Degradable cell micro-nano patch and application thereof in stem cell treatment and tissue repair

Abstract

The invention relates to a degradable cell micro-nano patch and application thereof in stem cell treatment and tissue repair, the micro-nano patch is composed of a drug carrying layer and a cell adhesion layer, the size is between 100 nanometers and 100 micrometers, and the thickness is between 10 nanometers and 10 micrometers. The degradable cell micro-nano patch can load and slowly release various biochemical factors, can be adsorbed on the surface of stem cells to be injected and migrated along with the cells, releases the factors around implanted cells, accurately regulates and controls the extracellular microenvironment, enhances the activity of the implanted cells and the capability of directionally differentiating the implanted cells into target cells, and has a good application prospect. The treatment effect of the stem cells is greatly improved, the stem cells are not prone to inactivation in the body, the stem cells are tightly attached to the cells when transplanted into the body together with the cells, and accurate and efficient regulation and control of the cells are achieved.

Description

Technical Field

[0001] The present invention relates to a degradable cell micro-nano patch and its application in stem cell therapy and tissue repair, belonging to the field of biomedicine. Background Art

[0002] Stem cell therapy is to transplant healthy stem cells into patients to repair or replace damaged cells or tissues, so as to achieve the purpose of curing diseases. Stem cells are pluripotent cells with self-renewal ability, which can be cultured and proliferated in vitro and differentiated into specific types of cells. At the same time, stem cells can release a variety of bioactive factors such as pro-cell growth factors, anti-inflammatory factors, immunomodulatory factors, and anti-apoptotic factors through paracrine action. These factors participate in various inflammatory responses and immune regulation mechanisms, can promote signal transduction between cells and between cells and the extracellular matrix, and thus have a positive impact on the physiological processes of the body. Therefore, stem cells (such as embryonic stem cells, induced pluripotent stem cells (iPSCs), or adult stem cells, neural stem cells, etc.) have broad application prospects as seed cells in the treatment of nervous system diseases, immune system diseases, and the fields of tissue engineering and regenerative medicine. However, stem cells face problems such as unsatisfactory treatment effects and large individual differences in clinical applications, and there are still many challenges in market-oriented applications. The main reasons for the above problems are that after stem cells are implanted into the body, their survival rate, activity, and efficiency of directional differentiation into target cells are relatively low in the complex microenvironment. Therefore, precisely regulating the behavior and function of stem cells, including proliferation, differentiation, and cytokine secretion, is the key to improving the application effect of stem cells in major diseases, tissue repair, etc.

[0003] Currently, in clinical trials, soluble biological factors or chemical factors are mainly used to regulate the behavior and function of stem cells. However, these factors are extremely easy to inactivate in the body and are easily degraded by organisms. In addition, when they are transplanted into the body together with cells, they will flow rapidly with body fluids, and at the same time, the cells will also migrate, which makes it difficult for these factors to accumulate around stem cells, difficult to effectively improve the stem cell microenvironment, and unable to precisely and efficiently regulate cells.

[0004] Therefore, there is an urgent need for a new material that can precisely and efficiently regulate the extracellular microenvironment of stem cells, further enhance the activity of implanted cells, be able to differentiate into target cells directionally, and improve the stem cell therapy effect. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a degradable cell micro-nano patch and its application in stem cell therapy and tissue repair.

[0006] The degradable cell micro-nano patch provided by the present invention can load and slowly release a variety of biochemical factors, and can be adsorbed on the surface of stem cells and migrate together with the cells during injection, release factors around the implanted cells, precisely regulate the extracellular microenvironment, enhance the activity of implanted cells and the ability to differentiate into target cells directionally, and greatly improve the therapeutic effect of stem cells.

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] A degradable cell micro-nano patch, the micro-nano patch is composed of a drug-loading layer and a cell adhesion layer, with a size between 100 nanometers and 100 micrometers and a thickness between 10 nanometers and 10 micrometers.

[0009] The present invention also provides a preparation method of the above-mentioned degradable cell micro-nano patch.

