Preparation method and application of multifunctional MOF (Metal Organic Framework) microneedle patch for adsorbing exosome

Abstract

The invention discloses a preparation method and application of a multifunctional MOF (Metal Organic Framework) microneedle patch for adsorbing exosomes, and belongs to the field of biomedical materials.The preparation method comprises the following specific steps: I, synthesizing ZIF-8 particles through a hydrothermal method, and preparing a PDMS (Polydimethylsiloxane) microneedle mold by adopting a photoresist technology; iI, preparing a PEGDA / GelMA mixed solution according to a specified proportion, adding ZIF-8 particles in different proportions into the mixed solution, uniformly mixing, and finally adding a photoinitiator LAP; compared with an existing microneedle patch preparation method, the photocrosslinking microneedle containing the ZIF-8 particles is originally designed, angiogenesis and collagen deposition of a wound surface can be effectively promoted, inflammatory response is inhibited, the effect of promoting wound healing is achieved, the method is suitable for industrialized production, and the obtained microneedle product is particularly suitable for treatment of the skin wound surface and has a good application prospect. And the composition can also be used for bone repair and other organ administration.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a preparation method of a multifunctional MOF microneedle patch for adsorbing exosomes and applications thereof. Background Art

[0002] In recent years, wound healing has attracted widespread attention due to its severe challenges and heavy economic burden. However, our current treatment measures for difficult wound healing are very limited, and we need to continue to explore effective treatments. The wound healing process is usually divided into three stages: inflammation, proliferation, and tissue remodeling. Excellent treatment methods can not only improve wound inflammation, but also accelerate angiogenesis, cell proliferation, and tissue remodeling. In order to solve the problem of difficult wound healing, people have developed a variety of treatment strategies, such as hydrogel films, electrospinning dressings, and various other wound patches. Among them, microneedles (MNs) are a promising medical technology that can deliver painless transdermal drugs through minimally invasive methods and are widely used in vaccination, cancer treatment, skin treatment, cosmetics, and other fields. In particular, MNs containing active ingredients have been used in wound healing research. Compared with traditional dressings, MNs patches can significantly increase the effect of promoting wound healing by increasing the contact area between them and the wound. However, for some MNs made of non-biodegradable materials, such as stainless steel, the needle body of MNs can easily form tiny pinholes at the wound site. These holes are likely to cause secondary physical damage and even cause serious microbial infection in the wound. These limitations restrict the development of MNs in the field of promoting wound healing. Therefore, hydrogel MNs with minimally invasive therapeutic effects that improve drug delivery and excellent biocompatibility are highly desirable. At present, the application potential of microneedles has yet to be explored in depth.

[0003] PEGDA and GelMA are hydrogel materials commonly used in the biomedical field. They have good biocompatibility and have broad application prospects in tissue engineering and wound repair. GelMA has good biodegradability and adjustable cross-linking degree, and has the characteristics of UV cross-linking. At the same time, it can better support cell adhesion and proliferation and promote tissue regeneration. The addition of PEGDA can further enhance the mechanical properties of the material. This double network structure of PEGDA and GelMA can continuously and stably release the loaded active ingredients, which helps to accelerate wound healing.

[0004] Studies have shown that zinc ions are also believed to contribute to antibacterial and wound healing effects. In recent years, zinc-based zeolite-imidazolate framework (ZIF-8MOF) has shown good antibacterial effects on wound models and bone repair models. Positively charged metal nanoparticles have a large specific surface area and high porosity, and the rough surface can increase the contact area with bacteria, thereby obtaining better antibacterial activity. Exosomes are a natural cell signal carrier that is negatively charged in itself. They can regulate local cell activities in the wound through the bioactive molecules such as proteins and RNA contained in them, thereby promoting wound healing. Therefore, we use electrostatic force interaction to make the positively charged microneedle patch adsorb negatively charged exosomes and deliver them directly to the wound site through the microneedle structure. In addition, zinc ions play an important role in the vascularization process, stimulating vascular endothelial cells to form blood vessels and inhibiting inflammatory responses. The loading of exosomes further enhances these functions, forming a synergistic effect, thereby showing better effects in the wound repair process. The author believes that loading ZIF-8 particles into PEGDA / GelMA microneedles gives the microneedles powerful antibacterial properties, while also having a good effect in promoting angiogenesis and inhibiting inflammation. The use of electrostatic force enables exosomes to self-load on the surface of the microneedle patch, further enhancing these functions and forming a synergistic effect. This combination is one of the reliable ways to treat wounds.

