Method for preparing recombinant collagen microspheres through photo-crosslinking

Abstract

The invention discloses a method for preparing recombinant collagen microspheres by photo-crosslinking, which comprises the following steps: dissolving recombinant collagen grafted with carbon-carbon double bonds and a photoinitiator in deionized water to serve as a dispersion phase, and taking fluorinated oil containing a surfactant as a continuous phase; respectively injecting the dispersed phase and the continuous phase into a micro-droplet generation chip by using a micro-injection pump of a microfluidic reaction device, and preparing emulsion microspheres under the shearing action; the emulsion microspheres are subjected to light curing under ultraviolet irradiation, washing and freeze drying are performed, and the recombinant collagen microspheres are obtained. According to the method, the recombinant collagen microspheres are prepared by utilizing a photo-crosslinking technology, the preparation process is simple, convenient and rapid, the operation is easy, not only are the dispersity and the size uniformity of the microspheres improved, but also the preparation effect is more advantageous. The microspheres prepared by the invention have good biocompatibility, can effectively promote the expression of local collagen genes after subcutaneous implantation, and are expected to be used as a facial filler in the medical beauty industry.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a method for preparing recombinant collagen microspheres through photo-crosslinking. Background Art

[0002] Collagen is a biological macromolecular protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. It is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics, and food industries.

[0003] Currently, the microsphere products used for medical and aesthetic filling on the market are mainly synthetic degradable materials. Biodegradable materials have been approved by the FDA for use in the human body due to their good biocompatibility, degradability and safety. This type of material has good compatibility with body fluids, undergoes catalytic hydrolysis in the body, and finally degrades into water and carbon dioxide that are non-toxic to the body through circulation.

[0004] The particle size, shape, and dispersibility of microspheres will directly affect their filling effect. If the microspheres are too large, it may increase the difficulty and pain during injection, and may also cause compression or irritation of local tissues, causing adverse reactions. Microspheres that are too small may be phagocytosed by macrophages, thus affecting their stability and duration of action in the body. Microspheres with more regular shapes and smoother surfaces are more injectable, and may also be easier to migrate and evenly distribute in the body, reducing friction and irritation with surrounding tissues. Microspheres with rough surfaces may be more easily identified as foreign bodies by the immune system, triggering immune rejection reactions.

[0005] In addition, chemical cross-linking agents such as (EDC / NHS / glutaraldehyde, etc.) residues often bring certain cytotoxicity, and UV cross-linking can effectively avoid this situation.

[0006] Therefore, in view of the above situation, there is an urgent need to develop a method for preparing recombinant collagen microspheres by photocrosslinking to overcome the shortcomings in current practical applications. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for preparing recombinant collagen microspheres by photocrosslinking in view of the deficiencies of the above-mentioned prior art. The method uses photocrosslinking technology to prepare recombinant collagen microspheres, has a simple and rapid preparation process and is easy to operate, which not only improves the dispersibility and size uniformity of the microspheres, but also has more advantages in preparation effect. The prepared microspheres have good biocompatibility and can effectively promote the expression of local collagen genes after subcutaneous implantation, and are expected to be used as facial fillers in the medical beauty industry.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing recombinant collagen microspheres by photocrosslinking, characterized in that it includes: dissolving recombinant collagen grafted with carbon-carbon double bonds and a photoinitiator in deionized water as a dispersed phase, using a fluorinated oil containing a surfactant as a continuous phase, using a microinjection pump of a microfluidic reaction device to inject the dispersed phase and the continuous phase into a droplet generation chip respectively, and obtaining emulsion microspheres through shearing; placing the emulsion microspheres under ultraviolet light for photocuring, washing, and freeze-drying to obtain recombinant collagen microspheres.

[0009] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the preparation method of the recombinant collagen grafted with carbon-carbon double bonds includes: dissolving the recombinant collagen in deionized water to obtain a recombinant collagen solution; then adding glycidyl methacrylate to the recombinant collagen solution, stirring, and reacting at 30°C to 60°C for 12h to 48h to obtain system A; dialyzing and freeze-drying the system A to obtain recombinant collagen grafted with carbon-carbon double bonds.

[0010] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the mass of the glycidyl methacrylate is 1 to 10 times the mass of the recombinant collagen; and the mass percentage of the recombinant collagen in the recombinant collagen solution is 0.1% to 20%.

