A decellularized matrix microparticle, a preparation method and application thereof, and a decellularized matrix microparticle gel material

Abstract

The application discloses a kind of acellular matrix microparticles and preparation method and application thereof, and acellular matrix microparticle gel material, belong to the technical field of biomaterial preparation.The technical problem to be solved is to simplify the preparation process and improve safety.The technical solution points to a kind of preparation method of acellular matrix microparticles, comprising the following steps: step one: preparing acellular matrix;Step two: the acellular matrix is immersed in organic solvent, crosslinking agent is added to crosslink, and crosslinked acellular matrix is obtained;Step three: the crosslinked acellular matrix is immersed in acid solution, and acid protease is added to enzymolysis, filtration, and acellular matrix microparticles are obtained.

Description

Technical Field

[0001] This invention relates to the field of biomaterial preparation technology, specifically to a decellularized matrix microparticle, its preparation method and application, and a decellularized matrix microparticle gel material. Background Technology

[0002] Decellularized extracellular matrix (dECM) is a biomaterial with a microscopic three-dimensional structure and activity obtained by selectively removing cellular components and immunogenic substances from tissues and organs through physical, chemical, and biological methods. In applications, decellularized matrix has become a cornerstone material for tissue engineering and regenerative medicine, and is widely used in dermal repair, hernia repair, ligament reconstruction, and other fields.

[0003] Decellularized matrix microparticles are micron- to nano-sized particulate materials obtained by processing decellularized matrix through certain methods. This morphological change expands the application scenarios of decellularized matrix, and its main characteristics are: (1) Micro- and nano-sized microparticles greatly increase the specific surface area of ​​the material, fully retain active sites, and enhance the interaction with cells. (2) The microparticle form of decellularized matrix becomes an injectable material, which can be precisely delivered to irregular or deep tissue defect areas through minimally invasive methods (such as needle injection). (3) Decellularized matrix microparticles can serve as drug carriers to achieve controlled release of active molecules and synergistically promote tissue repair.

[0004] Currently, the main preparation processes for decellularized matrix microparticles include grinding and emulsification cross-linking. Among them, grinding is further divided into direct grinding and cryogenic grinding.

[0005] The direct grinding method mainly involves directly grinding the decellularized matrix using equipment such as homogenizers or colloid mills. The decellularized matrix particles obtained by this method tend to have a larger particle size.

[0006] Relevant patent documents retrieved: The document, published in China (CN113769168A) on December 10, 2021, discloses a method for preparing decellularized matrix microparticle products for soft tissue filling and repair, wherein a homogenizing device is used to directly homogenize the decellularized matrix.

[0007] The document, published in China (CN120242158A) on July 4, 2025, discloses a method for preparing decellularized dermal matrix microparticles, wherein the decellularized matrix is ​​directly ground using a colloid mill.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) The particle size is relatively large. The relevant evidence is that the average particle size of the decellularized matrix microparticles prepared in CN113769168A is 500 μm. The D50 of the decellularized matrix microparticles obtained by colloid milling in CN120242158A is about 200 μm.

[0009] (2) Poor patient experience and easy to cause adverse reactions: Acellular matrix microparticles with an average particle size of more than 200 μm require the use of thicker needles during injection, causing greater pain to patients. Moreover, large-sized acellular matrix microparticles are more likely to cause adverse reactions such as nodules and granulomas.

[0010] (3) High requirements for raw materials: Direct grinding method is only suitable for relatively hard decellularized materials such as decellularized dermis. It is not easy to crush relatively soft materials such as decellularized submucosa of small intestine and decellularized bladder basement membrane into microparticles.

[0011] The cryo-grinding method involves freezing and embrittlement of decellularized matrix in a low-temperature environment (such as liquid nitrogen), followed by mechanical impact and grinding, and then sieving to obtain decellularized matrix microparticles.

[0012] Relevant patent documents retrieved: The document, published in China (CN116672503A) on September 1, 2023, discloses a method for preparing bioactive decellularized matrix microparticles. The method involves freezing a decellularized matrix in liquid nitrogen and then ball milling it in a cold trap circulating medium at a temperature below -50°C to obtain decellularized matrix microparticles.

[0013] The document, published in China (CN120267899A) on July 8, 2025, discloses a process for preparing decellularized matrix microparticles, wherein the granulation process is completed by grinding with a low-temperature pulverizer.