[0005] The present invention aims to construct a multifunctional microneedle material for wound repair and treatment. It is based on PEGDA / GelMA, which is loaded with ZIF-8 particles, and uses electrostatic force to adsorb exosomes, exerting antibacterial, angiogenic and anti-inflammatory effects, and is prepared through a series of steps and processes. The multifunctional microneedle is expected to have excellent biocompatibility, biodegradability, broad-spectrum antibacterial properties, angiogenic activity and inflammatory response inhibition, and has great potential for clinical transformation. At present, there are no reports on the preparation of microneedles using PEGDA / GelMA and ZIF-8 particles as raw materials at the same time, and the use of electrostatic force to complete exosome loading. Summary of the invention

[0006] The purpose of the present invention is to solve the defects in the prior art and propose a method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes, wherein the specific steps of the preparation method are as follows:

[0009] Ⅰ. Synthesize ZIF-8 particles by hydrothermal method and prepare PDMS microneedle mold by photoresist technology;

[0010] II. Prepare a PEGDA / GelMA mixed solution according to the prescribed ratio, add different proportions of ZIF-8 particles to the mixed solution and mix well, and finally add the photoinitiator LAP;

[0011] III. Pour the mixed solution into a microneedle mold, centrifuge to remove bubbles, and then place the mold under an ultraviolet light source to cross-link and obtain microneedles;

[0012] IV. Place the microneedle into the exosome solution for exosome adsorption.

[0013] As a further solution of the present invention, the morphological parameters of the PDMS microneedle mold in step I are needle height 400-1500 μm, base diameter 150-500 μm, needle tip diameter 5-50 μm, center spacing 500-2000 μm, and array number greater than 5×5.

[0014] As a further embodiment of the present invention, the molecular weight of the PEGDA in step II is 600, the mass fraction of the GelMA is 10%, wherein the mixing ratio of the PEGDA / GelMA mixed solution is PEGDA:GelMA=2:1;

[0015] The amount of the photoinitiator LAP added is 0.05% of the total mass of the solution.

[0016] As a further solution of the present invention, the parameters of the centrifugal degassing in step III are a rotation speed of 3000 to 10000 rpm, a time of 5 to 15 min, and repeated three times.

[0017] As a further solution of the present invention, the specific steps of placing the mold under an ultraviolet light source and cross-linking to obtain microneedles are as follows:

[0018] S1.1: The wavelength of the UV light source is set to 200-400 nm, the power is 50-500 W, the cross-linking time is 1-3 min, and the mold is placed at room temperature for 24 hours;

[0019] S1.2: When the microneedles are fully dried and stabilized, remove the microneedle array from the PDMS mold.

[0020] As a further embodiment of the present invention, the specific steps of exosome adsorption in step IV are as follows:

[0021] S2.1: Suspend the exosomes at a concentration of 1 mg / mL in 1 mL of PBS solution with a pH of 7;

[0022] S2.2: Add the exosome solution into the composite microneedle scaffold and incubate at 4°C overnight to load the exosomes onto the microneedle surface.

[0023] Applications of multifunctional MOF microneedle patches for adsorbing exosomes include:

[0024] The multifunctional MOF microneedle patch for adsorbing exosomes specifically uses a photo-crosslinked microneedle containing ZIF-8 particles, the microneedle is an array formed by a plurality of conical needles, and is made of a hydrogel prepolymer cured by ultraviolet light;

[0025] The chemical composition of the hydrogel prepolymer is polyethylene glycol diacrylate PEGDA, methacrylated gelatin GelMA, and photoinitiator LAP;

[0026] In the photo-cross-linked microneedle containing ZIF-8 particles, by adjusting the loading amount of ZIF-8 particles, a microneedle with both excellent antibacterial effect and good biocompatibility can be obtained, and exosomes can be autonomously loaded on the surface of the microneedle.

[0027] As a further embodiment of the present invention, the results of in vivo wound experiments using the exosome-adsorbing multifunctional MOF microneedle patch showed that the exosome-adsorbing multifunctional MOF microneedle patch containing photo-crosslinked microneedles of ZIF-8 and exosomes can effectively promote angiogenesis and collagen deposition on the wound surface, inhibit inflammatory response, and play a role in promoting wound healing. The exosome-adsorbing multifunctional MOF microneedle patch has certain application potential in wound treatment.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] Compared with the previous microneedle patch preparation method, the present invention combines the clinical needs of dressings, and originally designs a photocross-linked microneedle containing ZIF-8 particles, and prepares a series of microneedles with different chemical compositions; through a variety of in vivo and in vitro experiments to evaluate its performance, a microneedle product with good mechanical properties, broad-spectrum antibacterial properties, angiogenic effects and anti-inflammatory effects is selected, which can effectively promote angiogenesis and collagen deposition in the wound surface, inhibit inflammatory response, and promote wound healing; by optimizing the preparation process and parameters, a set of microneedle preparation methods with strong stability and good repeatability is obtained, which is suitable for factory production, and the obtained microneedle products are particularly suitable for the treatment of skin wounds, and can also be used for bone repair and drug delivery to other organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0031] Figure 1 This is a flowchart of the method for preparing the multifunctional MOF microneedle patch for adsorbing exosomes proposed in the present invention;

