Preparation method of intercellular substance-containing artificial tissue

Abstract

The invention relates to a preparation method of an intercellular substance-containing artificial tissue, which comprises the following steps: introducing a phenylboronic acid group on a protein vesicle by a method of copolymerization of N-isopropylacrylamide and 3-acrylamido phenylboronic acid; the preparation method comprises the following steps: constructing aggregate micro-droplets by taking polydiene dimethyl ammonium chloride and carboxylated amylose as primitives; based on a biological orthogonal reaction of phenylboronic acid and polysaccharide, polysaccharide aggregate droplets are used as intercellular substances to adhere phenylboronic acid modified protein vesicles together to form an aggregate phase with a microstructure similar to that of a biological tissue. According to the method, the problems that the aggregate microdroplets cannot stably exist in an aqueous solution for a long time and the application of the aggregate microdroplets is limited to the micro-scale are solved, a stable aggregate phase can be formed on the macro-scale, and biological enzyme reaction under low concentration is enriched and activated; optimization thoughts in the aspects of consumption saving, enrichment and recovery, enzyme activity regulation and control and the like are provided for some high-value biological enzymes in the using process.

Description

Technical Field

[0001] The invention belongs to the technical field of artificial cell / tissue model application, and in particular relates to a method for preparing artificial tissue containing intercellular matrix. Background Art

[0002] Bionic materials refer to artificial materials developed from the perspective of biomaterials, relying on the interpretation of the physical structure and life processes of organisms. As a type of biomimetic material, artificial cells are based on the "bottom-up" research method of synthetic biology, starting from nanoscale building blocks, and constructing microscale cell-like structural components, playing an important role in understanding life behavior and developing advanced materials. In recent years, various artificial cell models have been developed based on the self-assembly of different macromolecules, and due to the rapid development of the field of artificial cells, this field is no longer limited to mimicking the structure and function of living cells. Some artificial cell-based materials have emerged, showing their potential in bridging the gap between life sciences and materials sciences, including disease treatment, enzyme catalytic regulation, ultrasensitive immunoassays, modular microreactor components, gene transfection, and cell repair. However, due to the relatively single composition and simple application strategy of artificial cell materials, their development is still in its infancy. Therefore, the development of multi-component hybrid artificial cell materials can broaden their application scenarios and scope.

[0003] Organisms usually rely on proliferation and differentiation to ensure the diversity of cell structure and function. Biological tissues are complex three-dimensional structures composed of interconnected cell groups with similar morphology or functions. The cells in these cell groups communicate with each other and exhibit new functions and behaviors. Therefore, in order to broaden the application scenarios and scope of artificial cell materials, their structure needs to move towards more complex tissue structures (artificial tissues). So far, artificial tissues are aggregates or aggregates formed by artificial cells in order to play a role at the macro level. In such a chemical system, the formation of artificial tissues can be triggered by driving forces such as chemical mediation, 3D printing technology, optical tweezers, magnetic fields, and acoustic fields. However, most artificial tissues lack intercellular matrix, which not only has a gap with biological tissues in structure, but also limits its development prospects as a new material. Therefore, artificial tissues composed of a variety of artificial cells with different properties and functions can provide a broader space and potential for building artificial tissues that are closer to biological tissues in structure and function. Summary of the invention

[0004] The present invention provides ideas for the field of artificial cells as bionic materials, realizes the purpose of developing new materials inspired by nature, provides a preparation method of artificial tissue containing intercellular matrix, is closer to the structure of biological tissue on a microscopic scale, forms a stable condensate phase on a macroscopic scale, and enriches and activates biological enzyme reactions at low concentrations, providing optimization ideas for saving dosage, enriching and recovering, regulating enzyme activity, etc. for some high-value biological enzymes during use.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing artificial tissue containing intercellular matrix, the method comprising:

[0007] Step 1: Phenylboronic acid functionalization of protein vesicles:

[0008] (1) Preparation of phenylboronic acid functionalized polymer: 3-acrylamidophenylboronic acid was selected as a functional monomer and dissolved in anhydrous acetonitrile with N-isopropylacrylamide at a molar ratio of 1:30-60, and deoxygenated by bubbling for 30-90 min. After deoxygenation, copolymerization was performed at 60-80° C. for 6-9 h to prepare a polymer containing phenylboronic acid groups;

[0009] (2) Preparation of phenylboronic acid functionalized moieties: incubating amino-modified bovine serum albumin with a polymer containing phenylboronic acid groups in a phosphate buffer solution at pH 6 to 8 overnight, dialyzing and freeze-drying for use;

[0010] (3) Preparation of phenylboronic acid functionalized protein vesicles: The phenylboronic acid functionalized unit was prepared at 5-10 mg mL -1 The aqueous solution is mixed and then the oil phase (isooctanol) is added. After vigorous shaking, the aqueous phase is transferred for standby use. The purpose of transferring the aqueous phase is to remove the oil phase to facilitate subsequent assembly and enzymatic reaction.