[0010] The preparation method of the above-mentioned degradable cell micro-nano patch includes the following steps:

[0011] 1) Prepare an aqueous solution of a cellophilic molecule with a mass concentration of 0.1-15%, and spin-coat the aqueous solution of the cellophilic molecule on a mold with an array structure to form a cell adhesion layer, obtaining a mold carrying the cell adhesion layer;

[0012] 2) Dissolve a degradable biomaterial in a solvent, prepare a degradable biomaterial solution with a mass concentration of 0.1-20%, and incorporate biochemical factors to obtain a precursor solution of the drug-loading layer;

[0013] 3) Continuously spin-coat the precursor solution of the drug-loading layer on the mold carrying the cell adhesion layer, and form a drug-loading layer after curing, obtaining a mold with a cell adhesion layer and a drug-loading layer;

[0014] 4) Prepare a water-soluble polymer solution with a mass concentration of 1-20%, and spin-coat the water-soluble polymer solution on a glass slide or silicon wafer substrate to obtain a polymer-coated substrate;

[0015] 5) Fit the drug-loading layer of the mold with a cell adhesion layer and a drug-loading layer to the water-soluble polymer-coated substrate, apply pressure to make the surface of the mold and the substrate in full contact, and remove the mold;

[0016] 6) Immerse the substrate obtained in step 5) in an aqueous solution for 1-100 minutes, remove the substrate under ultrasonic assistance, separate the micro-nano patch, and centrifuge to collect the micro-nano patch to obtain the degradable cell micro-nano patch.

[0017] Preferably according to the present invention, in step 1), the cellophilic molecule is selected from one or more of laminin, polylysine, RGD polypeptide, sodium polystyrene sulfonate, and polyallylamine hydrochloride.

[0018] Preferably according to the present invention, in step 1), the surface structure shape of the mold is a cylindrical, triangular prism, cube, hexagonal prism, or octagonal prism structure; the diameter or side length of the surface array structure unit of the mold is between 100 nanometers and 100 micrometers, and the height is between 10 nanometers and 10 micrometers.

[0019] Preferably according to the present invention, in step 1), the mold composition is PDMS or PMMA.

[0020] Preferably according to the present invention, in step 1), the cell adhesion layer is 1 - 20 layers.

[0021] Preferably according to the present invention, in step 2), the biodegradable biomaterial is selected from one or more of polylactic acid (PLA), poly (lactic - co - glycolic acid) (PLGA), polycaprolactone (PCL), chitosan, methacrylated hydrogel, alginate, methacrylated hyaluronic acid, and methacrylated gelatin.

[0022] Preferably according to the present invention, in step 2), the solvent is selected from one or more of water, methanol, ethanol, dichloromethane, acetone, dimethylformamide, or dimethylacetamide.

[0023] Preferably according to the present invention, in step 2), the biochemical factor is selected from one or more of the differentiation - promoting drug retinoic acid, ascorbic acid, neurotrophic factor, bone morphogenetic protein, fibroblast growth factor, ginkgolide B, ganglioside, baicalein, or the anti - inflammatory drug curcumin.

[0024] Preferably according to the present invention, in step 2), the concentration of the biochemical factor in the drug - loaded layer precursor solution is 0.1 - 5%.

[0025] Preferably according to the present invention, in step 3), the curing is by natural evaporation, photocuring, or thermal curing.

[0026] Preferably according to the present invention, in step 3), the drug - loaded layer is 1 - 80 layers.

[0027] Preferably according to the present invention, in step 4), the water - soluble polymer is polyvinyl alcohol or polyvinylpyrrolidone.

[0028] Preferably according to the present invention, in step 5), the applied pressure magnitude is 0.1 - 100 MPa, and the application time is 1 - 60 minutes.

[0029] Preferably according to the present invention, in step 6), the ultrasonic wave is carried out according to the existing technology.

[0030] Application of the above - mentioned biodegradable cell micro - nano patch in the preparation of stem cell therapeutics and tissue repair agents

[0031] Preferably according to the present invention, the specific application method is as follows:

[0032] (1) Incubate the above-mentioned degradable cell micro-nano patch and stem cells at a mass ratio of 1:2 - 20:1 for 10 - 200 minutes at 4 - 37 °C to allow the micro-nano patch to adsorb onto the surface of the stem cells;

[0033] (2) Centrifuge at 500 - 2000 rpm, collect the stem cells adsorbed with the micro-nano patch, and resuspend them in the culture medium to obtain a stem cell therapeutic agent or a tissue repair agent.

