Enzyme-residue-free extracellular matrix microsphere as well as preparation method and application thereof

Abstract

The invention provides an enzyme-residue-free extracellular matrix microsphere as well as a preparation method and application thereof. An elutable sacrificial phase material capable of being subjected to liquid-liquid phase separation with an extracellular matrix is introduced in an extracellular matrix pelletizing process, and transglutaminase is confined in a sacrificial phase, so that the extracellular matrix microsphere is prepared. And the transglutaminase and the extracellular matrix form a spatially separated structure in the balling process. In the gel forming stage, transglutaminase catalyzes protein in an extracellular matrix on a two-phase interface to generate a cross-linking reaction, and microspheres with a three-dimensional network gel structure are formed; after cross-linking is completed, transglutaminase is removed by eluting the sacrificial phase material, and meanwhile, a porous network with a communicated structure is formed in situ in the microsphere, so that the obtained extracellular matrix microsphere still keeps good structural integrity and mechanical property while realizing no residue of enzyme with catalytic activity; the scaffold can provide stable support for cell adhesion, migration and three-dimensional culture, and is applicable to cell delivery, organoid construction, tissue engineering scaffolds and the like.

Description

Technical Field

[0001] This invention relates to the field of biomaterials and tissue engineering technology, and in particular to an enzyme-free extracellular matrix microsphere, its preparation method, and its application. Background Technology

[0002] Extracellular matrix (ECM) has been widely used in recent years in the fields of three-dimensional cell culture, tissue engineering scaffolds and injectable biomaterials because it retains collagen, glycosaminoglycans and a variety of bioactive components, and can provide cells with a three-dimensional support structure that is close to the in vivo microenvironment.

[0003] Existing research indicates that some extracellular matrix (ECM) materials, after being neutralized to physiological conditions, can form physical gels through the self-assembly of collagen fibers. However, these gels typically exhibit low mechanical strength and insufficient structural stability, making them unsuitable for applications such as microsphere molding, injection delivery, and long-term culture. Therefore, in practical applications, cross-linking treatment of ECM materials is usually necessary to improve their structural stability and mechanical properties.

[0004] In existing technologies, transglutaminase (TG enzyme) catalyzes the formation of ε-(γ-glutamyl)-lysine covalent isopeptide bonds between glutamine residues and lysine residues in proteins, thereby constructing stable protein hydrogel networks. Due to its mild cross-linking conditions and high reaction specificity, TG enzyme is widely used for enzymatic cross-linking of collagen and extracellular matrix materials.

[0005] However, existing TG enzyme crosslinking methods typically involve directly incorporating the TG enzyme into the protein solution, allowing the enzyme to be uniformly distributed throughout the gel system and participate in the crosslinking reaction. While this method can achieve effective crosslinking of protein materials, after crosslinking, the TG enzyme is easily embedded or adsorbed into the formed gel network and cannot be removed, resulting in enzyme residues in the final hydrogel or microsphere material.

[0006] Because transglutaminase (TG) possesses sustained catalytic activity, residual TG enzymes in the material may further react with proteins containing glutamine and lysine residues in the surrounding environment during use or implantation, potentially impacting the material's structural stability, biocompatibility, and application safety. Therefore, effectively preventing TG enzyme residues in the final ECM material while ensuring sufficient cross-linking has become a pressing issue in the relevant technical field. Summary of the Invention

[0007] To address the problems of enzyme residues and difficulties in removal when using transglutaminase to crosslink the extracellular matrix in existing technologies, which may pose biosafety risks during in vivo application, the present invention aims to provide a method for preparing porous extracellular matrix microspheres without enzyme residues. This method ensures sufficient crosslinking of the extracellular matrix and improves its mechanical properties while effectively removing the crosslinking enzymes, thereby obtaining porous extracellular matrix microspheres suitable for safe injection and implantation.