[0011] Step 2: Preparation of polysaccharide aggregate droplets:

[0012] (1) Synthesis of carboxylated amylose: Amylose is carboxylated on its surface using the chloroacetic acid method. Amylose and sodium hydroxide are dissolved in water under heating conditions of 60 to 90° C., and then chloroacetic acid is added to react for 1.5 to 3.0 hours. After dialysis, the mixture is freeze-dried for use.

[0013] (2) Preparation of polysaccharide aggregate droplets: Polydimethylammonium chloride (PDDA) and carboxylated amylose (Cm-Am) were prepared at 1-4 mg mL -1 The aqueous solution is shaken in a ratio of 1 to 8:2 to form a white emulsion;

[0014] Step 3: Preparation of artificial tissue containing interstitial cells (condensate phase):

[0015] The phenylboronic acid functionalized protein vesicle solution obtained in step one and the polysaccharide aggregate droplets obtained in step two are mixed in a volume ratio of 1 to 6:2, and the pH of the system is adjusted to 6 to 8 using a phosphate buffer solution. The system is allowed to stand for 30 to 60 minutes at room temperature to obtain an artificial tissue containing intercellular matrix (aggregate phase).

[0016] Step 4: Test of dormant enzyme reaction activation effect: The dormant enzyme reaction activation in this part is tested by lactate dehydrogenase (LDH). The LDH enzymatic reaction time is 5 to 60 minutes. The test results show that LDH is 0.05 to 0.15 μg mL -1 The catalytic reaction cannot be carried out within the concentration range, so the preparation process of step three is carried out in the solution within this concentration range to form a coacervate phase to enrich LDH, and then the substrates NADH (0.1-1.0 mM) and sodium pyruvate (1.0-5.0 mM) are added to test whether the enzyme reaction is activated.

[0017] Furthermore, in step 1 (2), the mass ratio of the amino-modified bovine serum albumin to the polymer containing phenylboronic acid groups is 2:1-4, and the molecular weight cut-off of the dialysis bag used for dialysis is 30-50 kDa.

[0018] Furthermore, in step 1 (3), the preparation of phenylboronic acid functionalized protein vesicles is specifically as follows: using a pipette to draw the building block solution into a centrifuge tube, then adding phosphate buffer, controlling the pH to 6-8, mixing evenly, adding isooctyl alcohol oil phase and shaking vigorously for 30-120s to obtain emulsion vesicles. In addition, if there is a requirement to transfer the aqueous phase, a final concentration of 0.02-0.1 mg μL -1 Cross-linking agent (NHS-PEG 16 -NHS), and after the cross-linking reaction overnight, the phase was transferred to the aqueous phase by gradient dialysis using different concentrations of ethanol.

[0019] Furthermore, in step 1 (3), the water-oil ratio is 1:20-40.

[0020] Furthermore, in step 2 (1), the ratio of amylose, sodium hydroxide, water and chloroacetic acid is 100-200 mg: 300-500 mg: 3-10 mL: 200-400 mg; and the molecular weight cut-off of the dialysis bag used for dialysis is 3.0-5.0 kDa.

[0021] Furthermore, in step 2 (1), the molecular weight of the amylose is 10 to 25 kDa.

[0022] Furthermore, in step 2 (2), the pH range of the mixed solution is controlled to be 6 to 8, which can be controlled by using a phosphate buffer.

[0023] Furthermore, in step 3, the concentration of the phenylboronic acid functionalized protein vesicle solution is 0.1 to 2.0 mg mL -1 .

[0024] Compared with the prior art, the artificial tissue containing interstitial cells (condensate phase) of the present invention has the following beneficial effects:

[0025] (1) Inspired by the structure of natural biological tissues, protein vesicles are adhered together using coacervates, which not only forms an artificial tissue model containing intercellular matrix that is closer to biological tissues, but also solves the problem that coacervate droplets cannot exist stably in aqueous solution for a long time. The two artificial cells complement each other and enhance each other's structure and function.