[0034] Preferably according to the present invention, in step (1), the stem cells include neural stem cells, adipose stem cells, mesenchymal stem cells, induced pluripotent stem cells, and umbilical cord blood stem cells.

[0035] Technical features and advantages of the present invention:

[0036] 1. The degradable cell micro-nano patch provided by the present invention can load and slowly release a variety of biochemical factors, and can adsorb on the surface of stem cells and migrate with the cells during injection, release factors around the implanted cells, precisely regulate the extracellular microenvironment, enhance the activity of implanted cells and the ability to differentiate into target cells directionally, and greatly improve the therapeutic effect of stem cells.

[0037] 2. The degradable cell micro-nano patch of the present invention is not easily inactivated in vivo. When transplanted into the body together with cells, it closely adheres to the cells, realizing precise and efficient regulation of the cells.

[0038] 3. The degradable cell micro-nano patch of the present invention is made of a biodegradable material, can be gradually degraded into harmless substances in the human body, and is excreted from the body through the metabolic system, causing basically no damage to the body.

[0039] 4. The degradable cell micro-nano patch of the present invention can achieve single-cell drug delivery, realize stable drug delivery to target cells, reduce the flow of drugs with body fluids, and reduce the side effects on non-target tissues.

[0040] 5. The degradable cell micro-nano patch of the present invention can deliver different types of drugs, and thus has wide applicability in anti-inflammatory, nerve repair, and trauma diseases. Description of the Drawings

[0041] Figure 1 SEM and AFM of the micron-sized PLGA retinoic acid-loaded degradable cell micro-nano patch in Example 1.

[0042] Figure 2 Fluorescence image of the micron-sized PLGA retinoic acid-loaded degradable cell micro-nano patch in Example 1.

[0043] Figure 3It is the retinoic acid release curve of the micron-scale PLGA-loaded retinoic acid degradable cell micro-nano patch in Example 1.

[0044] Figure 4 It is the self-assembly fluorescence image of the micron-scale PLGA-loaded retinoic acid degradable cell micro-nano patch and neural stem cells in Example 1. Specific implementation manners

[0045] The following examples are further specific descriptions of the present invention to illustrate the characteristics of the present invention, but the implementation manners of the present invention are by no means limited thereto.

[0046] Example 1

[0047] The preparation method of the micron-scale PLGA-loaded retinoic acid degradable cell micro-nano patch is as follows:

[0048] 1) Cast the PDMS prepolymer and curing agent on the Su8 photoresist template, and then cure and demold to obtain a PDMS template with a column array structure on the surface, where the surface structure is column arrays with a side length of 8 μm, the center distance between cylinders is 12 μm, and the column height is 6 μm.

[0049] 2) Configure the cationic polymer poly(allylamine hydrochloride) (PAH) into an aqueous solution of poly(allylamine hydrochloride) with a mass concentration of 0.5%, and spin-coat the aqueous solution of poly(allylamine hydrochloride) on the PDMS array structure in step 1) to obtain a mold with 5 layers of cell adhesion layers;

[0050] 3) Dissolve poly(lactic-co-glycolic acid) PLGA in acetone to prepare a PLGA solution with a mass concentration of 12%, and then incorporate retinoic acid to make the mass concentration of retinoic acid reach 1% to obtain a drug-loaded layer precursor solution;

[0051] 4) Continuously spin-coat the drug-loaded layer precursor solution on the mold with the cell adhesion layer, and form 50 layers of drug-loaded layers after drying and curing to obtain a mold with a cell adhesion layer and a drug-loaded layer;

[0052] 5) Prepare a polyvinyl alcohol solution with a mass concentration of 3%, and spin-coat the solution on a glass substrate to obtain a polyvinyl alcohol-coated substrate;

[0053] 6) Fit the drug-loaded layer of the mold with the cell adhesion layer and the drug-loaded layer to the water-soluble polymer-coated substrate, apply a pressure of 0.1 Mpa, and pressurize for 5 minutes to make the surface of the mold and the substrate in full contact, and remove the mold;

[0054] 7) At 25 °C, immerse the substrate obtained in step 6) in water for 30 minutes, separate the substrate and the micro-nano patch under ultrasonic assistance, and collect the micro-nano patch by centrifugation to obtain a micron-sized PLGA retinoic acid-degradable cell micro-nano patch.