[0014] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Although cryo-grinding is a commonly used method, it has the following disadvantages: (1) it requires a large amount of cooling medium or energy; (2) the efficiency of preparing microparticles is low and it is difficult to produce on a large scale; (3) the impact and grinding process can easily cause compression damage to the structure of the decellularized matrix.

[0015] The emulsification crosslinking method involves converting the decellularized matrix into a solution state, emulsifying it in the oil phase to form microdroplets, and then crosslinking and solidifying it to obtain decellularized matrix microparticles.

[0016] Relevant patent documents retrieved: The document, published in China (CN116492510A) on July 28, 2023, discloses a tissue filling and repair material based on decellularized matrix microspheres. The decellularized matrix microspheres are prepared by dissolving a decellularized matrix as an aqueous phase, adding it to an oil phase for emulsification, and then cross-linking and curing.

[0017] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The disadvantage of the emulsification crosslinking method is that it introduces a large amount of oily organic solvents and emulsifiers. These substances are not easy to remove completely, resulting in residues in the decellularized matrix microspheres, which affects the safety of the material. Summary of the Invention

[0018] The purpose of this invention is to provide: A method for preparing decellularized matrix microparticles, and related technologies, to solve technical problems such as simplifying the preparation process and improving safety, or a combination thereof.

[0019] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0020] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0021] The definition of the standard chemical term can be found in the reference "Feng Xiangsheng, Pan Yingen. Clinical Application and Basic Research of Xenogeneic Acellular Dermal Matrix [M]. People's Medical Publishing House, 2011".

[0022] Unless otherwise stated, conventional methods within the scope of the art, such as methods for preparing decellularized matrix, shall be used.

[0023] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0024] In a first aspect, the present invention provides a method for preparing decellularized matrix microparticles, comprising the following steps: Step 1: Preparation of decellularized matrix; Step 2: Immerse the decellularized matrix in an organic solvent, add a cross-linking agent to cross-link it, and obtain a cross-linked decellularized matrix; Step 3: Immerse the cross-linked decellularized matrix in an acidic solution, add acidic protease for enzymatic hydrolysis, and filter to obtain decellularized matrix microparticles.

[0025] Step one includes the specific preparation steps of the decellularized matrix.

[0026] The specific preparation steps for the decellularized matrix are selected from: obtaining mammalian tissue by decellularization followed by freeze-drying.

[0027] The preferred specific steps for preparing the decellularized matrix are as follows: (1-1) Take fresh mammalian tissue, clean it, remove the fascia, lymph nodes and large pieces of fat, and cut it into small pieces; (1-2) Use organic solvents to further remove fat; (1-3) Perform virus inactivation procedures; (1-4) Perform decellularization treatment; (1-5) Freeze-dry to obtain the product.

[0028] This includes: organic solvents, virus inactivation, decellularization treatment, etc.

[0029] The organic solvent is selected from ethyl acetate.

[0030] Among them, virus inactivation is selected from: virus inactivation by soaking in peracetic acid solution.

[0031] Among them, decellularization treatment is selected from: decellularization treatment using enzymes.

[0032] The enzymes are preferably trypsin, DNA enzymes, and RNA enzymes.

[0033] The preferred method for decellularization is to first use trypsin for decellularization, followed by decellularization using DNase and RNase.

[0034] Step two includes: the composition of the cross-linking agent, the concentration of the cross-linking agent, cross-linking, the composition of the organic solvent, the amount of organic solvent used, and the post-treatment steps of the cross-linked decellularized matrix.

[0035] The crosslinking agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2-chloro-1-methylpyridinium iodide (CMPI), and N,N'-carbonyldiimidazole (CDI).

[0036] The concentration of the crosslinking agent is selected from 20-200 μmol / L in the organic solvent.

[0037] The crosslinking is selected from the following: the crosslinking time is 10-30 min.

[0038] The organic solvent is selected from at least one of anhydrous ethanol, n-propanol, and isopropanol. Using at least one of anhydrous ethanol, n-propanol, and isopropanol in the crosslinking process can achieve the desired crosslinking effect and obtain decellularized matrix microparticles within the desired particle size range.

[0039] The preferred composition of the organic solvent is anhydrous ethanol.