[0011] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the mass percentage of recombinant collagen grafted with carbon-carbon double bonds in the dispersed phase is 0.1% to 20%, and the mass percentage of the photoinitiator is 0.05% to 0.5%; the mass percentage of the surfactant in the fluorinated oil containing a surfactant is 0.1% to 20%, and the fluorinated oil is 3M Novec7700 fluorinated oil.

[0012] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the photoinitiator is LAP, I2959 or riboflavin.

[0013] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the surfactant is Tween-60, Tween-80, Span 60 or Span 80.

[0014] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the microfluidic reaction device includes a droplet generation chip and two microinjection pumps, the two microinjection pumps are respectively connected to the two inlets of the droplet generation chip through pipelines, and the outlet of the droplet generation chip is connected to a silicone capillary.

[0015] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the droplet generation chip is a "Y"-shaped droplet generation chip, the material of the pipeline is polytetrafluoroethylene, and the material of the droplet generation chip is PDMS.

[0016] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the flow rate of the continuous phase is 2 to 10 times the flow rate of the dispersed phase.

[0017] The above-mentioned method for preparing recombinant collagen microspheres by photocrosslinking is characterized in that the recombinant collagen is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen; and the average particle size of the recombinant collagen microspheres is 40μm to 60μm.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. The present invention uses photocrosslinking technology to prepare recombinant collagen microspheres, which has a simple and rapid preparation process and is easy to operate. Compared with the traditional chemical crosslinking agent preparation method, the photocrosslinking method not only improves the dispersibility and size uniformity of the microspheres, but also has more advantages in preparation effect. The microspheres prepared by the present invention have good biocompatibility and can effectively promote the expression of local collagen genes after subcutaneous implantation, and are expected to be used as facial fillers in the medical beauty industry.

[0020] 2. The present invention uses a microinjection pump to adjust the flow rate of the fluid. By precisely controlling the flow rate, the desired flow conditions can be obtained in the microchannel, thereby affecting the size of particles or droplets in the fluid, achieving precise control of the characteristics of the microspheres, and further realizing its application in a variety of soft tissue filling and repair fields to meet personalized and precise clinical needs.

[0021] 3. The photo-crosslinking method of the present invention for preparing recombinant collagen microspheres does not require a crosslinking agent, which effectively avoids the potential hazards that may be caused by the crosslinking agent, such as inducing allergic reactions or toxicity to tissues, making the recombinant collagen microspheres safer in clinical applications.

[0022] 4. Compared with traditional collagen filling materials, the recombinant collagen microspheres prepared by the present invention can more effectively absorb and retain water due to their larger specific surface area, thereby improving the filling effect. At the same time, the adjustability of the elastic modulus enables the material to better adapt to different application requirements, thereby providing a more natural filling effect. In addition, the larger specific surface area helps to improve cell adhesion and growth, providing good support for tissue regeneration.

[0023] 5. The preparation method of the present invention is simple and has good industrial production potential. The simple preparation process can not only reduce production costs, but also ensure the consistency and quality of the product, thereby meeting the market demand for high-quality filling materials.

[0024] 6. The use of recombinant collagen microspheres prepared by the present invention effectively avoids the immunogenicity problem of collagen from animal sources. This feature not only improves the safety of the product, but also reduces the risk of allergic and rejection reactions that patients may face when using collagen filling materials.

[0025] 7. Both the mobile phase and dispersed phase materials of the present invention can be of medical grade, which further ensures the quality and safety of the product and avoids the potential health risks that may be caused by non-medical grade materials.

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic structural diagram of the microfluidic reaction device of the present invention.

[0028] Figure 2 This is the Fourier infrared spectrum image of the recombinant collagen with grafted carbon-carbon double bonds according to Example 1 of the present invention.

[0029] Figure 3 This is an optical microscope picture of the recombinant collagen microspheres prepared in Example 1 of the present invention.

[0030] Figure 4 This is a SEM image of the recombinant collagen microspheres prepared in Example 1 of the present invention.

[0031] Figure 5 This is the MTT test result of the recombinant collagen microspheres prepared in Example 1 of the present invention.

[0032] Figure 6 The results are as follows: The recombinant collagen microspheres prepared in Example 1 of the present invention promote the migration of HSF cells.

[0033] Figure 7 The collagen gene expression of rats was shown after the recombinant collagen microspheres prepared in Example 1 of the present invention were implanted subcutaneously in rats for eight weeks. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are all medical grade and, unless otherwise specified, can be obtained from commercial channels.

