Multifunctional microcapsule for wound repair and preparation method thereof

Abstract

The invention discloses a drug-loaded core-shell microcapsule for wound repair and a preparation method thereof, the method is based on a microfluidic electrospray technology, liquid drops are sprayed into collection liquid through electrostatic force, and the core-shell microcapsule with uniform size and containing various active components is generated through ultraviolet polymerization. The particle size of the microcapsule can be accurately adjusted by changing preparation conditions (internal and external phase flow velocity or voltage), the preparation method is simple and easy to implement, and the requirements on the preparation conditions are low; the microcapsule prepared by the invention can load two active components, can adjust inflammatory response and enhance collagen deposition along with the degradation time sequence release of the microcapsule, thereby accelerating wound healing, and has comprehensive functions, obvious curative effect and strong applicability.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a core-shell microcapsule for wound repair and a preparation method thereof. Background Art

[0002] Wounds often occur in daily life and surgical operations, and wound remodeling and regeneration have always been key areas of concern in the medical field. In the past few decades, a large number of wound dressings, such as patches and stents, have emerged to promote wound healing. However, solid patches that completely cover the wound actually hinder the permeability of the wound, which is harmful to the healing process. In addition, uncontrolled drug release and delivery also pose challenges, and the therapeutic effect still needs to be improved.

[0003] At present, microfluidic technology has been used to prepare microcapsules loaded with antimicrobial drugs, growth factors or cells for wound healing. However, the existing technology still has shortcomings in terms of precise control of drug release, multifunctional integration, mechanical properties and adaptability. For example, the drug release rate is difficult to meet the needs of different stages of wound healing, the single function is difficult to adapt to the complex wound environment, the mechanical stability and wound adhesion ability of microcapsules are poor, and the preparation process is complicated and the cost is high. In response to these defects, we propose an integrated preparation technology based on a microfluidic electrospray device. By constructing core-shell structure microcapsules, we can achieve precise drug release, multifunctional integration (such as antibacterial, anti-inflammatory and angiogenic), and optimize the mechanical properties of the material to improve its stability and adaptability. At the same time, it simplifies the preparation process and reduces costs, providing a more efficient solution for wound healing. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a multifunctional microcapsule for wound repair and a preparation method thereof. The microcapsule of the present invention has a flexible and adjustable degradation rate and drug release characteristics, and can accurately regulate drug release according to different stages of wound healing to meet the dynamic needs during the wound repair process.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: A multifunctional microcapsule for wound repair, the microcapsule is a core-shell structure, the shell structure is obtained by cross-linking reaction of an external phase solution and calcium ions, and the core structure is obtained by photopolymerization reaction of methacryloyl gelatin in an internal phase solution, the external phase solution is a sodium alginate aqueous solution containing black phosphorus nanosheets and curcumin nanoparticles, and the internal phase solution is an aqueous solution including vascular endothelial growth factor, methacryloyl gelatin and a photoinitiator. The photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP).

[0006] The natural active ingredient curcumin has good antioxidant properties, and its water solubility and bioavailability are improved through nano-sizing. In addition, black phosphorus has excellent biocompatibility and outstanding photothermal conversion efficiency, which enables it to quickly heat and eliminate bacteria under the irradiation of near-infrared light.

[0007] Furthermore, the outer diameter of the microcapsule is 370-430 μm, and the inner diameter is 170-280 μm. Here, the outer diameter refers to the outer diameter of the microcapsule shell, and the inner diameter refers to the diameter of the microcapsule core.

[0008] Furthermore, the microcapsules are prepared in a one-step process using a microfluidic electrospray device.