[0040] The amount of organic solvent used is selected from the mass-volume ratio of decellularized matrix to organic solvent as 1g: 50-200mL.

[0041] The post-processing step of the cross-linked decellularized matrix is ​​selected from: transferring the cross-linked decellularized matrix into an acidic aqueous solution, filtering it, washing it with pure water, and using the washed cross-linked decellularized matrix in step three.

[0042] Acidic aqueous solution is used to terminate the crosslinking reaction, and pure water is used to wash away excess crosslinking agent.

[0043] The acidic aqueous solution is selected from at least one of hydrochloric acid solution and acetic acid solution.

[0044] Among them, the acidic aqueous solution is selected from those with a pH of 1.5-3.0.

[0045] The volume ratio of the acidic aqueous solution to the organic solvent is 1-4:1.

[0046] Step three includes: the composition of the acidic solution and acidic protease, the amount of acidic protease used, the enzymatic hydrolysis process, the filtration step, the pretreatment process of the cross-linked decellularized matrix, and the post-treatment process after filtration.

[0047] The acidic solution is selected from at least one of hydrochloric acid solution and acetic acid solution.

[0048] Among them, the acidic solution is selected from the acidic solution with a pH of 1.5-3.0.

[0049] The acidic protease is composed of pepsin.

[0050] The amount of acidic protease used is selected from a concentration of 0.5-5 mg / mL in acidic solution. The enzyme activity of the acidic protease is preferably 1200-3000 U / g.

[0051] The enzymatic hydrolysis process is selected from the following conditions: the hydrolysis temperature is 20-25℃ (room temperature), and the hydrolysis time is 24-120h.

[0052] The filtration steps are selected from the following: first, filtering with a filter membrane with a pore size of 100-500μm and collecting the filtered material; then filtering with a filter membrane with a pore size of 1-5μm and collecting the unfiltered material.

[0053] The pretreatment process of the cross-linked decellularized matrix is ​​selected from the following: first, the cross-linked decellularized matrix is ​​cut, and then immersed in an acidic solution, wherein the cut size is no greater than 5mm×5mm.

[0054] The post-treatment process after filtration is selected from: adding an alkaline solution for neutralization after filtration, and then adding pure water for washing.

[0055] The alkaline solution is a sodium hydroxide solution and / or a disodium hydrogen phosphate solution.

[0056] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: A method for preparing decellularized matrix microparticles includes the following steps: Step 1: Preparation of decellularized matrix; Step 2: Immerse the decellularized matrix in an organic solvent, add a cross-linking agent to cross-link it, and obtain a cross-linked decellularized matrix; the concentration of the cross-linking agent in the organic solvent is 20-200 μmol / L; Step 3: Immerse the cross-linked decellularized matrix in an acidic solution, add acidic protease for enzymatic hydrolysis, and filter to obtain decellularized matrix microparticles.

[0057] This technical solution not only solved the technical problems of "simplifying the preparation process and improving safety", but also further solved the technical problem of "increasing the yield of decellularized matrix microparticles".

[0058] Secondly, the present invention provides: a decellularized matrix microparticle, prepared by the above-described preparation method.

[0059] The decellularized matrix microparticles have a particle size of 5-45 μm.

[0060] Thirdly, the present invention provides the application of the above-mentioned decellularized matrix microparticles in the preparation of gel materials.

[0061] Fourthly, the present invention provides: a decellularized matrix microparticle gel material, obtained by gelling the aforementioned decellularized matrix microparticles, or obtained by mixing the aforementioned decellularized matrix microparticles with additives.

[0062] The gelation conditions are as follows: decellularized matrix microparticles are dispersed in a solution with a pH of 2.0-4.0, the concentration of decellularized matrix microparticles is ≥5.0 mg / mL, and then the pH of the solution is adjusted to 6.0-8.0 to obtain the decellularized matrix microparticle gel material.

[0063] The preferred concentration of decellularized matrix microparticles is 5-100 mg / mL. If the concentration of decellularized matrix microparticles exceeds 100 mg / mL, the microparticles cannot be uniformly dispersed, but a gel can still be formed.

[0064] The additives include a solution and / or gel of at least one of sodium hyaluronate, carboxymethyl cellulose, carboxymethyl chitosan, and collagen.

[0065] The mass ratio of the additive to the decellularized matrix microparticles is 10:1 to 1000:1.

