Method for preparing recombinant collagen microspheres through microfluidics

Abstract

The invention discloses a method for preparing recombinant collagen microspheres by microfluidics, which comprises the following steps: dissolving recombinant collagen in deionized water as a dispersion phase, taking petroleum ether containing a surfactant as a continuous phase, and respectively injecting the dispersion phase and the continuous phase into a microdroplet generation chip by using a micro-injection pump of a microfluidic reaction device, emulsion microspheres are prepared under the shearing action; and putting the emulsion microspheres into a stationary liquid containing a cross-linking agent, curing, washing, and freeze-drying to obtain the recombinant collagen microspheres with uniform size. The recombinant collagen microspheres are prepared by using a microfluidic technology, the preparation process is simple, convenient and rapid, the operation is easy, and the prepared microspheres 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 by microfluidic control. Background Art

[0002] Collagen is a structural protein mainly found in skin, bones, tendons and other tissues, accounting for about 30% of the total protein in the human body. It is a triple helix structure formed by three polypeptide chains, which provides strength and elasticity to the tissue. Collagen plays a key role in tissue repair, cell growth and wound healing, and is widely used in medicine, cosmetics and biomaterials. Its biocompatibility and degradability make it have important application potential in tissue engineering and regenerative medicine.

[0003] Currently, the microsphere products used for medical and aesthetic filling on the market are mainly based on 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] Microfluidics is a new method for preparing monodisperse microspheres developed in recent years. By controlling the flow rate of the continuous phase and the dispersed phase, droplets and microspheres of uniform size can be prepared. Compared with traditional preparation methods, microspheres prepared by microfluidics have uniform particle size, controllable size, and monodispersity, which is of great significance in drug delivery, biomedicine, etc., and therefore have attracted much attention.

[0005] The particle size, shape, smoothness 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 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.

[0006] Therefore, in view of the above situation, there is an urgent need to develop a method for preparing recombinant collagen microspheres using microfluidic technology 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 microfluidics to address the deficiencies of the above-mentioned prior art. The method uses microfluidics technology to prepare recombinant collagen microspheres, has a simple and rapid preparation process and is easy to operate. 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 microfluidics, characterized in that it includes: dissolving recombinant collagen in deionized water as a dispersed phase, using petroleum ether 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 in a fixing liquid containing a cross-linking agent for solidification, washing, and freeze-drying to obtain recombinant collagen microspheres of uniform size.

[0009] The above-mentioned method for preparing recombinant collagen microspheres by microfluidics is characterized in that the mass percentage of recombinant collagen in the dispersed phase is 0.1% to 20%, and the mass percentage of surfactant in the continuous phase is 0.1% to 20%.

[0010] The above-mentioned method for preparing recombinant collagen microspheres by microfluidics is characterized in that the fixing liquid containing the crosslinking agent is a solution obtained by adding the crosslinking agent to the continuous phase, and the mass percentage of the crosslinking agent in the fixing liquid is 0.1% to 10%.

[0011] The above-mentioned method for preparing recombinant collagen microspheres by microfluidics is characterized in that the cross-linking agent is glutaraldehyde, genipin, tetrakishydroxymethylphosphine chloride or four-arm PEG succinimidyl carbonate.

[0012] The above-mentioned method for preparing recombinant collagen microspheres by microfluidics is characterized in that the petroleum ether is of pharmaceutical grade and has a boiling range of 30°C to 60°C, 60°C to 90°C or 90°C to 120°C.

[0013] The above-mentioned method for preparing recombinant collagen microspheres by microfluidics 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 microfluidics 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 microfluidics 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 microfluidics 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 microfluidics 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] The present invention uses microfluidic technology to prepare recombinant collagen microspheres, which has a simple and rapid preparation process and is easy to operate. Compared with the traditional emulsification preparation method, microfluidic technology can accurately control the reaction conditions and ensure the uniform size of the microspheres by finely adjusting the flow rate and pressure of the fluid, thereby avoiding the problem of uneven size distribution that may occur in the traditional emulsification method. Microspheres of uniform size help to improve the stability of the filling effect and can achieve a more consistent and natural effect in clinical applications.

[0020] After subcutaneous implantation, microspheres can effectively promote local collagen gene expression. This is due to their good adjustability and bioactivity. For facial fillers in the field of medical beauty, microspheres can not only improve the appearance of the skin immediately, but also achieve lasting effects by promoting collagen production.