[0009] The present invention also provides a method for preparing a multifunctional microcapsule for wound repair, comprising the following steps: 1) preparing an inner and outer phase solution and a collecting solution: the outer phase solution is an aqueous solution of sodium alginate (ALG) containing black phosphorus nanosheets (BP) and curcumin nanoparticles, the inner phase solution is an aqueous solution including vascular endothelial growth factor (VEGF), methacryloyl gelatin (GelMA) and a photoinitiator (HMPP), and the collecting solution is an aqueous solution of calcium chloride; 2) Assembling a coaxial microfluidic electrospray chip: performing hydrophobic modification on the inner phase capillary and hydrophilic modification on the outer phase capillary; 3) Preparation of core-shell structure microcapsules: Monodisperse double emulsion droplets of uniform size are prepared by using the microfluidic electrospray chip constructed in step 2), and the double emulsion droplets are collected in a fixed container pre-filled with calcium chloride collection solution, and irradiated with ultraviolet light at the same time.

[0010] Furthermore, in step 2), the microfluidic electrospray chip is assembled from a glass capillary, a glass slide, a spotting needle and quick-drying glue, wherein the glass capillary is assembled by coaxially nesting an outer phase capillary and an inner phase capillary.

[0011] Furthermore, in step 2), the outer phase capillary tube diameter is 300-350 μm, and the inner phase capillary tube diameter is 80-100 μm.

[0012] Furthermore, in step 1), the mass concentration of sodium alginate in the external phase solution is 1.8 wt %, the concentration of black phosphorus nanosheets is 0-1.0 mg / mL, and the concentration of curcumin nanoparticles is 0.1 mg / mL.

[0013] Furthermore, the mass concentration of methacryloyl gelatin in the inner phase solution of step 1) is 5 wt %, and the concentration of vascular endothelial growth factor is 200 ng / mL. The collection solution of step 1) is a calcium chloride aqueous solution with a mass concentration of 2%.

[0014] Furthermore, in step 3), the flow rate range of the inner phase is 8-40 μL / min, the flow rate range of the outer phase is 40-200 μL / min, and the voltage range is 4-12 kV.

[0015] Furthermore, in step 3), the fixed container is a round glass culture dish, and the collection distance is 3-11 cm.

[0016] The size of the microcapsules can be adjusted by changing the flow rates of the internal and external phases, the collection distance and the voltage.

[0017] Beneficial effects: 1) Compared with the traditional complex microsphere preparation method, the present invention adopts a one-step method of microfluidic electrospray device to prepare microcapsules, avoiding the subsequent complex multi-step reaction and tedious post-processing process, and significantly improving the preparation efficiency. Under near-infrared irradiation, the microcapsules have a certain bactericidal effect and release drugs in a timed manner, which is of great significance for the acceleration of wound healing.

[0018] 2) The present invention relies on microfluidic technology and uses a microfluidic chip to prepare droplets. It has a simplified channel design and a small number of construction steps, does not require complex mechanical processing procedures, and has a simple process. The size of the prepared monodisperse double emulsion droplets and the polymer microspheres after solidification can be controlled by adjusting the flow rate of the inner and outer phases or the diameter of the capillary, and the operation is convenient.

[0019] 3) The microcapsules of the present invention have flexible and adjustable degradation rates and drug release characteristics, and can accurately regulate drug release according to different stages of wound healing to meet the dynamic needs of the wound repair process. Through this intelligent response mechanism, the microcapsules can quickly release anti-infective drugs in the early stage and slowly release factors that promote tissue regeneration in the subsequent stage, thereby significantly improving the wound healing effect, shortening the treatment cycle and reducing the number of medications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention is a process flow chart of the preparation of multifunctional microcapsules for wound repair; Figure 2 These are actual pictures of the electrospray microfluidic device, where Figure a is a real picture of the electrospray microfluidic device, and Figure b is a partial enlarged picture of the microfluidic electrospray device.

[0021] Figure 3 Generate images of double emulsion droplets in real time.

[0022] Figure 4 This is a light microscopy image of monodisperse multifunctional microcapsules.