[0066] The decellularized matrix microparticle gel material of the present invention exhibits microscopic aggregation of decellularized matrix microparticles and macroscopic formation of gel.

[0067] In this invention, Examples 1-6 at least support the protection scope of the technical solution "A method for preparing decellularized matrix microparticles, comprising the following steps: Step 1: preparing a decellularized matrix; Step 2: immersing the decellularized matrix in an organic solvent, adding a crosslinking agent for crosslinking, to obtain a crosslinked decellularized matrix; Step 3: immersing the crosslinked decellularized matrix in an acidic solution, adding an acidic protease for enzymatic hydrolysis, filtering, and thus obtaining decellularized matrix microparticles".

[0068] The term "organic solvent" is derived from the foregoing explanation and / or the common characteristic of anhydrous ethanol in Examples 1-6. Therefore, those skilled in the art can reasonably infer that organic solvents, their subordinate concepts, essentially equivalent technical means, and technical means that can replace organic solvents within the scope of conventional and common knowledge based on existing technology should all fall within the protection scope of the above-mentioned technical solutions. For example, replacing the organic solvent with isopropanol, n-propanol, etc., while keeping everything else unchanged, still falls within the protection scope of the above-mentioned technical solutions of this invention.

[0069] The term "crosslinking agent" is a generalization derived from the common characteristics of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2-chloro-1-methylpyridinium iodide (CMPI), and N,N'-carbonyldiimidazole (CDI) as explained above and / or in Examples 1-6. Therefore, those skilled in the art can reasonably infer that the crosslinking agent, its subordinate concepts, the essentially equivalent technical means, and the technical means that can replace the crosslinking agent within the scope of conventional and common knowledge based on existing technology should all fall within the protection scope of the above-mentioned technical solutions. For example, replacing the crosslinking agent with glutaraldehyde, genipor, etc., while keeping everything else unchanged, still falls within the protection scope of the above-mentioned technical solutions of this invention.

[0070] The term "acidic solution" is derived from the aforementioned explanation and / or the common characteristic of "acidic solution" in the hydrochloric acid solution, acetic acid solution, etc., described in Examples 1-6. Therefore, those skilled in the art can reasonably infer that acidic solution, its subordinate concept, the technical means essentially equivalent to acidic solution, and technical means that can replace acidic solution within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of the above-mentioned technical solutions. For example, replacing acidic solution with phosphoric acid solution, citric acid solution, etc., while keeping everything else unchanged, still falls within the protection scope of the above-mentioned technical solutions of this invention.

[0071] The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has better technical effects in terms of equipment requirements, production costs, safety, and particle size controllability.

[0072] The method used in this invention does not involve a grinding process, does not require special equipment or a large amount of cooling medium, and can achieve large-scale, mass production under the condition of using conventional equipment. It has lower equipment requirements and reduces production costs.

[0073] The method used in this invention does not introduce oily organic solvents and emulsifiers that are difficult to remove, thus avoiding the adverse effects that these substances may cause and making it safer.

[0074] The method used in this invention can control the degree of cross-linking of the cross-linked decellularized matrix, thereby regulating the average particle size of the resulting decellularized matrix microparticles to a certain extent and meeting different application requirements.

[0075] The decellularized matrix microparticles obtained by this invention have a small particle size, making them suitable for injection with extremely fine needles and improving the patient's experience.

[0076] 2. Compared with the prior art, the present invention provides a technical solution with a different technical concept. The difference between the technical concept of the present invention and the prior art includes, but is not limited to, preparing decellularized matrix microparticles by enzymatic hydrolysis after cross-linking the decellularized matrix.

[0077] By further optimizing the cross-linking and enzymatic hydrolysis steps, this invention successfully prepared decellularized matrix microparticles with an average particle size of less than 25 μm, and the yield of the prepared decellularized matrix microparticles was further improved. Attached Figure Description

[0078] Figure 1 This is a photograph of the decellularized matrix microparticles obtained in Example 2 dispersed in an aqueous solution.

[0079] Figure 2 This is a microscope image of the decellularized matrix microparticles obtained in Example 2.

[0080] Figure 3 This is a microscope image of the decellularized matrix microparticles obtained in Example 3.

[0081] Figure 4 This is a microscope image of the decellularized matrix microparticles obtained in Example 4.