[0035] like Figure 1 As shown, the microfluidic reaction device used in the present invention includes a droplet generation chip and two microinjection pumps, the two microinjection pumps are respectively connected to the two inlets of the droplet generation chip through pipes with an inner diameter of 1 mm, and the outlet of the droplet generation chip is connected to a silicone capillary; the droplet generation chip is a "Y"-shaped droplet generation chip, the material of the pipe is polytetrafluoroethylene, and the material of the droplet generation chip is PDMS.

[0036] Example 1

[0037] The preparation method of the recombinant collagen microspheres of this embodiment specifically includes:

[0038] Step 1, prepare recombinant collagen grafted with carbon-carbon double bonds: dissolve 1g of recombinant collagen in 10mL of deionized water to obtain a recombinant collagen solution; then add 2g of glycidyl methacrylate to the recombinant collagen solution, stir, and react at 60°C for 24h to obtain system A; dialyze and freeze-dry the system A to obtain recombinant collagen grafted with carbon-carbon double bonds; the recombinant collagen is a large molecular type I recombinant collagen, which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit and is repeated multiple times, wherein the short amino acid sequence GAPGAPGSQGAPGL is repeated 52 times, and the collagen has good water solubility and a molecular weight of 78862Da, which is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0039] Step 2: Dissolve 1g of recombinant collagen grafted with carbon-carbon double bonds and 0.05g of photoinitiator LAP in 10mL of deionized water as the dispersed phase, suck it into a 5mL syringe, and fix the syringe on a microinjection pump; use a fluorinated oil containing a surfactant as the continuous phase, suck it into a 5mL syringe, and fix the syringe on a microinjection pump; set the flow rate ratio of the dispersed phase to the continuous phase to 1:4, and use a microinjection pump to inject the dispersed phase and the continuous phase into a "Y"-shaped droplet generation chip, respectively, with the flow rate of the continuous phase set to 200μL / min, and the flow rate of the dispersed phase set to 50μL / min. Prepare emulsion microspheres by shearing and export them through a silica capillary; place the exported emulsion microspheres under ultraviolet light irradiation at 365nm (50w / cm2 ) photocuring for 30 minutes, washing, freeze-drying, and obtaining recombinant collagen microspheres with an average particle size of 50 μm; the mass percentage of the surfactant in the fluorinated oil containing the surfactant is 5%, and the fluorinated oil is 3M Novec7700 fluorinated oil; the surfactant is Tween-80.

[0040] Figure 3 This is the Fourier infrared spectrum of the recombinant collagen grafted with carbon-carbon double bonds prepared in Example 1. It can be seen from the figure that 1450cm -1 The absorption peak at is the C=O double bond, indicating that the grafting of glycidyl methacrylate is successful.

[0041] Morphology determination

[0042] The recombinant collagen microspheres prepared in Example 1 were photographed using an optical microscope. Figure 3 As shown, the average particle size of the prepared recombinant collagen microspheres is 50 μm; the recombinant collagen microspheres of Example 1 were photographed using a field emission scanning electron microscope (GeminiSEM300, Car1 zeiss, Germany). Figure 4 As shown in the figure, it can be seen that the diameter of the collagen microspheres is about 50 μm, and there are dense pores on the surface.

[0043] Biocompatibility

[0044] The extract was prepared using the method recommended by GB / T 16886.5-2017 and the MTT and cell scratch methods were used for in vitro cell compatibility tests. The results are shown in Figure 5 and Figure 6 The results showed that the recombinant collagen microsphere extract prepared in Example 1 can effectively promote the proliferation and migration of human epidermal fibroblasts (HSF), and the 24h cell proliferation rate is 129%, indicating that it has good biocompatibility.

[0045] Collagen (abbreviated as collagen) is an important component of the skin and plays a major role in supporting and elasticity. With age or the influence of the external environment (ultraviolet rays), the loss of subcutaneous soft tissue leads to the formation of wrinkles and folds. Subcutaneous injection of microspheres of specific materials stimulates the body to produce a foreign body reaction, thereby forming a fibrous capsule composed of collagen to achieve the purpose of facial rejuvenation.

[0046] like Figure 7 As shown, the recombinant type I collagen microspheres of this example increased the mRNA expression of type I (2.48 times) and type III collagen (1.71 times) 8 weeks after being implanted in the skin, indicating that the microspheres not only have a filling effect themselves, but also can promote the body to produce collagen.