[0021] Compared with traditional collagen filling materials, microspheres have a larger specific surface area and can more effectively absorb and retain moisture, thereby enhancing the filling effect. At the same time, the elastic modulus is adjustable, allowing the microspheres to flexibly adapt to different needs and provide a more natural filling experience.

[0022] The use of recombinant collagen avoids the immunogenicity problems that may be caused by animal-derived collagen, making this material safer in clinical applications, reducing patients' rejection of filling materials, and improving the user experience.

[0023] In addition, the mobile phase and dispersed phase materials used in the microspheres are all medical grade, which ensures their quality and safety. Medical grade materials have undergone rigorous safety assessments, effectively avoiding potential health risks that may be caused by non-medical grade materials.

[0024] 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

[0025] Figure 1 It is a schematic structural diagram of the microfluidic reaction device of the present invention.

[0026] Figure 2 This is an optical microscope picture of the recombinant collagen microspheres prepared in Example 1 of the present invention.

[0027] Figure 3 This is a SEM image of the recombinant collagen microspheres prepared in Example 1 of the present invention.

[0028] Figure 4 This is the MTT test result of the recombinant collagen microspheres prepared in Example 1 of the present invention.

[0029] Figure 5 The results are as follows: The recombinant collagen microspheres prepared in Example 1 of the present invention promote the migration of HSF cells.

[0030] Figure 6 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

[0031] 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, unless otherwise specified, can be obtained from commercial channels.

[0032] 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.

[0033] Example 1

[0034] The preparation method of the recombinant collagen microspheres of this embodiment specifically includes:

[0035] Step 1: Dissolve 1g of recombinant collagen in 10mL of deionized water as a dispersed phase, and inhale it into a 5mL syringe, and fix the syringe on a microinjection pump; the recombinant collagen is a macromolecular 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.;

[0036] Step 2: Add 5 g of Tween-80 to 100 mL of petroleum ether (boiling range 30-60 ° C) and stir evenly as a continuous phase, inhale into a 5 mL syringe, and fix the syringe on a microinjection pump; take 20 mL of the continuous phase solution and add 200 μL of glutaraldehyde and stir evenly as a stationary liquid; 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 200 μL / min, and the flow rate of the dispersed phase set to 50 μL / min. The emulsion microspheres are prepared by shearing and exported through a silica gel capillary; the exported emulsion microspheres are placed in the stationary liquid for curing for 12 hours, and then the microspheres are washed with deionized water for multiple times, and freeze-dried to obtain recombinant collagen microspheres of uniform size.

[0037] Morphology determination

[0038] The recombinant collagen microspheres prepared in Example 1 were photographed using an optical microscope. Figure 2 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 3 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.

[0039] Biocompatibility

[0040] 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 4 and Figure 5 ,The results showed that the extract of the recombinant collagen microspheres prepared in Example 1 could effectively promote the proliferation and migration of human umbilical vein endothelial cells (HUVEC), and the 24h cell proliferation rate was 141%, indicating that it had good biocompatibility.

[0041] 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.

[0042] like Figure 6 As shown, the recombinant type I collagen microspheres of this example increased the mRNA expression of type I (2.01 times) and type III collagen (2.11 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.

[0043] Example 2

[0044] The preparation method of the recombinant collagen microspheres of this embodiment specifically includes:

[0045] Step 1: dissolve 2 g of recombinant collagen in 10 mL of deionized water as a dispersed phase, suck it into a 5 mL syringe, and fix the syringe on a microinjection pump; the recombinant collagen is recombinant type II collagen (recombinant type III collagen or recombinant type IV collagen may also be used);

[0046] 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.;

[0047] 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 Biogene Technology Co., Ltd.;

[0048] 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.;

[0049] Step 2: Add 0.1 g of Tween-60 to 100 mL of petroleum ether (boiling range 60-90 ° C) and stir evenly as a continuous phase, inhale it into a 5 mL syringe, and fix the syringe on a microinjection pump; take 20 mL of the continuous phase solution and add 20 mg of genipin and stir evenly as a stationary liquid; 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. The flow rate of the continuous phase is set to 250 μL / min, and the flow rate of the dispersed phase is set to 25 μL / min. The emulsion microspheres are prepared by shearing and exported through a silica gel capillary; the exported emulsion microspheres are placed in a stationary liquid for curing for 12 hours, and then the microspheres are washed with deionized water for multiple times, and freeze-dried to obtain recombinant collagen microspheres of uniform size.