[0023] Figure 5Relationship curves between the internal and external phase flow rates, collection distance and voltage in the microfluidic electrospray device and the microcapsule particle size, where (a) is the relationship between the collection distance and the microcapsule particle size, (b) is the relationship between the voltage and the microcapsule particle size, (c) is the relationship between the internal phase flow rate and the microcapsule particle size, and (d) is the relationship between the external phase flow rate and the microcapsule particle size.

[0024] Figure 6 The photothermal response diagram of the microcapsules, in which (a) the microcapsules with different black phosphorus nanosheet contents in the near infrared (1 wcm -2 ) under irradiation; Figure (b) shows the temperature change curve of microcapsules (black phosphorus nanosheet content 0.25 mg mL) under different laser intensities -1 ) is the temperature change curve of the microcapsule during 5 on / off cycles; Figure (c) is the temperature change curve of the microcapsule during 5 on / off cycles.

[0025] Figure 7 Figure 2 is the H&E staining result of each group, where Figure (a) is the H&E staining result of the control group; Figure (b) is the H&E staining result of the experimental group. DETAILED DESCRIPTION

[0026] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments and drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0027] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0028] Black phosphorus nanosheets were purchased from Pioneer Nanomaterials Technology Co., Ltd. The preparation of curcumin nanoparticles refers to the prior art, reference: Liu J, Chen Z, Wang J, et al. Encapsulation of curcumin nanoparticles with MMP9-responsive and thermos-sensitive hydrogel improves diabetic wound healing[J]. ACS Applied Materials& Interfaces, 2018, 10(19): 16315-16326.

[0029] Sodium alginate was purchased from Aladdin Methacryloyl gelatin was purchased from Aladdin 2-Hydroxy-2-methyl-1-phenyl-1-propanone was purchased from Aladdin Example 1

[0030] A multifunctional microcapsule for wound repair, the preparation process is as follows Figure 1 As shown, the following steps are included: (1) Preparation of internal and external phase solutions and collection solution: 1.1) External phase solution: composed of sodium alginate (ALG), black phosphorus (BP) nanosheets and curcumin nanoparticles (CNPs); the concentration of sodium alginate aqueous solution is 1.8%; the concentration of black phosphorus (BP) nanosheets in the mixed solution is 0.25 mg / mL, and the concentration of curcumin nanoparticles (CNPs) in the mixed solution is 0.1 mg / mL; 1.2) Inner phase solution: It is composed of 5% methacryloyl gelatin (GelMA) and 1% 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP) solution, wherein the concentration of vascular endothelial growth factor (VEGF) in the inner phase solution is 250 ng / mL, and the methacryloyl gelatin solid and 2-hydroxy-2-methyl-1-phenyl-1-propanone liquid are dissolved in the aqueous solution.

[0031] 1.3) Collection solution: 2% calcium chloride solution; (2) Assembling a coaxial microfluidic electrospray chip: Use a tube puller or an acetylene torch to pull two glass capillaries of different sizes. The diameter of the inner phase glass capillary is 80-100 μm, and the diameter of the outer phase glass capillary is 300-400 μm. According to the needs, the glass capillaries used for the inner phase and the outer phase are hydrophilic and hydrophobic respectively: the glass capillaries used for the inner phase solution are immersed in an acetone solution containing 5% (v / v) octadecyltrimethoxysilane (OTS) to achieve a hydrophobic surface; the glass capillaries used for the outer phase solution are hydrophilically modified using an ethanol solution containing 5% (v / v) 3-aminopropyltriethoxysilane (APTES). The entire microfluidic chip is assembled from the modified inner and outer phase glass capillaries, a glass slide, a cover glass, a spotting needle and quick-drying glue. The inner and outer phase glass capillaries are coaxially nested to ensure stable droplet generation.