[0047] Example 2

[0048] The preparation method of the recombinant collagen microspheres of this embodiment specifically includes:

[0049] Step 1, preparing recombinant collagen grafted with carbon-carbon double bonds: dissolving 2 g of recombinant collagen in 10 mL of deionized water to obtain a recombinant collagen solution; then adding 20 g of glycidyl methacrylate to the recombinant collagen solution, stirring, and reacting at 50° C. for 12 h to obtain system A; dialyzing and freeze-drying the system A to obtain recombinant collagen grafted with carbon-carbon double bonds; the recombinant collagen is recombinant type II collagen (recombinant type III collagen or recombinant type IV collagen may also be used);

[0050] The recombinant type III collagen is composed of a short amino acid sequence from natural human type III collagen as a repeating unit and repeated multiple times, wherein the short amino acid sequence (GERGAPGFRGPAGPNGIPGEKGPAGER) is repeated 30 times. The collagen has good water solubility and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0051] The recombinant type IV collagen is composed of the full-length α1 chain sequence of human type IV collagen, and the collagen has good water solubility and was purchased from Xi'an Juzi Bio-Gene Technology Co., Ltd.;

[0052] The recombinant type II collagen is composed of the full-length α1 chain sequence of human type II collagen, and the collagen has good water solubility and was purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0053] Step 2: Dissolve 2 g of recombinant collagen grafted with carbon-carbon double bonds and 0.03 g of photoinitiator I2959 in 10 mL of deionized water as the dispersed phase, inhale into a 5 mL syringe, and fix the syringe on a microinjection pump; use a fluorinated oil containing a surfactant as the continuous phase, inhale into a 5 mL syringe, and fix the syringe on a microinjection pump; set the flow rate ratio of the dispersed phase to the continuous phase to 1:10, and use a microinjection pump to inject the dispersed phase and the continuous phase into a "Y"-shaped droplet generation chip, respectively, with the flow rate of the continuous phase set to 250 μL / min, and the flow rate of the dispersed phase set to 25 μL / min. Prepare emulsion microspheres by shearing and export them through a silica capillary; place the exported emulsion microspheres under ultraviolet irradiation at 365 nm (50 w / cm 2 ) photocuring for 30 minutes, washing, freeze-drying, and obtaining recombinant collagen microspheres with an average particle size of 60 μm; the mass percentage of the surfactant in the fluorinated oil containing the surfactant is 0.1%, and the fluorinated oil is 3M Novec7700 fluorinated oil; the surfactant is Span 60 or Span 80.

[0054] The recombinant collagen microspheres prepared in this embodiment are photographed by SEM using a field emission scanning electron microscope (GeminiSEM 300, Car1 zeiss, Germany). The results show that the diameter of the prepared recombinant collagen microspheres is about 60 μm, and there are dense pores on the surface; the extract is prepared by the GB / T16886.5-2017 recommended method and the MTT and cell scratch methods are used to perform in vitro cell compatibility tests. The results show that the recombinant collagen microsphere extract prepared in this embodiment can effectively promote the proliferation and migration of human epidermal fibroblasts (HSF), and the 24h cell proliferation rate is 129.65%, indicating that it has good biocompatibility. The recombinant collagen microspheres of this embodiment will increase the expression of type I (1.55 times) and type III collagen (2.32 times) mRNA after being implanted in the skin for 8 weeks, indicating that the microspheres not only have a filling effect themselves, but also can promote the body to produce collagen.

[0055] Example 3

[0056] The preparation method of the recombinant collagen microspheres of this embodiment specifically includes:

[0057] Step 1, preparing recombinant collagen grafted with carbon-carbon double bonds: dissolving 0.01 g of recombinant collagen in 10 mL of deionized water to obtain a recombinant collagen solution; then adding 0.01 g of glycidyl methacrylate to the recombinant collagen solution, stirring, and reacting at 30° C. for 48 hours to obtain system A; dialyzing and freeze-drying the system A to obtain recombinant collagen grafted with carbon-carbon double bonds; the recombinant human-like collagen is a large molecular type I recombinant collagen CF-5, which is composed of type I collagen from human, has good water solubility, and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0058] Step 2: Dissolve 0.01g of recombinant collagen grafted with carbon-carbon double bonds and 0.005g of photoinitiator riboflavin in 10mL of deionized water as the dispersed phase, suck it into a 5mL syringe, and fix the syringe on a microinjection pump; use a fluorinated oil containing a surfactant as the continuous phase, suck it into a 5mL syringe, and fix the syringe on a microinjection pump; set the flow rate ratio of the dispersed phase to the continuous phase to 1:2, and use a microinjection pump to inject the dispersed phase and the continuous phase into a "Y"-shaped droplet generation chip, respectively, with the flow rate of the continuous phase set to 100μL / min, and the flow rate of the dispersed phase set to 50μL / min. Prepare emulsion microspheres by shearing and export them through a silica capillary; place the exported emulsion microspheres under ultraviolet light irradiation at 365nm (50w / cm 2) photocuring for 30 minutes, washing, freeze-drying, and obtaining recombinant collagen microspheres with an average particle size of 40 μm; the mass percentage of the surfactant in the fluorinated oil containing the surfactant is 20%, and the fluorinated oil is 3M Novec7700 fluorinated oil; the surfactant is Tween-60.