[0050] The recombinant collagen microspheres prepared in this embodiment were photographed by SEM using a field emission scanning electron microscope (GeminiSEM 300, Car1 zeiss, Germany). The results showed that the diameter of the prepared recombinant collagen microspheres was about 40 μm, and there were dense pores on the surface; the extract was prepared by the GB / T16886.5-2017 recommended method and the MTT and cell scratch methods were used for in vitro cell compatibility tests. The results showed that the extract of the recombinant collagen microspheres prepared in this embodiment can effectively promote the proliferation and migration of human umbilical vein endothelial cells (HUVEC), and the 24h cell proliferation rate was 133.25%, indicating that it has good biocompatibility. The recombinant collagen microspheres of this embodiment will increase the expression of type I (1.36 times) and type III collagen (1.55 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.

[0051] Example 3

[0052] The preparation method of the recombinant collagen microspheres of this embodiment specifically includes:

[0053] Step 1: Dissolve 0.01 g of recombinant collagen in 10 mL of deionized water as a dispersed phase, and inhale it into a 5 mL syringe, and fix the syringe on a microinjection pump; the recombinant human-like collagen is a large molecular type I recombinant collagen CF-5, which is composed of type I human collagen, has good water solubility, and is purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0054] Step 2: Add 20g of Span 80 to 100mL of petroleum ether (boiling range 90-120°C) and stir evenly as the continuous phase, inhale into a 5mL syringe, and fix the syringe on a microinjection pump; take 20mL of the continuous phase solution and add 2g of four-arm PEG succinimidyl carbonate (or tetrakis(hydroxymethyl)phosphine chloride) and stir evenly as the stationary liquid; 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 the "Y"-shaped droplet generation chip respectively, the flow rate of the continuous phase is set to 100μL / min, and the flow rate of the dispersed phase is set to 50μL / min. The emulsion microspheres are prepared by shearing and exported through a silica gel capillary; the exported emulsion microspheres are placed in the stationary liquid for curing for 12h, and the microspheres are washed with deionized water for multiple times, and freeze-dried to obtain recombinant collagen microspheres of uniform size.

[0055] The recombinant collagen microspheres prepared in this embodiment were photographed by SEM using a field emission scanning electron microscope (GeminiSEM 300, Car1 zeiss, Germany). The results showed that the diameter of the prepared recombinant collagen microspheres was about 60 μm, and there were dense pores on the surface; the extract was prepared by the GB / T16886.5-2017 recommended method and the MTT and cell scratch methods were used to perform in vitro cell compatibility tests. The results showed that the extract of the recombinant collagen microspheres prepared in this embodiment can effectively promote the proliferation and migration of human umbilical vein endothelial cells (HUVEC), and the 24h cell proliferation rate was 129.36%, indicating that it has good biocompatibility. The recombinant collagen microspheres of this embodiment will increase the expression of type I (1.46 times) and type III collagen (2.45 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.

[0056] 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 microfluidics, characterized in that: include: Recombinant collagen is dissolved in deionized water as a dispersed phase, petroleum ether containing a surfactant is used as a continuous phase, and 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 latex microspheres are prepared by shearing; the latex microspheres are solidified in a fixing liquid containing a cross-linking agent, washed, and freeze-dried to obtain recombinant collagen microspheres of uniform size.

2. The method for preparing recombinant collagen microspheres by microfluidics according to claim 1, characterized in that: The mass percentage of the recombinant collagen in the dispersed phase is 0.1% to 20%, and the mass percentage of the surfactant in the continuous phase is 0.1% to 20%.

3. The method for preparing recombinant collagen microspheres by microfluidics according to claim 1, characterized in that: The stationary liquid containing the cross-linking agent is a solution obtained by adding the cross-linking agent into the continuous phase, and the mass percentage of the cross-linking agent in the stationary liquid is 0.1% to 10%.

4. A method for preparing recombinant collagen microspheres by microfluidics according to claim 1 or 3, characterized in that: The cross-linking agent is glutaraldehyde, genipin, tetrakishydroxymethylphosphine chloride or four-arm PEG succinimidyl carbonate.

5. The method for preparing recombinant collagen microspheres by microfluidics according to claim 1, characterized in that: The petroleum ether is of pharmaceutical grade, with a boiling range of 30°C to 60°C, 60°C to 90°C or 90°C to 120°C.

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

7. The method for preparing recombinant collagen microspheres by microfluidics according to claim 1, 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 microfluidics 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 microfluidics 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 microfluidics 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, and the average particle size of the recombinant collagen microspheres is 40 μm to 60 μm.

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