[0055] SEM and AFM images of the micron-sized PLGA retinoic acid-degradable cell micro-nano patch are as Figure 1 shown, Figure 1 showing that the prepared micron-sized PLGA retinoic acid-degradable cell micro-nano patch has a diameter of about 8 μm and a thickness of about 300 nm.

[0056] The fluorescence image of the micron-sized PLGA retinoic acid-degradable cell micro-nano patch is as Figure 2 shown, Figure 2 The red fluorescence shows the cell adhesion layer, and the green shows the PLGA drug-loading layer. It can be seen from the fluorescence image that the patch consists of a cell adhesion layer and a drug-loading layer.

[0057] Example 2

[0058] Application of the micron-sized PLGA retinoic acid-degradable cell micro-nano patch:

[0059] (1) Incubate the degradable cell micro-nano patch of Example 1 with stem cells at a mass ratio of 1:5 at 37 °C for 60 minutes to allow the micro-nano patch to adsorb on the cell surface;

[0060] (2) Centrifuge at 2000 rpm, collect the neural stem cells adsorbed with the micro-nano patch, and resuspend them in the culture medium to obtain a micron-sized PLGA retinoic acid-degradable cell micro-nano patch.

[0061] Figure 3 This is the retinoic acid release curve of the micron-sized PLGA retinoic acid-degradable cell micro-nano patch. It can be seen from the retinoic acid release curve that the release rate of retinoic acid is relatively fast within the first 50 h, the release rate is relatively slow after 50 h, and the final release rate reaches about 60% after 350 h.

[0062] Figure 4 This is the self-assembly fluorescence image of the micron-sized PLGA retinoic acid-degradable cell micro-nano patch and neural stem cells; it can be seen from the fluorescence image that the drug-loaded patch adheres tightly to the neural stem cells, and drug release on neural stem cells can be achieved.

[0063] Example 3

[0064] Preparation method of the micron-sized PLA retinoic acid-degradable cell micro-nano patch, the steps are as follows:

[0065] 1) Casting PDMS prepolymer and curing agent on Su8 photoresist template, then curing and demolding to obtain a PDMS template with a columnar structure on the surface, wherein the surface structure is a columnar structure with a side length of 8 μm, the center distance between the columns is 12 μm, and the column height is 6 μm.

[0066] 2) preparing a cationic polymer poly(allylamine hydrochloride, PAH) into a poly(allylamine hydrochloride) aqueous solution with a mass concentration of 0.5%, and using a spin coater to spin coat the poly(allylamine hydrochloride) aqueous solution on the PDMS array structure in step 1) to obtain a mold with 5 cell-carrying adhesion layers;

[0067] 3) dissolving PLA in acetone to prepare a PLA solution with a mass concentration of 15%, and then adding retinoic acid to make the mass concentration of retinoic acid reach 1%, thereby obtaining a drug-carrying layer precursor solution;

[0068] 4) Continue to spin-coat the drug-carrying layer precursor solution on the mold carrying the cell adhesion layer, and form 80 layers of drug-carrying layers after drying and curing, thereby obtaining a mold with a cell adhesion layer and a drug-carrying layer;

[0069] 5) preparing a polyvinyl alcohol solution with a mass concentration of 3%, and spin coating the solution on a glass substrate to obtain a polyvinyl alcohol-coated substrate;

[0070] 6) Lay the drug-carrying layer of the mold with the cell adhesion layer and the drug-carrying layer on the substrate coated with the water-soluble polymer, apply a pressure of 1 MPa for 5 minutes to make the mold surface and the substrate completely contact, and remove the mold;

[0071] 7) Soaking the substrate obtained in step 6) in water at 25° C. for 30 minutes, separating the substrate and the micro-nano patch with the assistance of ultrasound, and collecting the micro-nano patch by centrifugation to obtain a micron-scale PLGA-loaded retinoic acid-degradable cell micro-nano patch.