[0032] Figure 2 Electron microscope images of ZIF-8 particles at different magnifications for the preparation method of the multifunctional MOF microneedle patch for adsorbing exosomes proposed in the present invention;

[0033] Figure 3 The morphology of the microneedles at different magnifications in the method for preparing the multifunctional MOF microneedle patch for adsorbing exosomes proposed in the present invention;

[0034] Figure 4 This is a result diagram of the in vitro biocompatibility evaluation of the microneedle MN in the preparation method of the multifunctional MOF microneedle patch for adsorbing exosomes proposed in the present invention;

[0035] Figure 5 This is a result diagram of the evaluation of the broad-spectrum antibacterial activity of the microneedle MN in the preparation method of the multifunctional MOF microneedle patch adsorbing exosomes proposed in the present invention;

[0036] Figure 6 This is a confocal imaging diagram of the microneedle adsorbing fluorescently labeled exosomes in the preparation method of the multifunctional MOF microneedle patch for adsorbing exosomes proposed in the present invention;

[0037] Figure 7 This is a graph showing the results of in vitro angiogenic and anti-inflammatory experiments of microneedles in the method for preparing the multifunctional MOF microneedle patch for adsorbing exosomes proposed in the present invention;

[0038] Figure 8 This is a diagram showing the results of the in vivo treatment evaluation of skin wounds using microneedles according to the method for preparing the multifunctional MOF microneedle patch for adsorbing exosomes proposed in the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0040] Example 1

[0041] Reference Figure 1-6 This embodiment discloses a method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes. The specific steps of the preparation method are as follows:

[0042] ZIF-8 particles were synthesized by hydrothermal method, and PDMS microneedle molds were prepared by photoresist technology.

[0043] Specifically, the morphological parameters of the PDMS microneedle mold are needle height 400-1500 μm, base diameter 150-500 μm, needle tip diameter 5-50 μm, center spacing 500-2000 μm, and array number greater than 5×5.

[0044] A PEGDA / GelMA mixed solution was prepared according to the prescribed ratio, and different ratios of ZIF-8 particles were added to the mixed solution and mixed evenly, and finally a photoinitiator LAP was added.

[0045] Specifically, the molecular weight of PEGDA is 600, the mass fraction of GelMA is 10%, wherein the mixing ratio of the PEGDA / GelMA mixed solution is PEGDA:GelMA=2:1, and the added amount of the photoinitiator LAP accounts for 0.05% of the total mass of the solution.

[0046] The mixed solution is poured into a microneedle mold, centrifuged to remove bubbles, and then the mold is placed under an ultraviolet light source to cross-link and obtain microneedles.

[0047] Specifically, the parameters of centrifugal degassing are a rotation speed of 3000 to 10000 rpm, a time of 5 to 15 minutes, and repeated three times.

[0048] Specifically, the wavelength of the ultraviolet light source is set to 200-400 nm, the power is 50-500 W, the cross-linking time is 1-3 min, the mold is placed at room temperature for 24 hours, and when the microneedles are fully dried and stabilized, the microneedle array is separated from the PDMS mold.

[0049] The microneedle was placed into the exosome solution for exosome adsorption.

[0050] Specifically, exosomes were suspended in 1 mL of PBS solution with a pH of 7 at a concentration of 1 mg / mL, the exosome solution was added to the composite microneedle scaffold, and incubated overnight at 4°C to load the exosomes onto the microneedle surface.

[0051] Example 2

[0052] Reference Figure 1 as well as Figure 7-8 This embodiment discloses an application of a multifunctional MOF microneedle patch for adsorbing exosomes, including: the multifunctional MOF microneedle patch for adsorbing exosomes specifically uses a photo-crosslinked microneedle containing ZIF-8 particles, the microneedle is an array formed by a plurality of conical needles, and is prepared by ultraviolet light curing of a hydrogel prepolymer, the chemical composition of the hydrogel prepolymer is polyethylene glycol diacrylate PEGDA and methacrylated gelatin GelMA, and a photoinitiator LAP.