[0008] To achieve the above objectives, as one aspect of the present invention, a method for preparing enzyme-free extracellular matrix microspheres is provided, comprising the following steps: (1) A microsphere system is formed by mixing extracellular matrix with sacrificial phase material, wherein the sacrificial phase material and the extracellular matrix are capable of liquid-liquid phase separation, and the sacrificial phase material also includes transglutaminase that has been pre-introduced and confined in the sacrificial phase material; (2) Under gelation conditions, the transglutaminase catalyzes the cross-linking reaction of proteins in the extracellular matrix at the two-phase interface between the extracellular matrix and the sacrificial phase material to form microspheres with a three-dimensional network gel structure; (3) Remove the sacrificial phase material from the microspheres and remove the transglutaminase at the same time, so that a porous network with a connected structure is formed in situ in the microspheres, thereby obtaining extracellular matrix microspheres without transglutaminase residue.

[0009] Alternatively, in combination with any of the above aspects, in another implementation of this aspect, in step (1), the extracellular matrix material is prepared from mammalian tissue as raw material after decellularization.

[0010] Specifically, the extracellular matrix material is selected from tissues of pig, cattle, sheep or human origin and obtained through decellularization. It can be selected from one or more of the following sources in combination: submucosal matrix of small intestine (SIS), decellularized dermal matrix (ADM), bladder matrix (UBM), decellularized adipose tissue matrix (DAT), amniotic matrix (AM), decellularized skin matrix, decellularized cartilage matrix, decellularized cardiac matrix or myocardial matrix (H-ECM), decellularized liver matrix (L-ECM), decellularized kidney matrix, or any combination of the above matrix materials.

[0011] Optionally, in combination with any of the above aspects, in another implementation of this aspect, in step (1), the concentration of the extracellular matrix in the microsphere system is 2 mg / mL-20 mg / mL; the concentration of the sacrificial phase material in the microsphere system is 2 mg / mL-200 mg / mL; and the concentration of the transglutaminase is 10-200 U / g, calculated based on the weight of the extracellular matrix.

[0012] Optionally, in conjunction with any of the above aspects, in another implementation of this aspect, 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, and polyethylene glycol, wherein the liquid-liquid phase separation is initiated by incompatibility between polymeric materials, volume repulsion effect, charge interaction, or a combination thereof.

[0013] Alternatively, in combination with any of the above aspects, in another implementation of this aspect, in step (1), the microsphere system is prepared by using microfluidics, membrane emulsification, mechanical stirring, electrostatic spraying, or a combination thereof.

[0014] Alternatively, in combination with any of the above aspects, in another implementation of this aspect, in step (2), the gelation conditions are temperature and time conditions that can initiate the transglutaminase-catalyzed crosslinking reaction.

[0015] Alternatively, in conjunction with any of the above aspects, in another implementation of this aspect, in step (3), the method for removing the sacrificial phase is one or a combination of water washing, buffer washing, and solvent replacement.

[0016] Alternatively, in combination with any of the above aspects, in another implementation of this aspect, in step (3), during the removal of the sacrificial phase material from the microspheres, the removal of the sacrificial phase material forms a porous network with interconnected structures in situ inside the microspheres, and provides channels for further removal of transglutaminase.

[0017] As another aspect of the present invention, an enzyme-free extracellular matrix microsphere is also provided, which is prepared according to the preparation method described in any of the above aspects.

[0018] Alternatively, in combination with any of the above aspects, in another implementation of this aspect, the average particle size of the extracellular matrix microspheres is 5~300 μm.

[0019] Alternatively, in combination with any of the above aspects, in another implementation of this aspect, the Young's modulus of the extracellular matrix microspheres is above 3000 Pa.

[0020] As another aspect of the present invention, an application of the enzyme-free extracellular matrix microspheres of the above-mentioned aspects in the fields of three-dimensional cell culture, organoid construction, stem cell expansion, or tissue engineering is also provided. Compared with the prior art, the present invention has at least the following beneficial effects: 1) This invention achieves enzyme-free extracellular matrix microspheres by confining transglutaminase in the sacrificial phase and eluting it along with the sacrificial phase and through interconnected macropores after cross-linking, which significantly improves the biosafety of the material and is especially suitable for in vivo injection and implantation applications; 2) The extracellular matrix microspheres obtained have a large-pore structure that is interconnected, which is conducive to cell adhesion, migration and nutrient exchange, and is suitable for three-dimensional cell culture and tissue engineering applications. 3) Through enzymatic cross-linking, a stable three-dimensional network structure is formed. The extracellular matrix microspheres maintain good structural integrity and mechanical properties while maintaining good biological activity.