[0072] Example 4

[0073] The preparation method of GelMA micron-scale degradable drug-loaded cell patch is as follows:

[0074] 1) Casting PDMS prepolymer and curing agent on Su8 photoresist template, then curing and demolding to obtain a PDMS template with a columnar structure on the surface, wherein the surface structure is a columnar structure with a side length of 8 μm, the center distance between the columns is 12 μm, and the column height is 6 μm.

[0075] 2) Prepare an aqueous solution of poly(allylamine hydrochloride) (PAH), a cationic polymer, with a mass concentration of 0.5%. Spin-coat the aqueous solution of poly(allylamine hydrochloride) on the PDMS array structure in step 1) using a spin coater to obtain a mold with a cell adhesion layer of 1 layer.

[0076] 3) Dissolve methacrylated gelatin (GelMA) in a 0.25% w / w LAP photoinitiator, and add homovanillic acid to a concentration of 40 μM to obtain a drug-loaded layer precursor solution.

[0077] 4) Continuously spin-coat the drug-loaded layer precursor solution on the mold with the cell adhesion layer at a spin speed of 2000 rpm for 30 seconds. After spin-coating, cure it with a 405 nm ultraviolet lamp at 30 mW / cm² for 1 minute to obtain a mold with a cell adhesion layer and a drug-loaded layer. 2

[0078] 5) Prepare a 3% polyethylene glycol (PVA) solution by mass concentration, and spin-coat this solution on a glass substrate to obtain a PVA-coated substrate.

[0079] 6) Bond the drug-loaded layer of the mold with the cell adhesion layer and the drug-loaded layer to the water-soluble polymer-coated substrate, apply a pressure of 1 MPa, and pressurize for 5 minutes to make the mold surface and the substrate in full contact, then remove the mold.

[0080] 7) Immerse the substrate obtained in step 6) in water at 25°C for 30 minutes, separate the substrate and the micro-nano patch under ultrasonic assistance, and collect the micro-nano patch by centrifugation.

[0081] Example 5

[0082] 1) Cast a PDMS prepolymer and a curing agent on a SU-8 photoresist template, and then cure and demold to obtain a PDMS template with a columnar structure on the surface. The surface structure is a columnar array with a side length of 8 μm, a center distance of 12 μm between cylinders, and a column height of 6 μm.

[0083] 2) Prepare an aqueous solution of poly(allylamine hydrochloride) (PAH), a cationic polymer, with a mass concentration of 0.5%. Spin-coat the aqueous solution of poly(allylamine hydrochloride) on the PDMS array structure in step 1) using a spin coater to obtain a mold with a cell adhesion layer of 20 layers.

[0084] 3) Dissolve methacrylated gelatin (GelMA) in water to prepare a 12% PLGA solution, and then incorporate ganglioside to a mass concentration of 1% to obtain a drug-loaded layer precursor solution. ​

[0085] 4) Continue to spin-coat the drug-carrying layer precursor solution on the mold carrying the cell adhesion layer, and form 50 layers of drug-carrying layers after drying and curing, thereby obtaining a mold with a cell adhesion layer and a drug-carrying layer;

[0086] 5) preparing a polyvinyl alcohol solution with a mass concentration of 3%, and spin coating the solution on a glass substrate to obtain a polyvinyl alcohol-coated substrate;

[0087] 6) Lay the drug-carrying layer of the mold with the cell adhesion layer and the drug-carrying layer on the substrate coated with the water-soluble polymer, apply a pressure of 1 MPa for 5 minutes to make the mold surface and the substrate completely contact, and remove the mold;

[0088] 7) Soak the substrate obtained in step 6) in water at 25° C. for 30 minutes, separate the substrate and the micro-nano patch with the assistance of ultrasound, and collect the micro-nano patch by centrifugation.

Claims

1. A degradable cell micro-nano patch, which consists of a drug-carrying layer and a cell adhesion layer, with a size between 100 nanometers and 100 micrometers and a thickness between 10 nanometers and 10 micrometers.