[0082] Figure 5 This is a microscope image of the decellularized matrix microparticles obtained in Example 5.

[0083] Figure 6 This is a microscope image of the decellularized matrix microparticles obtained in Example 6.

[0084] Figure 7 This is a photograph of the decellularized matrix microparticles obtained in Example 7.

[0085] Figure 8 This is a microscope image of the aggregated decellularized matrix particles obtained in Example 7.

[0086] Figure 9 This is a photograph of the decellularized matrix that was not completely micronized, as shown in Comparative Example 1. Detailed Implementation

[0087] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0088] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0089] In the following examples, EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0090] Example 1 Preparation of decellularized porcine small intestinal submucosa: Fresh pig small intestine (approximately 4 kg) was collected, cleaned, and the submucosa of the small intestine was separated using a scraper. Fascia, lymph nodes, and large pieces of adipose tissue were then scraped off, leaving approximately 1.3 kg. This was cut into small segments of about 5 cm. After rinsing with pure water, the segments were soaked in ethyl acetate and shaken for 120 min for further defatting. After rinsing with pure water, the segments were soaked in 0.1% peracetic acid solution and shaken for 60 min for virus inactivation. After rinsing with pure water, the segments were soaked in phosphate buffer (pH=7.4) containing 0.25% trypsin and shaken for 60 min for decellularization. After rinsing with pure water, the segments were soaked in phosphate buffer (pH=7.4) containing 4 U / mL DNase and 1 U / mL RNase and shaken for 8 h for further decellularization. After rinsing with pure water, the segments were freeze-dried to obtain 117.6 g of decellularized pig small intestinal submucosa.

[0091] Example 2 Cross-linking of decellularized porcine small intestinal submucosa: Take 5.0 g of the decellularized porcine small intestinal submucosa obtained in Example 1, soak it in 250 mL of anhydrous ethanol, add 9.6 mg (50 μmol) of EDC, shake for 10 min, filter and collect the cross-linked decellularized porcine small intestinal submucosa, transfer it to 1000 mL of acetic acid solution with pH 2.5, filter and collect the cross-linked decellularized porcine small intestinal submucosa again, and then wash it 5 times with pure water. The final cross-linked decellularized porcine small intestinal submucosa is still a small segment with a length of about 5 cm.

[0092] Cross-linked decellularized porcine small intestinal submucosa enzymatic microparticles: The cross-linked decellularized porcine small intestinal submucosa obtained after washing with pure water was cut into small pieces with an area of ​​approximately 3 mm × 3 mm. These pieces were then soaked in 1000 mL of acetic acid solution with a pH of 2.5, and pepsin (enzyme activity 1200 U / g) was added to a concentration of 2 mg / mL. The mixture was then enzymatically hydrolyzed at room temperature for 120 h. The hydrolysate was filtered through a 50-mesh filter, and the filtered material was collected. The filtered material was then filtered again through a 5 μm pore size filter membrane, and the unfiltered material was collected. The pH was adjusted to 7.0 with 1 mol / L sodium hydroxide solution, and the mixture was washed with pure water to obtain decellularized porcine small intestinal submucosa microparticles.

[0093] like Figure 1 The image shown is a macroscopic photograph of the decellularized porcine small intestinal submucosal microparticles dispersed in an aqueous solution. The morphology of the decellularized porcine small intestinal submucosal microparticles observed under a microscope is as follows: Figure 2 As shown. The particle size was measured using ImageJ software under three different microscope fields of view. The minimum value was 5.5 μm, the maximum value was 44.8 μm, and the average value was 24.6 μm.

[0094] Example 3 Cross-linking of decellularized porcine small intestinal submucosa: Take 5.0 g of the decellularized porcine small intestinal submucosa obtained in Example 1, soak it in 1000 mL of anhydrous ethanol, add 19.2 mg (100 μmol) of EDC, shake for 10 min, filter and collect the cross-linked decellularized porcine small intestinal submucosa, transfer it to 4000 mL of hydrochloric acid solution with pH 1.5, filter and collect the cross-linked decellularized porcine small intestinal submucosa again, and then wash it 5 times with pure water.