[0059] The recombinant collagen microspheres prepared in this embodiment are photographed by SEM using a field emission scanning electron microscope (GeminiSEM 300, Car1 zeiss, Germany). The results show that the diameter of the prepared recombinant collagen microspheres is about 40 μm, and there are dense pores on the surface; the extract is prepared by the GB / T16886.5-2017 recommended method and the MTT and cell scratch methods are used to perform in vitro cell compatibility tests. The results show that the recombinant collagen microsphere extract prepared in this embodiment can effectively promote the proliferation and migration of human epidermal fibroblasts (HSF), and the 24h cell proliferation rate is 148%, indicating that it has good biocompatibility. The recombinant collagen microspheres of this embodiment will increase the expression of type I (1.33 times) and type III collagen (2.41 times) mRNA after being implanted in the skin for 8 weeks, indicating that the microspheres not only have a filling effect themselves, but also can promote the body to produce collagen.

[0060] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing recombinant collagen microspheres by photocrosslinking, characterized in that: include: Recombinant collagen grafted with carbon-carbon double bonds and a photoinitiator are dissolved in deionized water as a dispersed phase, and a fluorinated oil containing a surfactant is used as a continuous phase. The dispersed phase and the continuous phase are respectively injected into a droplet generation chip using a microinjection pump of a microfluidic reaction device, and emulsion microspheres are obtained through shearing action; the emulsion microspheres are placed under ultraviolet light for photocuring, washed, and freeze-dried to obtain recombinant collagen microspheres.

2. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 1, characterized in that: The preparation method of the recombinant collagen grafted with carbon-carbon double bonds comprises: dissolving the recombinant collagen in deionized water to obtain a recombinant collagen solution; then adding glycidyl methacrylate to the recombinant collagen solution, stirring, and reacting at 30° C. to 60° C. for 12 h to 48 h to obtain system A; dialyzing and freeze-drying the system A to obtain the recombinant collagen grafted with carbon-carbon double bonds.

3. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 2, characterized in that: The mass of the glycidyl methacrylate is 1 to 10 times the mass of the recombinant collagen; the mass percentage of the recombinant collagen in the recombinant collagen solution is 0.1% to 20%.

4. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 1, characterized in that: The mass percentage of the recombinant collagen grafted with carbon-carbon double bonds in the dispersed phase is 0.1% to 20%, and the mass percentage of the photoinitiator is 0.05% to 0.5%; the mass percentage of the surfactant in the fluorinated oil containing the surfactant is 0.1% to 20%, and the fluorinated oil is 3M Novec7700 fluorinated oil.

5. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 1, characterized in that: The photoinitiator is LAP, I2959 or riboflavin.

6. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 1, characterized in that: The surfactant is Tween-60, Tween-80, Span 60 or Span 80.

7. A method for preparing recombinant collagen microspheres by photocrosslinking according to any one of claims 1 to 6, characterized in that: The microfluidic reaction device comprises a microdroplet generation chip and two microinjection pumps. The two microinjection pumps are respectively connected to two inlets of the microdroplet generation chip through pipelines, and the outlet of the microdroplet generation chip is connected to a silica gel capillary.

8. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 7, characterized in that: The droplet generation chip is a "Y"-shaped droplet generation chip, the material of the pipeline is polytetrafluoroethylene, and the material of the droplet generation chip is PDMS.

9. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 1, characterized in that: The flow rate of the continuous phase is 2 to 10 times the flow rate of the dispersed phase.

10. The method for preparing recombinant collagen microspheres by photocrosslinking according to claim 1, characterized in that: The recombinant collagen is recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen or recombinant human-like collagen; the average particle size of the recombinant collagen microspheres is 40 μm to 60 μm.

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