Nanoscale natural extracellular matrix material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a nanoscale natural extracellular matrix material as well as a preparation method and application thereof. The invention provides a nano-scale natural extracellular matrix material which is prepared by the following steps: ball-milling a freeze-dried natural extracellular matrix material, performing enzyme digestion and centrifugation, and taking supernate, and the centrifugal force of the centrifugation is 6000 to 21000 g. According to the invention, a preparation process is optimized, and the nano-scale small intestinal submucosa material is prepared and has good solubility; the release content of active ingredients such as growth factors can be remarkably increased, the active ingredients can continuously act in vivo, repair and regeneration of focuses are promoted, and the problem that the active ingredients in a traditional SIS material are insufficiently released is solved. The material can be used for safe and effective perfusion treatment of cavity organ inflammation. The preparation method is simple, can be popularized to other extracellular matrix materials, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a nano-scale natural extracellular matrix material and a preparation method and application thereof. Background Art

[0002] Hollow organs have lumens or cavities that allow the flow, storage or exchange of substances. Drug lavage is an important method for treating inflammation of hollow organs, but some lavage fluids may cause allergic reactions or other side effects, especially when the fluid contains drug ingredients. For example, 5-aminosalicylic acid, a commonly used enema drug for the treatment of colitis in clinical practice, can cause adverse symptoms including diarrhea, headache, nausea, vomiting, rash, etc. with long-term use, and is likely to induce chronic hepatitis and chronic tubulointerstitial nephritis. Therefore, it is necessary to develop a lavage drug with high biosafety.

[0003] Natural extracellular matrix (ECM) materials have significant advantages in anti-inflammatory aspects. On the one hand, after decellularization, ECM materials have low immunogenicity and are not likely to cause immune rejection reactions in the host; their composition and structure are similar to the natural ECM of the human body, which can promote cell adhesion, growth and functional expression. On the other hand, the bioactive components of ECM materials, such as growth factors and anti-inflammatory factors, can promote tissue repair and regeneration, regulate immune function, etc. These advantages make ECM materials have broad application potential in anti-inflammatory and tissue repair.

[0004] Small intestinal submucosa (SIS) is an ECM material that has been reported to be used in a variety of applications such as tissue repair and anti-inflammatory. However, traditional SIS materials have two problems when used for lavage drugs to treat inflammation of hollow organs: (1) Poor solubility; SIS is mainly composed of macromolecular components (such as collagen, elastin, glycosaminoglycans, etc.), with high molecular weight and complex cross-linked structure, and is usually not easy to dissolve quickly in water or other solvents. (2) Insufficient release of active ingredients such as growth factors; solubility problems make it difficult for active ingredients such as growth factors and cytokines in SIS materials to be effectively released, difficult to form sufficient concentrations at the treatment site, and difficult to achieve effective enrichment, affecting the effect of tissue repair and regeneration. Perfusion therapy usually requires efficient and rapid delivery of soluble factors and drugs. The physical properties of SIS limit its application effect in perfusion therapy of hollow organs. Therefore, the existing SIS is not suitable for perfusion therapy of hollow organ diseases such as colitis.

[0005] In summary, developing an ECM material with excellent solubility to achieve safe and effective perfusion treatment of hollow organ inflammation is a challenge facing this field. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a nano-scale natural extracellular matrix material, aiming to improve the solubility of ECM and achieve safe and effective perfusion treatment of cavity organ inflammation.

[0007] The present invention provides a nano-scale natural extracellular matrix material, which is prepared by a method comprising the following steps:

[0008] Step 1, ball milling the freeze-dried natural extracellular matrix material;

[0009] Step 2, the ball-milled material is digested with enzymes and centrifuged, and the supernatant is obtained;

[0010] The centrifugal force of the centrifugation is 6000-21000g.

[0011] Preferably, the centrifugal force of the centrifugation is 15000g.

[0012] Preferably, the enzymatic digestion is performed using a hydrochloric acid-pepsin solution with a pH of 1 to 3 at 30 to 40° C. for 24 to 72 hours;

[0013] And / or, the pH needs to be adjusted to 7-9 after the enzyme digestion; and / or, the centrifugation time is 45-60 min; and / or, the ball milling conditions are: ball milling 3-5 times at a power of 30-45 Hz, each time for 5-10 min.