[0021] The above summary provides a simplified overview of some concepts, which will be further described in detail in the following specific embodiments. The above summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to help determine the scope of the claimed subject matter. The claimed subject matter is not limited to embodiments that address any or all the shortcomings pointed out in the background art.

[0022] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by means of embodiments thereof. Attached Figure Description

[0023] The accompanying drawings, incorporated in and forming part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application. To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without any creative effort. These drawings and textual descriptions are not intended to limit the scope of the concept of this application in any way, but rather to illustrate the concepts of this application to those skilled in the art by referring to specific embodiments.

[0024] Figure 1 This is a schematic diagram illustrating the preparation principle of the enzyme-free extracellular matrix microspheres in Example 1 of the present invention; Figure 2 This is a schematic diagram illustrating the reaction principle of transglutaminase-catalyzed cross-linking of the extracellular matrix in Example 1 of the present invention; Figure 3 The images show a microscope image and particle size distribution of the extracellular matrix microspheres obtained in Example 1 of this invention. Figure 4 This is a rheological diagram of the extracellular matrix microspheres obtained in Example 1 of the present invention; Figure 5The elution kinetics curve of TG enzyme in the extracellular matrix microspheres obtained in Example 1 of this invention; Figure 6 This is a biocompatibility characterization of the extracellular matrix microspheres obtained in Example 2 of the present invention. Detailed Implementation

[0025] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0026] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element. It should be further understood that, as used herein, the singular forms "a," "an," and "the" are intended to also include the plural forms, unless the context indicates otherwise. Furthermore, the terms "or," "and / or," "including at least one of the following," etc., as used herein, can be interpreted as inclusive, or mean any one or any combination thereof. Exceptions to this definition only arise when combinations of elements, functions, steps, or operations are inherently mutually exclusive in some manner.

[0027] It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit the scope of this application. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer. Example

[0028] 1) Preparation of extracellular matrix (ECM)-polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer (F127) microspheres 1.1) Preparation of extracellular matrix solution Weigh 10 mg of decellularized porcine small intestinal mucosal matrix into a centrifuge tube, add 1 mL of hydrochloric acid (0.01 M) to dissolve it, and then adjust the pH of the digestion solution to 7.4 with 0.1 M NaOH. Add 10×PBS solution at a volume ratio of 10:1 to achieve ion balance and form a pre-gel solution, and store it at 4°C.

[0029] 1.2) Preparation of F127 solution Weigh 160 mg of F127 into a centrifuge tube, add 1 mL of deionized water, dissolve in an ice bath, then add 8 U of transglutaminase to the dissolved F127 solution, dissolve and store at 4°C.

[0030] 1.3) Microfluidic preparation of ECM-F127 microspheres ECM solution and F127 solution were mixed at a volume ratio of 8:2, denoted as dispersed phase A1. A surfactant (FE-surf) was dissolved in fluorinated oil to prepare a 2% (w / w) continuous phase B1. The prepared solutions were connected to a microfluidic chip via pipes. The flow rate of oil phase B1 was adjusted to 40 μL / min, and the flow rate of aqueous phase A1 was adjusted to 4 μL / min. Stable monodisperse droplets were prepared using the microfluidic chip. Microspheres were collected for 2 hours and crosslinked in a 37°C water bath for 4 hours, followed by overnight incubation at room temperature. The schematic diagram of the preparation process is shown below. Figure 1 As shown, the microspheres prepared are as follows Figure 3 As shown, it can be clearly seen that F127 separates from ECM in the ECM microspheres. Subsequently, by washing the uncrosslinked F127, a porous structure can be formed.

[0031] 2) Characterization of sacrificial phase removal and enzyme residue The cross-linked microspheres were subjected to sacrificial phase removal treatment. First, the surfactant on the surface of the microspheres was removed by multiple washings. Then, the microspheres were placed in a dialysis bag with a molecular weight cutoff of 50 kDa and dialyzed in deionized water at 4 °C, with magnetic stirring to improve mass exchange efficiency. During dialysis, the washing solution was changed every 12 hours, and washing solution samples were collected at each stage.