[0095] Cross-linked decellularized porcine small intestinal submucosa enzymatic microparticles: The cross-linked decellularized porcine small intestinal submucosa obtained after washing with pure water was cut into small pieces with an area of ​​approximately 3 mm × 3 mm. These pieces were then immersed in 1000 mL of acetic acid solution with a pH of 3.0, and pepsin (enzyme activity 3000 U / g) was added to a concentration of 5 mg / mL. The mixture was then enzymatically hydrolyzed at room temperature for 72 h. The hydrolysate was filtered through a 50-mesh filter, and the filtered material was collected. This filtered material was then further filtered through a 5 μm pore size filter membrane, and the unfiltered material was collected. The pH was adjusted to 7.0 with 1 mol / L sodium hydroxide solution, and the mixture was washed with pure water to obtain decellularized porcine small intestinal submucosa microparticles.

[0096] The morphology of decellularized porcine small intestinal submucosa microparticles observed under a microscope is as follows: Figure 3 As shown. The particle size was measured using ImageJ software under three different microscope fields of view. The minimum value was 5.0 μm, the maximum value was 36.2 μm, and the average value was 19.8 μm.

[0097] Example 4 Cross-linking of decellularized porcine small intestinal submucosa: Take 5.0 g of the decellularized porcine small intestinal submucosa obtained in Example 1, soak it in 1000 mL of anhydrous ethanol, add 3.8 mg (19.8 μmol) of EDC, shake and react for 30 min, filter and collect the cross-linked decellularized porcine small intestinal submucosa, transfer it to 4000 mL of acetic acid solution with pH 3.0, filter and collect the cross-linked decellularized porcine small intestinal submucosa again, and then wash it 5 times with pure water.

[0098] Cross-linked decellularized porcine small intestinal submucosa enzymatic microparticleization: The cross-linked decellularized porcine small intestinal submucosa obtained after washing with pure water was cut into small pieces with an area of ​​approximately 3 mm × 3 mm. These pieces were then soaked in 1000 mL of hydrochloric acid solution with a pH of 1.5, and pepsin (enzyme activity 3000 U / g) was added to a concentration of 2 mg / mL. The mixture was then enzymatically hydrolyzed at room temperature for 72 h. The hydrolysate was filtered through a 50-mesh filter, and the filtered material was collected. The filtered material was then filtered again through a 5 μm pore size filter membrane, and the unfiltered material was collected. The pH was adjusted to 7.0 with 1 mol / L sodium hydroxide solution, and the mixture was washed with pure water to obtain decellularized porcine small intestinal submucosa microparticles.

[0099] The morphology of decellularized porcine small intestinal submucosa microparticles observed under a microscope is as follows: Figure 4 As shown, the particle size was measured using ImageJ software under three different microscope fields of view. The minimum value was 5.0 μm, the maximum value was 27.6 μm, and the average value was 11.2 μm.

[0100] Example 5 Referring to Example 3, the cross-linking agent EDC was replaced with an equimolar amount of 2-chloro-1-methylpyridinium iodide (CMPI), while all other conditions remained unchanged. The morphology of the decellularized porcine small intestinal submucosa microparticles observed under a microscope is as follows. Figure 5 As shown. The particle size was measured using ImageJ software under three different microscope fields of view. The minimum value was 5.8 μm, the maximum value was 43.5 μm, and the average value was 17.3 μm.

[0101] Example 6 Referring to Example 3, the crosslinking agent EDC was replaced with an equimolar amount of N,N'-carbonyldiimidazole (CDI), while all other conditions remained unchanged. The morphology of the decellularized porcine small intestinal submucosa microparticles observed under a microscope is as follows. Figure 6 As shown. The particle size was measured using ImageJ software under three different microscope fields of view. The minimum value was 5.0 μm, the maximum value was 22.8 μm, and the average value was 9.5 μm.

[0102] Example 7 The decellularized porcine small intestinal submucosa microparticles obtained in Example 3 were lyophilized, and 50 mg was weighed and dispersed in 10 mL of hydrochloric acid solution with a pH of 3.0. The pH was adjusted to 7.0 with sodium hydroxide solution, and a gel was formed after standing at room temperature (e.g., ...). Figure 7 As shown), the aggregation of microparticles in the submucosa of decellularized porcine small intestine can be observed under a microscope (e.g. Figure 8 (As shown).

