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CN-121991876-A - Extracellular matrix microsphere without enzyme residues, preparation method and application thereof

CN121991876ACN 121991876 ACN121991876 ACN 121991876ACN-121991876-A

Abstract

The invention provides an extracellular matrix microsphere without enzyme residue, a preparation method and application thereof, which are characterized in that an elutable sacrificial phase material capable of being separated from an extracellular matrix in a liquid-liquid phase manner is introduced in the process of balling the extracellular matrix, and transglutaminase is limited in the sacrificial phase, so that the transglutaminase and the extracellular matrix form a structure which is separated from each other in space in the process of balling. In the gel forming stage, transglutaminase catalyzes protein in extracellular matrix to produce cross-linking reaction in two-phase interface to form microsphere with three-dimensional network gel structure, and after cross-linking, the transglutaminase is eliminated through eluting sacrificial phase material, and the porous network with communicated structure is formed inside the microsphere.

Inventors

  • YU ZIYI
  • CHEN KAIHAO
  • ZHANG JING
  • ZHANG YANG
  • ZHENG HAIPING

Assignees

  • 南京工业大学

Dates

Publication Date
20260508
Application Date
20260212

Claims (10)

  1. 1. A method for preparing extracellular matrix microspheres without enzyme residues, which is characterized by comprising the following steps: (1) Mixing an extracellular matrix with a sacrificial phase material to form a microsphere system, wherein the sacrificial phase material is capable of liquid-liquid phase separation from the extracellular matrix, and wherein the sacrificial phase material further comprises transglutaminase pre-incorporated and confined in the sacrificial phase material; (2) Under the gel forming strip, the transglutaminase catalyzes proteins in the extracellular matrix to carry out a crosslinking reaction at a two-phase interface of the extracellular matrix and the sacrificial phase material to form microspheres with a three-dimensional network gel structure; (3) And removing the sacrificial phase material in the microsphere and simultaneously removing the transglutaminase, so that a porous network with a communication structure is formed in situ in the microsphere, thereby obtaining the extracellular matrix microsphere without transglutaminase residues.
  2. 2. The method according to claim 1, wherein in the step (1), the extracellular matrix material is prepared from a mammalian tissue as a raw material by decellularization treatment.
  3. 3. The method according to claim 1, wherein in the step (1), the concentration of the extracellular matrix in the microsphere system is 2mg/mL-20mg/mL, the concentration of the sacrificial phase material in the microsphere system is 2mg/mL-200mg/mL, and the concentration of the transglutaminase is 10 to 200U/g based on the weight of the extracellular matrix.
  4. 4. The method of claim 1, wherein in step (1), the sacrificial phase material is selected from one or more of polyethylene oxide-polypropylene oxide-polyethylene oxide block copolymer, sodium alginate, carboxymethyl cellulose, hydroxypropyl cellulose, dextran, polyethylene glycol, wherein the liquid-liquid phase separation is induced by incompatibility between polymeric materials, volume exclusion effect, charge effect, or a combination thereof.
  5. 5. The method of claim 1, wherein in step (1), the microsphere system is prepared by using a microfluidic method, a membrane emulsification method, a mechanical agitation method, an electrostatic spraying method, or a combination thereof.
  6. 6. The method according to claim 1, wherein in the step (2), the gelling conditions are temperature and time conditions capable of initiating the transglutaminase-catalyzed crosslinking reaction.
  7. 7. The method of claim 1, wherein in step (3), the sacrificial phase is removed by one or a combination of water washing, buffer washing, and solvent displacement.
  8. 8. An extracellular matrix microsphere free from enzyme residues, which is prepared according to the preparation method of any one of claims 1 to 7.
  9. 9. The method according to claim 8, wherein the average particle diameter of the extracellular matrix microsphere is 5 to 300 μm and/or the Young's modulus of the extracellular matrix microsphere is 3000Pa or more.
  10. 10. Use of the enzyme-residue-free extracellular matrix microsphere according to claim 9 in the fields of cell three-dimensional culture, organoid construction, stem cell expansion or tissue engineering.