[0014] Preferably, the natural extracellular matrix material is small intestinal submucosa material.

[0015] Preferably, the freeze-dried natural extracellular matrix material in step 1 is obtained by freeze-drying the small intestine of an animal after washing, mechanical treatment, defatting, digestion, and decellularization.

[0016] Preferably, the mechanical treatment includes mechanically scraping the serosa, mucosa and muscularis tissue;

[0017] and / or, the degreasing is carried out in a solution of methanol and chloroform mixed in a volume ratio of 1:1 for 24 hours;

[0018] and / or, the digestion is carried out in a trypsin solution at 2 to 6° C. for 10 to 15 h;

[0019] And / or, the decellularization is performed by immersing the cells in a protein denaturing detergent solution for 2-6 hours.

[0020] Preferably, the animal source of the small intestinal submucosal material is selected from pigs, cattle and sheep.

[0021] The present invention provides a method for preparing the nano-scale natural extracellular matrix material described in any one of the above items, which comprises the following steps:

[0022] Step 1, ball milling the freeze-dried natural extracellular matrix material;

[0023] Step 2, the ball-milled material is digested with enzymes and centrifuged, and the supernatant is obtained;

[0024] The centrifugal force of the centrifugation is 6000-21000g.

[0025] The present invention provides use of the nano-scale natural extracellular matrix material described in any one of the above items in preparing a tissue repair drug.

[0026] Preferably, the tissue repair drug includes a drug for treating inflammation of hollow organs.

[0027] Preferably, the cavity organ inflammation includes colitis, appendicitis, cholecystitis, gastritis, and enteritis.

[0028] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for the terms in this document apply to the terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art would give them should be given based on the disclosure and context.

[0029] The term "natural extracellular matrix material" refers to processed or unprocessed extracellular matrix materials of natural origin, including small intestinal submucosal material, dermal matrix, pericardial matrix, bladder matrix, liver matrix and adipose tissue matrix.

[0030] The term "tissue repair drug" refers to drugs used to promote the regeneration, repair and functional recovery of damaged tissues. It is not limited to drugs that directly promote tissue regeneration, but also includes drugs that reduce tissue damage through mechanisms such as anti-inflammatory.

[0031] The present invention provides a nano-scale natural extracellular matrix material and a preparation method thereof. The present invention optimizes the preparation process and prepares a nano-scale small intestinal submucosal material, which has good solubility; can significantly increase the release content of active ingredients such as growth factors, these active ingredients can continue to act in the body, promote the repair and regeneration of lesions, and solve the problem of insufficient release of active ingredients in traditional SIS materials; the material also has excellent biocompatibility, can be well combined with human tissues and cells, and reduce immune rejection reactions. At the same time, as a natural biomaterial, compared with conventional drugs, it has fewer side effects and can provide a safer treatment plan. The nano-scale small intestinal submucosal material prepared by the present invention can be used for safe and effective perfusion treatment of hollow organ inflammation. The preparation method of the present invention is simple and convenient, highly scalable, and can be extended to other extracellular matrix materials, with broad clinical application prospects.

[0032] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0033] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are pictures of nano-sized small intestinal submucosa (nSIS) and small intestinal submucosa (SIS) before and after dissolution in saline;

[0035] Figure 2 The absorbance curves of nano-sized small intestinal submucosa (nSIS) and small intestinal submucosa (SIS) dissolved in normal saline;

[0036] Figure 3 The graph shows the ELISA test results of the growth factor content in the nanoscale small intestinal submucosa (nSIS) and small intestinal submucosa (SIS) prepared under different centrifugal forces. DETAILED DESCRIPTION

[0037] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0038] Example 1 A perfusable nanoscale small intestinal submucosal material

[0039] The perfusable nanoscale small intestinal submucosal material of this embodiment is prepared by the following method:

[0040] 1. Preparation of small intestinal submucosa (SIS) materials

[0041] 1) Cleaning and sorting: take the pig small intestine, clean it, and cut it into sections;