[0032] A microbial transglutaminase activity assay kit was used to detect the transglutaminase activity in each washing solution sample. The release of transglutaminase during the washing process was characterized by measuring its catalytic activity in the washing solution. To eliminate the influence of sample volume and detection conditions at different washing stages on the results, the transglutaminase activity detected in each washing solution was normalized and expressed as the proportion of the enzyme content detected in each washing solution to the total enzyme content in the system.

[0033] The results are as follows Figure 5As shown, with the increase of washing times, the proportion of transglutaminase detected in each washing solution gradually decreased, and the enzyme content in the fourth washing solution was lower than the detection threshold, indicating that no catalytically active transglutaminase residue was detected in the prepared porous extracellular matrix microspheres.

[0034] 3) Characterization of the mechanical properties of extracellular matrix microspheres To evaluate the mechanical properties of extracellular matrix microspheres, rheological characterization was performed on the dialyzed microspheres. The results are as follows: Figure 4 As shown, the storage modulus (G′) of the washed microspheres reached 3000 Pa, indicating that the extracellular matrix microspheres have certain mechanical strength and structural stability.

[0035] 4) Extracellular matrix microspheres without enzyme residue are used for subcutaneous filling. The enzyme-free, porous extracellular matrix microspheres prepared above were aseptically processed and then injected into the subcutaneous tissue of experimental animals using a syringe (22G needle). The microspheres exhibited good injectability during injection, did not clog the syringe, and remained in the subcutaneous tissue to create a certain volume of filling effect. The microspheres of this invention can be used as a soft tissue filler for injectable filling applications. Example

[0036] 1) Preparation of extracellular matrix (ECM)-dextran DEX microspheres 1.1) Preparation of extracellular matrix solution Weigh 10 mg of decellularized porcine small intestinal mucosal matrix into a centrifuge tube, add 1 mL of hydrochloric acid (0.01 M) to dissolve it, and then adjust the pH of the digestion solution to 7.4 with 0.1 M NaOH. Add 10×PBS solution at a volume ratio of 10:1 to achieve ion balance and form a pre-gel solution, and store it at 4°C.

[0037] 1.2) Preparation of dextran solution Weigh 150 mg of DEX into a centrifuge tube, add 1 mL of deionized water, dissolve in an ice bath, then add 8 U of transglutaminase to the dissolved DEX solution, dissolve and store at 4°C.

[0038] 1.3) Microfluidic preparation of ECM-DEX microspheres ECM solution and DEX solution were mixed at a volume ratio of 8:2 and denoted as dispersed phase A2. Surfactant (FE-surf) was dissolved in fluorinated oil to prepare a continuous phase B1 with a mass fraction of 2%. The prepared solutions were connected to the microfluidic chip through pipes. The flow rate of oil phase B1 was adjusted to 40 μL / min and the flow rate of aqueous phase A1 was adjusted to 4 μL / min. Stable monodisperse droplets were prepared by the microfluidic chip. Microspheres were collected for 2 hours and crosslinked in a 37°C water bath for 4 hours, and then left to stand at room temperature overnight.

[0039] 2) Characterization of sacrificial phase removal and enzyme residue The cross-linked microspheres were subjected to sacrificial phase removal treatment. First, the surfactant on the surface of the microspheres was removed by multiple washings. Then, the microspheres were placed in a dialysis bag with a molecular weight cutoff of 50 kDa and dialyzed in deionized water at 4 °C, with magnetic stirring to enhance the mass exchange efficiency. During dialysis, the washing solution was changed every 12 hours, and washing solution samples were collected at each stage.

[0040] A microbial transglutaminase activity assay kit was used to detect the transglutaminase activity in each washing solution sample. The release of transglutaminase during the washing process was characterized by measuring its catalytic activity in the washing solution. To eliminate the influence of sample volume and detection conditions at different washing stages on the results, the transglutaminase activity detected in each washing solution was normalized and expressed as the proportion of the enzyme content detected in each washing solution to the total enzyme content in the system.

