 0.5% (w / v) photoinitiator LAP solution to obtain 5 mL of 20% (w / v) RHCⅢ-MA-EDC solution, then mix the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution in a volume ratio of 10:1;

[0102] (4) Then, the mixed recombinant human type I / III collagen solution was slowly added to the mixed solution of 10 mL rapeseed oil and 2 mL span 80 through a syringe at a speed of 1000 rpm, stirred for 5 minutes, and then cured by ultraviolet light for 5 minutes. The supernatant was removed by centrifugation, PBS was added and shaken to remove the remaining oil phase, and PBS buffer was added three times to remove the oil phase. Finally, PBS buffer was added for washing, and recombinant human type I / III collagen microgels with a type I / III collagen ratio of 10:1 were collected by filtration;

[0103] (5) Weigh 2 g of carboxylated chitosan powder and dissolve it in PBS phosphate buffer at room temperature to prepare 20% (w / v) carboxylated chitosan hydrogel;

[0104] (6) The recombinant human type I / III collagen microgel with a type I to type III collagen ratio of 10:1 prepared at 1000 rpm was mixed evenly with 20% (w / v) carboxylated chitosan hydrogel, and the bubbles were removed by negative pressure. The microgel was then stored in a refrigerator at 4 °C. Example 7

[0105] Human skin fibroblasts (HSFs) were cultured in carboxylated chitosan hydrogels containing recombinant human type I / III collagen microgels.

[0106] HSFs culture medium contains the following components: complete culture medium containing 500 mL basal culture medium, 25 mL fetal bovine serum, 5 mL fibroblast growth supplement, and 5 mL penicillin / streptomycin solution. Resuspend HSFs with the above complete culture medium. 5 The cells were inoculated at a density of 10 cells / dish in a culture dish containing cells. The experimental group added 5 mL of the above culture medium and mixed evenly with the hydrogel dressing prepared in Example 5, and the blank group was a complete culture medium. Then, the cells were placed in a 37°C, 5% CO2 incubator for culture. After 24 h of culture, the cells were stained with acridine orange / ethidium bromide and observed under a fluorescent inverted microscope.

[0107] The results are as follows Fig. 9 As shown, the HSFs cells were in regular green cobblestone shape with good morphology and normal condition, indicating that the dressing had good cell compatibility. Example 8

[0108] Human umbilical vein endothelial cells (HUVECs) were cultured in carboxylated chitosan hydrogels containing recombinant human type I / III collagen microgels.

[0109] HUVECs cells use endothelial cell culture medium, which contains the following components: 15 mL fetal bovine serum, 1 mL penicillin-streptomycin mixture (100×), and 84 mL F12 culture medium per 100 mL complete culture medium. Resuspend HUVECs cells in this culture medium and incubate the resuspended cells at 10 5 The cells were inoculated in a cell culture dish at a density of 10 cells / medium. The experimental group added 5 mL of the above culture medium and mixed evenly with the hydrogel dressing prepared in Example 5, and the blank group was a complete culture medium. Then, the cells were placed in a 37 °C, 5% CO2 incubator for culture. After 24 h of culture, the cells were taken out, washed with PBS, stained with acridine orange / ethidium bromide, and observed under a fluorescent inverted microscope.

[0110] The results are as follows Fig.10 As shown, HUVECs cells are in regular green cobblestone shape with good morphology and normal state, indicating that the dressing has good cell compatibility. Example 9

[0111] Repair of skin wounds in SD rats by carboxylated chitosan hydrogel containing recombinant human type I / III collagen microgel.

[0112] Male SD rats weighing 300-320 g were selected for skin wound repair experiments to evaluate the effects of carboxylated chitosan hydrogels containing different ratios of type I / III recombinant human collagen on rat wound repair, and compared with the results of pure carboxylated chitosan gels without recombinant human collagen microgels and blank groups. First, the hydrogel dressings prepared in Examples 4 and 5 were sterilized by ultraviolet irradiation for standby use. Secondly, the wound model was simulated on the back of the rat after anesthesia with 10% chloral hydrate intraperitoneal injection. In the experimental group, the wound on the back of the rat was smeared according to the experimental design, and within the selected time period, carboxylated chitosan hydrogels containing microgels with a type I / III ratio of 5:5, carboxylated chitosan hydrogels containing microgels with a type I / III ratio of 8:2, and carboxylated chitosan hydrogels without microgels were used, and then wrapped with sterile medical gauze, and the wounds in the blank group were only wrapped with sterile medical gauze.