[0042] 2) Mechanical scraping: scrape the serosa, mucosa and muscle tissue, soak and clean with saline;

[0043] 3) Degreasing: After washing, dry the pig intestines, place them in a methanol / chloroform mixed solution with a volume ratio of 1:1 for degreasing for 24 hours, and then wash them;

[0044] 4) Digestion: The cleaned pig small intestine was immersed in a 0.25% mass concentration trypsin solution at 4°C for 12 h and then washed;

[0045] 5) Decellularization: Soak the material in 0.5% sodium dodecyl sulfate (SDS) solution for 6 h and wash;

[0046] 6) Freeze drying: Spread out and freeze dry in a freeze dryer at -60°C for 24 hours for later use;

[0047] 7) Ball milling: Cut the freeze-dried material into pieces, put it into a ball mill, and ball mill it three times at a power of 30 Hz, each time for 5 minutes.

[0048] 2. Preparation of nanoscale small intestinal submucosal (nSIS) materials

[0049] 1) Enzyme digestion: The small intestinal submucosal material was placed in a hydrochloric acid-pepsin solution with a pH of 2 and digested in a shaker at 37°C for 48 hours;

[0050] 2) Adjusting pH: Using NaOH solution, adjust the pH of the hydrochloric acid-pepsin solution to 7;

[0051] 3) Centrifugation: centrifuge the digestion solution at 15000g for 45 minutes and collect the supernatant;

[0052] 4) Freeze-drying: Use a freeze dryer at -60°C for 24 hours to obtain nano-scale small intestinal submucosal material.

[0053] The technical solution of the present invention is further described below through experiments. The samples SIS and nSIS tested in the following experimental examples are prepared by the method of Example 1.

[0054] Experimental Example 1 Solubility of Materials

[0055] 1. Experimental Methods

[0056] (1) Take the same mass of SIS and nSIS materials and dissolve them in physiological saline respectively, and observe the dissolution phenomenon.

[0057] (2) Take the same mass of SIS and nSIS materials and dissolve them in physiological saline respectively, and use a UV-visible spectrophotometer to measure the absorbance curve.

[0058] 2. Experimental Results

[0059] Dissolve pictures such as Figure 1 As shown: When dissolved in normal saline, the SIS solution is unevenly white and a lot of undissolved flocculent matter can be observed, while the nSIS solution is transparent and has good light transmittance, and no undissolved matter can be observed with the naked eye.

[0060] The absorbance curve results are as follows Figure 2As shown: compared with the absorbance curve of normal saline, the absorbance curve of SIS dissolved in normal saline deviates far from it, while the absorbance curve of nSIS dissolved in normal saline is close to it, which proves that the solubility of nSIS is greatly improved compared with SIS.

[0061] Experimental Example 2: Release of Growth Factors

[0062] 1. Experimental Methods

[0063] nSIS was prepared according to the method of Example 1, except that the centrifugal forces during centrifugation were 6000g, 9000g, 12000g, 15000g, 18000g and 21000g, respectively. Thus, nSIS prepared under different centrifugal forces were obtained, and the nSIS materials and SIS materials were used to detect the content of growth factors VEGF, FGF-2 and TGF-β released by the materials using ELISA method.

[0064] 2. Experimental Results

[0065] The results are as follows Figure 3 As shown: From the perspective of the content levels of the three growth factors, compared with SIS, the levels of growth factors in nSIS prepared under different centrifugal forces are significantly improved. When the centrifugal force reaches 15000g, the contents of the three growth factors VEGF, FGF-2 and TGF-β all reach a high level, and when the centrifugal force is increased to 18000g or 21000g, the changes in the content of growth factors begin to show a downward trend. This shows that compared with SIS, the content of growth factors released by nSIS prepared under 6000-21000g centrifugal force is increased, and 15000g is the optimal centrifugal condition.

[0066] The above experimental examples show that, compared with natural extracellular matrix materials, the nano-scale small intestinal submucosal material prepared by the present invention has better solubility in aqueous solution, and the release of active ingredients such as growth factors is significantly increased.