[0032] (3) Preparation of core-shell microcapsules: The inner and outer phase aqueous solutions were drawn into syringes of suitable specifications and installed on two peristaltic pumps respectively. The glass syringe was connected to the coaxial microfluidic electrospray device through a polyethylene pipe, and the flow rate of the outer phase was set to 40μL / min and the flow rate of the inner phase was set to 8μL / min. The peristaltic pump was then started and a voltage of 8kV was applied at the same time. In the microfluidic channel, when the inner and outer phase aqueous solutions converge at the coaxial spray head, the double emulsion is stretched and eventually broken by the combined action of interfacial tension and electric field force, thereby forming monodisperse double emulsion droplets.

[0033] The double emulsion droplets were collected in a fixed container pre-filled with calcium chloride collection solution and irradiated with ultraviolet light at a collection distance of 5 cm.

[0034] Figure 2 These are actual pictures of the electrospray microfluidic device, where Figure a is a real picture of the electrospray microfluidic device, and Figure b is a partial enlarged picture of the microfluidic electrospray device.

[0035] Figure 3 Real-time generation of images of double emulsion droplets and multifunctional microcapsules.

[0036] Figure 4 This is a light microscope picture of monodisperse multifunctional microcapsules. It can be seen from the picture that the microcapsules have a core-shell structure, an average particle size of 396.6 microns, an average inner diameter of 221.1 microns, and a relatively uniform particle size distribution.

[0037] Figure 5 Relationship curves between the internal and external phase flow rates, collection distance and voltage in the microfluidic electrospray device and the microcapsule particle size, where (a) is the relationship between the collection distance and the microcapsule particle size, (b) is the relationship between the voltage and the microcapsule particle size, (c) is the relationship between the internal phase flow rate and the microcapsule particle size, and (d) is the relationship between the external phase flow rate and the microcapsule particle size.

[0038] from Figure 5 It can be seen that by controlling the collection distance, voltage and other factors unchanged and increasing the external phase flow rate, the particle size of the monodisperse double emulsion droplets increases due to the increase in the amount of fluid passing through a single shear; by controlling the internal and external phase flow rates, collection distance and other factors unchanged and increasing the voltage, the electric field force on the liquid increases and the particle size of the monodisperse double emulsion droplets decreases; by controlling the internal and external phase flow rates, voltage and other factors unchanged and increasing the collection distance, the electric field strength is relatively weakened when the collection distance increases, the initial stretching and deformation of the droplets decreases, and the particle size of the generated microspheres increases.

[0039] Therefore, the droplet size of the monodisperse double emulsion can be adjusted by changing the flow rate of the internal and external phases, the collection distance and the voltage. Example 2 Photothermal response experiment of black phosphorus nanosheet-loaded microcapsules

[0040] In order to make the microspheres have a wider application, first, we washed the multifunctional microcapsules for wound repair prepared in Example 1 with deionized water three times and placed them under 808 nm laser intensity (1 W cm -1), the maximum temperature of the microcapsules increased sharply with the increase of the concentration of black phosphorus nanosheets, indicating that the microcapsules have strong photothermal conversion ability. In addition, the photothermal behavior of the microcapsules can also be controlled by adjusting the laser power. When the laser intensity increases, the maximum temperature of the microcapsules increases significantly. In order to further verify its photothermal stability, the performance of the microcapsules was tested through 5 switching cycles. It can be found that the temperature change caused by each switching cycle is almost the same, indicating that the microcapsules have excellent photothermal stability. Figure 6 The photothermal response effect diagram of microcapsules. Among them, (a) shows the effect of different concentrations of black phosphorus nanosheets (BP nanosheet content is 0 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL) on the photothermal response ability of microcapsules, (b) shows the effect of different laser intensities on the photothermal ability of microcapsules. (c) is the test of the photothermal stability of microcapsules. Example 3: Experiment on the effect of multifunctional microcapsules on wound healing in mice