[0113] The rats were observed daily and the wound morphology was recorded on days 0, 7, and 10. Fig.11 As shown in the results, firstly, all composite dressings containing recombinant human type Ⅰ / Ⅲ collagen microgels had better repair effects than carboxylated chitosan hydrogel dressings without microgels; secondly, in carboxylated chitosan hydrogel dressings containing microgels, adjusting the ratio of type Ⅰ / Ⅲ recombinant human collagen in the microgels can help the wound repair process. Among them, the microgels with a type Ⅰ / Ⅲ ratio of 5:5 from 0 to 7 days and a type Ⅰ / Ⅲ ratio of 8:2 from 7 to 10 days had better repair effects than the carboxylated chitosan hydrogel dressings with a type Ⅰ / Ⅲ ratio of 5:5 from 0 to 10 days. The results showed that in the late stage of wound healing, the increase in the content of recombinant type Ⅰ collagen in the microgels had a better healing effect on the wounds on the back of rats at this stage, with no symptoms of redness, swelling and inflammation in the wounds, and the necrotic scab tissue had spontaneous scab dissolution, and the scars formed were also smaller.

Claims

1. A method for preparing recombinant human type I / III collagen microgel, characterized in that: The following steps are involved: 1) Add methacrylic acid to the recombinant human type I collagen solution and stir to prepare a methacrylic anhydride recombinant human type I collagen RHCⅠ-MA solution, then collect the supernatant of the RHCⅠ-MA solution by centrifugation, dilute, filter, dialyze, and freeze-dry to obtain a RHCⅠ-MA sponge-like solid sample; prepare a RHCⅢ-MA sponge-like solid sample by the same method; 2) The freeze-dried RHCⅠ-MA sponge-like solid sample was dissolved in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and reacted for 2-4 hours, and then the solution was filtered, dialyzed, and freeze-dried to obtain the RHCⅠ-MA-EDC sponge-like solid sample; the RHCⅢ-MA-EDC sponge-like solid sample was prepared by the same method; 3) dissolving the RHCⅠ-MA-EDC sponge-like solid sample in a photoinitiator with a concentration of 0.1%-1% (w / v) to obtain an RHCⅠ-MA-EDC solution, and similarly dissolving the RHCⅢ-MA-EDC sponge-like solid sample in a photoinitiator solution with a concentration of 0.1%-1% (w / v) to obtain an RHCⅢ-MA-EDC solution, and then uniformly mixing the RHCⅠ-MA-EDC solution and the RHCⅢ-MA-EDC solution at a volume ratio of 1:10-10:1; 4) The mixed solution obtained in step 3) is added dropwise to the mixed solution of the oil phase and the surfactant at different rotation speeds and stirred. After ultraviolet light irradiation for 3-10 min, the supernatant is removed by centrifugation, PBS is added and shaken evenly to remove the remaining oil phase, and finally PBS buffer is added to rinse it. The recombinant human type I / III collagen microgel with an adjustable type I / III collagen ratio is obtained by filtration and collection.

2. The method for preparing a recombinant human type I / III collagen microgel according to claim 1, characterized in that: In step 1), the amount of methacrylic acid added is 3%-12% of the volume of the recombinant human type I collagen solution.

3. The method for preparing a recombinant human type I / III collagen microgel according to claim 1, characterized in that: The photoinitiator in step 3) is irgacure 2959 or phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

4. The method for preparing a recombinant human type I / III collagen microgel according to claim 1, characterized in that: The oil phase in step 4) is one of liquid paraffin, rapeseed oil, mineral oil, and soybean oil; the surfactant is one of lecithin, Tween, and span 80.

5. The method for preparing a recombinant human type I / III collagen microgel according to claim 1, characterized in that: The different rotation speeds described in step 4) are 500 ~2000 rpm.

6. Recombinant human type I / III collagen microgel prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the recombinant human type I / III collagen microgel according to claim 6 in the preparation of skin damage repair materials or materials for promoting skin-related cell proliferation.

8. A carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel, characterized in that: The dressing is obtained by mixing the recombinant human type I / III collagen microgel described in claim 6 with carboxylated chitosan hydrogel to obtain a mixed hydrogel dressing that can be used for dynamic repair of skin wounds.

9. The method for preparing the carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel according to claim 8, characterized in that: The following steps are involved: S1 weighed carboxylated chitosan powder, stirred and dissolved it in PBS phosphate buffer to prepare 10%-20% (w / v) carboxylated chitosan hydrogel, and then added xanthan gum to adjust the viscosity of the carboxylated chitosan hydrogel; S2 mixes the recombinant human type I / III collagen microgel and the carboxylated chitosan hydrogel evenly, removes bubbles using negative pressure, and obtains a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel.

10. Use of the carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel according to claim 8 in the preparation of skin damage repair materials or materials for promoting skin-related cell proliferation.

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