[0041] 3) Enzyme-free extracellular matrix microspheres for 3D cell culture After aseptic treatment, the porous extracellular matrix microspheres described above are mixed with a suspension of cells to be cultured under aseptic conditions and suspended in a suitable culture medium. This mixture can then be placed in a low-adhesion culture plate or roller bottle for three-dimensional cell culture. In the three-dimensional environment of the porous microspheres, cells can adhere to the pore walls, enter the pores, and exhibit a three-dimensional distribution. This embodiment is not dependent on a specific cell type and is suitable for three-dimensional culture of various cell types.

[0042] 4) Cell compatibility evaluation To verify the biological safety of the enzyme-free extracellular matrix microspheres prepared in this invention, fibroblasts were selected as model cells to evaluate the in vitro cell compatibility of the extracellular matrix microspheres. Cells were co-cultured with the material, and cell viability was detected by the CCK-8 assay after 1, 3, and 7 days of culture.

[0043] The results are as follows Figure 6 As shown, the extracellular matrix microspheres prepared by the method of the present invention have high cell viability, no obvious cytotoxicity was observed, and good biocompatibility.

[0044] The sequence numbers of the embodiments in this application are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0045] In this application, the same or similar terms, concepts, technical solutions and / or application scenario descriptions are generally described in detail only when they appear for the first time. When they appear again, they are generally not repeated for the sake of brevity. When understanding the technical solutions and other contents of this application, the same or similar terms, concepts, technical solutions and / or application scenario descriptions that are not described in detail later can be referred to their previous relevant detailed descriptions.

[0046] In this application, the descriptions of the various embodiments have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0047] The technical features of the present application can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the present application.

[0048] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A method for preparing enzyme-free extracellular matrix microspheres, characterized in that, Includes the following steps: (1) A microsphere system is formed by mixing extracellular matrix with sacrificial phase material, wherein the sacrificial phase material and the extracellular matrix are capable of liquid-liquid phase separation, and the sacrificial phase material also includes transglutaminase that has been pre-introduced and confined in the sacrificial phase material; (2) Under gelation conditions, the transglutaminase catalyzes the cross-linking reaction of proteins in the extracellular matrix at the two-phase interface between the extracellular matrix and the sacrificial phase material to form microspheres with a three-dimensional network gel structure; (3) Remove the sacrificial phase material from the microspheres and remove the transglutaminase at the same time, so that a porous network with a connected structure is formed in situ in the microspheres, thereby obtaining extracellular matrix microspheres without transglutaminase residue.

2. The preparation method according to claim 1, characterized in that, In step (1), the extracellular matrix material is prepared from mammalian tissue as raw material after decellularization.

3. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the extracellular matrix in the microsphere system is 2 mg / mL-20 mg / mL; the concentration of the sacrificial phase material in the microsphere system is 2 mg / mL-200 mg / mL; and the concentration of the transglutaminase is 10-200 U / g, calculated based on the weight of the extracellular matrix.

4. The preparation method according to claim 1, characterized in that, 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, and polyethylene glycol, wherein the liquid-liquid phase separation is initiated by the incompatibility between polymer materials, volume repulsion effect, charge interaction, or a combination thereof.

5. The preparation method according to claim 1, characterized in that, In step (1), the microsphere system is prepared by microfluidic method, membrane emulsification method, mechanical stirring method, electrostatic spraying method or a combination thereof.

6. The preparation method according to claim 1, characterized in that: In step (2), the gelation conditions are the temperature and time conditions that can initiate the transglutaminase-catalyzed crosslinking reaction.

7. The preparation method according to claim 1, characterized in that, In step (3), the method for removing the sacrificial phase is one or a combination of water washing, buffer washing, and solvent replacement.

8. An extracellular matrix microsphere free of enzyme residue, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

9. The preparation method according to claim 8, characterized in that, The extracellular matrix microspheres have an average particle size of 5-300 μm and / or a Young's modulus of 3000 Pa or higher.

10. The application of the enzyme-free extracellular matrix microspheres according to claim 9 in the fields of three-dimensional cell culture, organoid construction, stem cell expansion, or tissue engineering.

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