[0041] Taking the multifunctional microcapsules for wound repair prepared in Example 1 as an example, 15 rats weighing 250-300 g were anesthetized by intraperitoneal injection of 10% (w / v) chloral hydrate, and a circular wound with a diameter of 1.5 cm was artificially made on the back; The rats with back trauma were randomly divided into a control group and an experimental group, with 3 rats in each group: the control group did not receive any treatment; the experimental group was treated with black phosphorus microcapsules loaded with CNPs and VEGF (microcapsules prepared in Example 1). Figure 7 The HE staining results shown in (a) show that the skin of the control group cracked, the wound surface was large, and almost no epithelialization occurred, indicating that the wound surface was serious; Figure 7 The wound surface of the experimental group in (b) is smaller, the epithelium is significantly regenerated, and the skin has basically healed. This shows that compared with the self-repair of the control group, the conductive microneedle patch has a good ability to promote wound repair.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A multifunctional microcapsule for wound repair, characterized in that: The microcapsule is a core-shell structure, wherein the shell structure is obtained by a cross-linking reaction between an external phase solution and calcium ions, and the core structure is obtained by a photopolymerization reaction of methacryloyl gelatin in an internal phase solution. The external phase solution is a sodium alginate aqueous solution containing black phosphorus nanosheets and curcumin nanoparticles, and the internal phase solution is an aqueous solution comprising vascular endothelial growth factor, methacryloyl gelatin and a photoinitiator.

2. The microcapsule according to claim 1, characterized in that The outer diameter of the microcapsule is 370-430 μm, and the inner diameter is 170-280 μm.

3. The method for preparing microcapsules according to claim 1 or 2, characterized in that: The microcapsule is prepared by a one-step method using a microfluidic electrospray device.

4. The preparation method according to claim 3, characterized in that: The steps include: 1) preparing an inner and outer phase solution and a collecting solution: the outer phase solution is a sodium alginate aqueous solution containing black phosphorus nanosheets and curcumin nanoparticles, the inner phase solution is an aqueous solution comprising vascular endothelial growth factor, methacrylated gelatin and a photoinitiator, and the collecting solution is a calcium chloride aqueous solution; 2) Assembling a coaxial microfluidic electrospray chip: performing hydrophobic modification on the inner phase capillary and hydrophilic modification on the outer phase capillary; 3) Preparation of core-shell structure microcapsules: Using the microfluidic electrospray chip constructed in step 2), the inner phase flow rate, outer phase flow rate and voltage are set to prepare monodisperse double emulsion droplets, and the double emulsion droplets are collected in a fixed container pre-filled with calcium chloride collection solution, while being irradiated with ultraviolet light.

5. The preparation method according to claim 4, characterized in that: Step 2) The microfluidic electrospray chip is assembled from a glass capillary, a glass slide, a spotting needle and quick-drying glue, wherein the glass capillary is assembled by coaxially nesting an outer phase capillary and an inner phase capillary.

6. The preparation method according to claim 4, characterized in that: Step 2) The outer capillary tube diameter is 300-350 μm, and the inner capillary tube diameter is 80-100 μm.

7. The preparation method according to claim 4, characterized in that: Step 1) The mass concentration of sodium alginate in the external phase solution is 1.8wt%, the concentration of black phosphorus nanosheets is 0-1.0 mg / mL, and the concentration of curcumin nanoparticles is 0.1 mg / mL.

8. The preparation method according to claim 4, characterized in that: The mass concentration of methacryloyl gelatin in the inner phase solution of step 1) is 5wt%, the concentration of vascular endothelial growth factor is 200ng / mL, and the collection solution of step 1) is a calcium chloride aqueous solution with a mass concentration of 2%.

9. The preparation method according to claim 4, characterized in that: In step 3), the flow rate range of the inner phase is 8-40 μL / min, the flow rate range of the outer phase is 40-200 μL / min, and the voltage range is 4-12 kV.

10. The preparation method according to claim 4, characterized in that: Step 3) The fixed container is a round glass culture dish with a collection distance of 3-11 cm.

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