Description

Extracellular matrix microsphere without enzyme residues, preparation method and application thereof Technical Field The invention relates to the technical field of biological materials and tissue engineering, in particular to an extracellular matrix microsphere without enzyme residues, a preparation method and application thereof. Background The extracellular matrix (Extracellular Matrix, ECM) can provide a three-dimensional support structure for cells, which approximates to an in-vivo microenvironment, due to its retention of collagen, glycosaminoglycan and various bioactive components, and has been widely used in the fields of three-dimensional culture of cells, tissue engineering scaffolds, injectable biomaterials, etc. in recent years. The existing research shows that part of extracellular matrix materials can form physical gel through the self-assembly action of collagen fibers after being neutralized to physiological conditions, but the gel is generally low in mechanical strength and insufficient in structural stability, and is difficult to meet the requirements of application scenes such as microsphere formation, injection delivery, long-term culture and the like. Therefore, in practical applications, it is generally necessary to crosslink the ECM material to improve its structural stability and mechanical properties. In the prior art, transglutaminase (TG enzyme) is capable of catalyzing the formation of epsilon- (gamma-glutamyl) -lysine covalent isopeptidic bonds between glutamine and lysine residues in proteins, thereby constructing a stable protein hydrogel network. Because of the mild crosslinking conditions and high reaction specificity, TG enzymes are widely used for enzymatic crosslinking of collagen and extracellular matrix materials. However, the existing TG enzyme crosslinking methods typically blend the TG enzyme directly into the protein solution, allowing the enzyme to be evenly distributed throughout the gel system and participate in the crosslinking reaction. Although this approach allows for efficient cross-linking of the proteinaceous material, TG enzymes are easily entrapped or adsorbed in the formed gel network after cross-linking is completed and cannot be removed, resulting in enzyme residues in the final hydrogel or microsphere material. Because transglutaminase has continuous catalytic activity, TG enzymes remaining in the material may react further with proteins containing glutamine and lysine residues in the surrounding environment during use or implantation of the material, thereby potentially affecting structural stability, biocompatibility and application safety of the material. Therefore, how to effectively avoid the TG enzyme residue in the final material while ensuring the ECM material to be sufficiently crosslinked has become a problem to be solved in the related art. Disclosure of Invention Aiming at the problems that enzyme residues are difficult to remove and biological potential safety hazards possibly occur in-vivo application when transglutaminase is adopted to crosslink extracellular matrixes in the prior art, the invention aims to provide a preparation method of porous extracellular matrix microspheres without enzyme residues, which ensures that the extracellular matrixes are fully crosslinked and the mechanical properties are improved, and simultaneously realizes effective removal of crosslinking enzyme, thereby obtaining the porous extracellular matrix microspheres suitable for safe injection and implantation. To achieve the above object, as one aspect of the present invention, there is provided a method for preparing extracellular matrix microspheres free from enzyme residues, comprising the steps of: (1) Mixing an extracellular matrix with a sacrificial phase material to form a microsphere system, wherein the sacrificial phase material is capable of liquid-liquid phase separation from the extracellular matrix, and wherein the sacrificial phase material further comprises transglutaminase pre-incorporated and confined in the sacrificial phase material; (2) Under the gel forming strip, the transglutaminase catalyzes proteins in the extracellular matrix to carry out a crosslinking reaction at a two-phase interface of the extracellular matrix and the sacrificial phase material to form microspheres with a three-dimensional network gel structure; (3) And removing the sacrificial phase material in the microsphere and simultaneously removing the transglutaminase, so that a porous network with a communication structure is formed in situ in the microsphere, thereby obtaining the extracellular matrix microsphere without transglutaminase residues. Alternatively, in another implementation manner of the present aspect, in any one of the foregoing aspects, in step (1), the extracellular matrix material is prepared from mammalian tissue as a raw material after a decellularization treatment. Specifically, the extracellular matrix material is selected from tissues of pig, c