[0067] Studies have reported that VEGF and FGF-2 play an important role in tissue repair by promoting angiogenesis, cell proliferation and tissue regeneration in the small intestinal submucosal tissue. Therefore, the nanoscale small intestinal submucosal material prepared by the present invention has a promising future in the preparation of tissue repair drugs. TGF-β is an anti-inflammatory growth factor that can play an immunomodulatory role by regulating the proliferation, differentiation and activation of immune cells. Therefore, the nanoscale small intestinal submucosal material prepared by the present invention has a promising future in the preparation of anti-inflammatory drugs.

[0068] It can be seen from the above embodiments and experimental examples that the present invention provides a nano-scale small intestinal submucosal material and a preparation method thereof. The present invention optimizes the preparation process and prepares a nano-scale small intestinal submucosal material, which has good solubility; it can significantly increase the release content of active ingredients such as growth factors, which can continue to act in the body, promote the repair and regeneration of lesions, and solve the problem of insufficient release of active ingredients in traditional SIS materials; the material also has excellent biocompatibility, can be well combined with human tissues and cells, and reduce immune rejection reactions. At the same time, as a natural biomaterial, compared with conventional drugs, it has fewer side effects and can provide a safer treatment plan. The nano-scale small intestinal submucosal material prepared by the present invention can be used for safe and effective perfusion treatment of hollow organ inflammation. The preparation method of the present invention is simple and convenient, highly scalable, and can be extended to other extracellular matrix materials, with broad clinical application prospects.

Claims

1. A nanoscale natural extracellular matrix material, characterized in that: It is prepared by a method comprising the following steps: Step 1, ball milling the freeze-dried natural extracellular matrix material; Step 2, the ball-milled material is digested with enzymes and centrifuged, and the supernatant is obtained; The centrifugal force of the centrifugation is 6000-21000g.

2. The nanoscale natural extracellular matrix material according to claim 1, characterized in that: The centrifugal force of the centrifugation is 15000g.

3. The nanoscale natural extracellular matrix material according to claim 1, characterized in that: The enzyme digestion is performed using a hydrochloric acid-pepsin solution with a pH of 1 to 3 at 30 to 40° C. for 24 to 72 hours; And / or, the pH needs to be adjusted to 7-9 after the enzyme digestion; and / or, the centrifugation time is 45-60 min; and / or, the ball milling conditions are: ball milling 3-5 times at a power of 30-45 Hz, each time for 5-10 min.

4. The nanoscale natural extracellular matrix material according to claim 1, characterized in that: The natural extracellular matrix material is small intestinal submucosa material.

5. The nanoscale natural extracellular matrix material according to claim 4, characterized in that: The freeze-dried natural extracellular matrix material described in step 1 is obtained by freeze-drying the small intestine of an animal after washing, mechanical treatment, defatting, digestion, and decellularization.

6. The nanoscale natural extracellular matrix material according to claim 5, characterized in that: The mechanical treatment includes mechanically scraping the serosa, mucosa and muscularis tissue; and / or, the degreasing is carried out in a solution of methanol and chloroform mixed in a volume ratio of 1:1 for 24 hours; and / or, the digestion is carried out in a trypsin solution at 2 to 6° C. for 10 to 15 h; And / or, the decellularization is performed by immersing the cells in a protein denaturing detergent solution for 2-6 hours.

7. The nanoscale natural extracellular matrix material according to claim 4, characterized in that: The animal source of the small intestinal submucosal material is selected from pigs, cattle and sheep.

8. The method for preparing the nano-scale natural extracellular matrix material according to any one of claims 1 to 7, characterized in that: The steps include: Step 1, ball milling the freeze-dried natural extracellular matrix material; Step 2, the ball-milled material is digested with enzymes and centrifuged, and the supernatant is obtained; The centrifugal force of the centrifugation is 6000-21000g.

9. Use of the nano-scale natural extracellular matrix material according to any one of claims 1 to 7 in the preparation of tissue repair drugs.

10. The use according to claim 9, characterized in that: The tissue repair drugs include drugs for treating inflammation of hollow organs.

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