[0103] Example 8 The decellularized porcine small intestinal submucosal microparticles obtained in Examples 2-6 were lyophilized, and 150 mg of each was weighed and dispersed in 5 mL of a 5 mg / mL sodium hyaluronate solution (molecular weight 1.1 million). After centrifugation sedimentation testing (3000 rpm, 30 min), no sedimentation occurred in any of the samples. This indicates that the decellularized matrix microparticles obtained in this invention have good suspension properties in sodium hyaluronate solutions of conventional concentrations used in hyaluronic acid injections.

[0104] Example 9 The samples obtained in Example 8 were respectively filled into 1mL pre-filled syringes, fitted with 34G needles (inner diameter 0.06-0.08mm), and injected at a speed of 30mm / min using a universal testing machine (Shenzhen Sansi Zongheng). The average pushing force of the samples of decellularized porcine small intestinal submucosal microparticles and sodium hyaluronate solution obtained in each example was measured as follows: Example 2: 11.87N; Example 3: 8.32N; Example 4: 9.65N; Example 5: 10.26N; Example 6: 8.91N. The above pushing forces are all within the range of easy injection. This indicates that the decellularized matrix microparticles obtained by this invention can be smoothly injected using the finest needles, effectively reducing the patient's pain, and can also be applied to injections in areas with thin skin.

[0105] Comparative Example 1 Referring to Example 2, the difference from Example 2 is that the amount of EDC added is 19.2 mg; after enzymatic hydrolysis, it was found that the decellularized matrix was not completely micronized, and a large number of flake-like substances were present, such as... Figure 9 As shown.

[0106] Similarly, based on Example 2, the only difference from Example 2 was the amount of EDC added. When the amount of EDC added was 14.4 mg, it was found after enzymatic hydrolysis that the decellularized matrix could not be completely micronized, and flocculent matter was obtained.

[0107] Comparative Example 2 Referring to Example 4, the difference from Example 4 is that the amount of EDC added is 1.0 mg; after enzymatic hydrolysis, when filtered using a 5 μm pore size filter membrane, almost no unfiltered material was collected, and decellularized matrix microparticles were not obtained.

[0108] Based on Example 4, the only difference from Example 4 is the amount of EDC added. When the amount of EDC added is 1.9 mg (10 μmol), after enzymatic hydrolysis, the unfiltered material collected after filtration using a 5 μm pore size membrane is only about 20% of the unfiltered material collected in Example 4.

[0109] Comparative Example 3 Referring to Example 2, the difference from Example 2 is that the crosslinking solvent, anhydrous ethanol, was replaced with an equal volume of purified water. After enzymatic hydrolysis, it was found that the decellularized matrix was not completely micronized, and a large number of flakes remained. This indicates that when using purified water as the crosslinking solvent, the degree of crosslinking cannot be controlled. The reason is that after reaching the set crosslinking time, even after the crosslinked decellularized matrix is ​​removed from the water, a large amount of crosslinking agent remains in the water-saturated decellularized matrix. The crosslinking reaction is still actually in progress, making it impossible to control the degree of crosslinking, which in turn leads to an excessively high degree of crosslinking and prevents micronization.

[0110] Comparative Example 4 The difference from Example 2 lies in adjusting the order of cross-linking and enzymatic hydrolysis, as follows: Take 5.0g of decellularized porcine small intestinal submucosa obtained in Example 1, cut it into small pieces with an area of ​​approximately 3mm × 3mm, then soak it in 1000mL of acetic acid solution with pH 2.5, add pepsin (enzyme activity 1200U / g) to a concentration of 2mg / mL, and enzymatically hydrolyze at room temperature for 120h; concentrate the enzymatic hydrolysis product to 200mL by ultrafiltration using a 50kDa ultrafiltration membrane and then freeze-dry it.

[0111] The lyophilized product was immersed in 250 mL of anhydrous ethanol, 9.6 mg of EDC was added, and the mixture was shaken for 10 min. The cross-linked product was collected by filtration and transferred to 1000 mL of acetic acid solution with pH 2.5. At this point, the sample was found to be in a blocky gel state, and no microparticles could be obtained.