2. The method for preparing the degradable cell micro-nano patch according to claim 1 comprises the following steps: 1) configuring the cytophilic molecule into a cytophilic molecule aqueous solution with a mass concentration of 0.1-15%, spin-coating the cytophilic molecule aqueous solution on a mold having an array structure to form a cell adhesion layer, thereby obtaining a mold carrying a cell adhesion layer; 2) dissolving the degradable biomaterial in a solvent to prepare a degradable biomaterial solution with a mass concentration of 0.1-20%, and adding biochemical factors to obtain a drug-carrying layer precursor solution; 3) Continue to spin-coat the drug-carrying layer precursor solution on the mold carrying the cell adhesion layer, and form the drug-carrying layer after solidification, thereby obtaining a mold with a cell adhesion layer and a drug-carrying layer; 4) preparing a water-soluble polymer solution with a mass concentration of 1-20%, and spin coating the water-soluble polymer solution on a glass sheet or a silicon wafer substrate to obtain a polymer-coated substrate; 5) laminating the drug-carrying layer of the mold with the cell adhesion layer and the drug-carrying layer to the substrate coated with the water-soluble polymer, applying pressure to make the mold surface and the substrate completely contact, and removing the mold; 6) Soaking the substrate obtained in step 5) in an aqueous solution for 1-100 minutes, removing the substrate with the assistance of ultrasound, separating the micro-nano patch, and collecting the micro-nano patch by centrifugation to obtain a degradable cell micro-nano patch.

3. The preparation method according to claim 2, characterized in that: In step 1), the cytotropic molecule is selected from one or more of laminin, polylysine, RGD polypeptide, sodium polystyrene sulfonate, and polyallylamine hydrochloride.

4. The preparation method according to claim 2, characterized in that: The mold surface structure is in the shape of a cylinder, triangular prism, tetrahedron, hexagonal prism, or octagonal prism; the diameter or side length of the mold surface array structure unit is between 100 nanometers and 100 micrometers, and the height is between 10 nanometers and 10 micrometers. The mold component is PDMS or PMMA, and the cell adhesion layer is 1-20 layers.

5. The preparation method according to claim 2, characterized in that: In step 2), the degradable biomaterial is selected from one or more of polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), chitosan, methacrylated hydrogel, alginate, methacrylated hyaluronic acid, and methacrylated gelatin.

6. The preparation method according to claim 2, characterized in that: In step 2), the solvent is selected from one or more of water, methanol, ethanol, dichloromethane, acetone, dimethylformamide or dimethylacetamide, the biochemical factor is selected from one or more of differentiation-promoting drugs retinoic acid, anti-hypertensive acid, neurotrophic factor, bone morphogenetic protein, fibroblast factor, ginkgolide B, ganglioside, baicalein or anti-inflammatory drug curcumin, and the concentration of the biochemical factor in the drug-carrying layer precursor solution is 0.1-5%.

7. The preparation method according to claim 2, characterized in that: In step 3), the curing is natural volatilization, light curing or thermal curing, and the drug-carrying layer is 1-80 layers.

8. The preparation method according to claim 2, characterized in that: In step 4), the water-soluble polymer is polyvinyl alcohol or polyvinyl pyrrolidone. In step 5), the applied pressure is 0.1-100 MPa and the application time is 1-60 minutes.

9. The use of the degradable cell micro-nano patch according to claim 1 in the preparation of stem cell therapeutic agents and tissue repair agents, the specific application method is as follows: (1) incubating the above-mentioned degradable cell micro-nano patch and stem cells at a mass ratio of 1:2-20:1 at 4-37° C. for 10-200 minutes, so that the micro-nano patch is adsorbed on the surface of the stem cells; (2) Centrifuging at 500-2000 rpm to collect stem cells adsorbed to the micro-nano patch, and resuspending them in a culture medium to obtain a stem cell therapeutic agent or a tissue repair agent.

10. The use according to claim 9, characterized in that: In step (1), the stem cells include neural stem cells, adipose stem cells, mesenchymal stem cells, induced pluripotent stem cells, and umbilical cord blood stem cells.

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