[0053] In the photo-cross-linked microneedle containing ZIF-8 particles, by adjusting the loading amount of ZIF-8 particles, a microneedle with both excellent antibacterial effect and good biocompatibility can be obtained, and exosomes can be autonomously loaded on the surface of the microneedle.

[0054] As a further scheme of the present invention, the results of in vivo wound experiments using the multifunctional MOF microneedle patch that adsorbs exosomes show that the multifunctional MOF microneedle patch that adsorbs exosomes and contains photo-crosslinked microneedles of ZIF-8 and exosomes can effectively promote angiogenesis and collagen deposition on the wound surface, inhibit inflammatory response, and play a role in promoting wound healing. The multifunctional MOF microneedle patch that adsorbs exosomes has certain application potential in wound treatment.

Claims

1. A method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes, characterized in that: The specific steps of the preparation method are as follows: Ⅰ. Synthesize ZIF-8 particles by hydrothermal method and prepare PDMS microneedle mold by photoresist technology; II. Prepare a PEGDA / GelMA mixed solution according to the prescribed ratio, add different ratios of ZIF-8 particles to the mixed solution and mix well, and finally add the photoinitiator LAP; III. Pour the mixed solution into a microneedle mold, centrifuge to remove bubbles, and then place the mold under an ultraviolet light source to cross-link and obtain microneedles; IV. Place the microneedle into the exosome solution for exosome adsorption.

2. The method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes according to claim 1, characterized in that: The morphological parameters of the PDMS microneedle mold in step I are needle height 400-1500 μm, base diameter 150-500 μm, needle tip diameter 5-50 μm, center spacing 500-2000 μm, and array number greater than 5×5.

3. The method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes according to claim 2, characterized in that: Step II: the molecular weight of the PEGDA is 600, the mass fraction of the GelMA is 10%, wherein the mixing ratio of the PEGDA / GelMA mixed solution is PEGDA:GelMA=2:1; The amount of the photoinitiator LAP added is 0.05% of the total mass of the solution.

4. The method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes according to claim 1, characterized in that: The parameters of the centrifugal degassing in step III are a rotation speed of 3000 to 10000 rpm, a time of 5 to 15 min, and repeated three times.

5. The method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes according to claim 1, characterized in that: The specific steps of placing the mold under a UV light source and cross-linking to obtain microneedles are as follows: S1.1: The wavelength of the UV light source is set to 200-400 nm, the power is 50-500 W, the cross-linking time is 1-3 min, and the mold is placed at room temperature for 24 hours; S1.2: When the microneedles are fully dried and stabilized, remove the microneedle array from the PDMS mold.

6. The method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes according to claim 1, characterized in that: The specific steps of exosome adsorption in step IV are as follows: S2.1: Suspend the exosomes at a concentration of 1 mg / mL in 1 mL of PBS solution with a pH of 7; S2.2: Add the exosome solution into the composite microneedle scaffold and incubate at 4°C overnight to load the exosomes onto the microneedle surface.

7. Application of a multifunctional MOF microneedle patch for adsorbing exosomes, for realizing the method for preparing a multifunctional MOF microneedle patch for adsorbing exosomes according to any one of claims 1 to 6, characterized in that: include: The multifunctional MOF microneedle patch for adsorbing exosomes specifically uses a photo-crosslinked microneedle containing ZIF-8 particles, the microneedle is an array formed by a plurality of conical needles, and is made of a hydrogel prepolymer cured by ultraviolet light; The chemical composition of the hydrogel prepolymer is polyethylene glycol diacrylate PEGDA, methacrylated gelatin GelMA, and photoinitiator LAP; In the photo-cross-linked microneedle containing ZIF-8 particles, by adjusting the loading amount of ZIF-8 particles, a microneedle with both excellent antibacterial effect and good biocompatibility can be obtained, and exosomes can be autonomously loaded on the surface of the microneedle.

8. The use of the multifunctional MOF microneedle patch for adsorbing exosomes according to claim 7, characterized in that: The results of in vivo wound experiments using the exosome-absorbed multifunctional MOF microneedle patch showed that the exosome-absorbed multifunctional MOF microneedle patch containing photo-crosslinked microneedles of ZIF-8 and exosomes can effectively promote angiogenesis and collagen deposition on the wound surface, inhibit inflammatory response, and play a role in promoting wound healing. The exosome-absorbed multifunctional MOF microneedle patch has certain application potential in wound treatment.

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