[0112] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing decellularized matrix microparticles, characterized in that, Includes the following steps: Step 1: Preparation of decellularized matrix; Step 2: Immerse the decellularized matrix in an organic solvent, add a cross-linking agent to cross-link it, the concentration of the cross-linking agent in the organic solvent is 20-200 μmol / L, to obtain the cross-linked decellularized matrix; Step 3: Immerse the cross-linked decellularized matrix in an acidic solution, add acidic protease for enzymatic hydrolysis, and filter to obtain decellularized matrix microparticles; The crosslinking agent mentioned in step two includes at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2-chloro-1-methylpyridinium iodide, and N,N'-carbonyldiimidazole; The crosslinking time in step two is 10-30 minutes; The mass-to-volume ratio of the decellularized matrix to the organic solvent in step two is 1g: 50-200mL; Step two also includes transferring the cross-linked decellularized matrix into an acidic aqueous solution, filtering it, washing it with pure water, and using the washed cross-linked decellularized matrix in step three.

2. The preparation method according to claim 1, characterized in that, The organic solvent mentioned in step two includes at least one of anhydrous ethanol, n-propanol, and isopropanol.

3. The preparation method according to claim 1, characterized in that, The acidic aqueous solution includes at least one of hydrochloric acid solution and acetic acid solution; and / or the pH of the acidic aqueous solution is 1.5-3.0; and / or the volume ratio of the acidic aqueous solution to the organic solvent is 1-4:

1.

4. The preparation method according to claim 1, characterized in that, In step three, the acidic solution includes at least one of hydrochloric acid solution and acetic acid solution; And / or the pH of the acidic solution is 1.5-3.0; And / or the enzymatic hydrolysis process is as follows: the enzymatic hydrolysis temperature is 20-25℃, and the enzymatic hydrolysis time is 24-120h; And / or the filtration process involves first using a filter membrane with a pore size of 100-500 μm to filter and collect the filtered material; then using a filter membrane with a pore size of 1-5 μm to filter and collect the unfiltered material. And / or in step three, the cross-linked decellularized matrix is ​​first cut and then immersed in an acidic solution, wherein the cut size is no greater than 5mm × 5mm; And / or after filtration in step three, add an alkaline solution for neutralization, and then add pure water for rinsing; The alkaline solution is a sodium hydroxide solution and / or a disodium hydrogen phosphate solution; And / or the acidic protease includes pepsin; And / or the enzyme activity of the acidic protease is 1200-3000 U / g and / or the concentration of the acidic protease in acidic solution is 0.5-5 mg / mL.

5. The preparation method according to claim 1, characterized in that, The preparation steps of the decellularized matrix in step one include the following steps: (1-1) Take fresh mammalian tissue, clean it, remove the fascia, lymph nodes and large pieces of fat, and cut it into small pieces; (1-2) Use organic solvents to further remove fat; (1-3) Perform virus inactivation procedures; (1-4) Perform decellularization treatment; (1-5) Freeze-dry to obtain the product.

6. The preparation method according to claim 5, characterized in that, The organic solvent mentioned in steps (1-2) includes ethyl acetate; And / or virus inactivation is performed by immersion in peracetic acid solution in steps (1-3); And / or in steps (1-4) an enzyme is used for decellularization, the enzyme being selected from trypsin, DNase and RNase.

7. A decellularized matrix microparticle prepared by any one of the preparation methods according to claims 1-6.

8. The decellularized matrix microparticles according to claim 7, characterized in that, The decellularized matrix microparticles have a particle size of 5-45 μm.

9. The use of the decellularized matrix microparticles according to claim 7 or 8 in the preparation of gel materials.

10. A decellularized matrix microparticle gel material, characterized in that, It is obtained by gelling decellularized matrix microparticles or by mixing decellularized matrix microparticles with additives, wherein the decellularized matrix microparticles are the decellularized matrix microparticles as described in claim 7 or 8.

11. The decellularized matrix microparticle gel material according to claim 10, characterized in that, The gelation conditions are as follows: decellularized matrix microparticles are dispersed in a solution with a pH of 2.0-4.0, the concentration of decellularized matrix microparticles is ≥5.0 mg / mL, and then the pH of the solution is adjusted to 6.0-8.0 to obtain the decellularized matrix microparticle gel material. And / or the additives include a solution and / or gel of at least one of sodium hyaluronate, carboxymethyl cellulose, carboxymethyl chitosan and collagen; And / or the mass ratio of the additive to the decellularized matrix microparticles is 10:1 to 1000:

1.

12. The decellularized matrix microparticle gel material according to claim 11, characterized in that, The concentration of decellularized matrix microparticles is 5-100 mg / mL.

Images