[0026] (2) The characteristics of the aggregate droplets themselves can enrich various biomacromolecules. The flexible membrane structure on the outside and the hollow structure on the inside of the protein vesicles also determine that they can load various biomacromolecules inside. The enzymes are loaded in a non-covalent manner to ensure the activity of the enzymes loaded inside. The combination of the two provides a broader space for the application of related enzymatic reactions.

[0027] (3) The problem that the application of condensate droplets is limited to the microscopic scale is solved. A stable condensate phase can be formed on the macroscopic scale. Through its ability to enrich biological enzymes, dormant enzymatic reactions at low concentrations can be reactivated, providing optimization ideas for saving dosage, enrichment and recovery, and regulating enzyme activity for some high-value biological enzymes during use. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the H NMR spectrum of phenylboronic acid functionalized polymer;

[0029] Figure 2 This is a microscope photo of phenylboronic acid functionalized protein vesicles;

[0030] Figure 3 This is an optical microscope photo of phenylboronic acid functionalized protein vesicles in a dry state;

[0031] Figure 4 This is a test diagram of the flexibility of glycosylated protein vesicles in water phase;

[0032] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of carboxylated amylose;

[0033] Figure 6 Optical microscope photos and size statistics of aggregate droplets;

[0034] Figure 7 This is a diagram showing the formation process of artificial tissue containing intercellular matrix;

[0035] Figure 8This is a digital photo of the condensed phase;

[0036] Fig. 9 Schematic diagram of the dormant enzyme activation process;

[0037] Fig.10 This is a test data graph for the activation effect of dormant enzyme reaction. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0039] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention can also be used to realize the present invention.

[0040] The present invention introduces phenylboronic acid groups on protein vesicles by copolymerizing N-isopropylacrylamide with 3-acrylamidophenylboronic acid; uses polydiallyl dimethyl ammonium chloride (PDDA) and carboxylated amylose (Cm-Am) as primitives to construct coacervate droplets; based on the bioorthogonal reaction of phenylboronic acid and polysaccharide, the polysaccharide coacervate droplets act as intercellular matrix to adhere the phenylborated protein vesicles together to form a coacervate phase (artificial tissue) with a microstructure similar to that of biological tissue; subsequently, the reaction activity of biological enzymes at low concentrations on a macro scale is regulated by the characteristics of the coacervate itself that enriches biological macromolecules. The present invention solves the problem that coacervate droplets cannot stably exist in aqueous solution for a long time and that their application is limited to the microscale, can form a stable coacervate phase on a macroscale, and through its ability to enrich biological enzymes, reactivates dormant enzymatic reactions at low concentrations, and provides optimization ideas for saving dosage, enriching and recycling, regulating enzyme activity, etc. for some high-value biological enzymes during use.

[0041] The method of the present invention can be applied to enrich a variety of different enzymes in the coacervate phase, such as amylase, glucose oxidase, catalase, etc., and the coacervate itself has the property of enzyme enrichment, and does not damage the enzyme activity; or different enzymes that can form enzyme cascade reactions are introduced at the same time to increase the selection space of substrates without changing the final product. In short, the introduction of different functional proteins will increase the function of the coacervate phase and bring more advantages.

[0042] Embodiment 1:

[0043] 1. Phenylboronic acid functionalization of protein vesicles

[0044] (1) Preparation of phenylboronic acid functionalized polymers: 3-acrylamidophenylboronic acid was selected as the functional monomer to copolymerize with N-isopropylacrylamide to prepare a polymer containing phenylboronic acid groups. The specific steps are as follows: 6 mg of the reactant chain transfer agent (2-dodecylthiocarbonylthio-2-methylpropionic acid N-hydroxysuccinimide ester), 0.7 mg of azobisisobutyronitrile, 217.5 mg of N-isopropylacrylamide, and 6.3 mg of 3-acrylamidophenylboronic acid were dissolved in 4 mL of anhydrous acetonitrile, and deoxygenated by bubbling for 30 minutes. After deoxygenation, the reaction was heated at 65°C for 8 hours. After the reaction was completed, the polymer was precipitated in a mixed solvent of petroleum ether and diethyl ether (2:1), and the polymer was obtained as a yellow powder after drying. Figure 1 The product H NMR spectrum can prove that the phenylboronic acid functionalized polymer was successfully synthesized.

[0045] (2) Preparation of phenylboronic acid functionalized moieties: 10 mg of aminated bovine serum albumin was dissolved in 10 mL of ultrapure water and slowly added dropwise to the phenylboronic acid functionalized polymer solution (10 mL, 1 mg mL -1 ) and reacted at room temperature overnight. The solution was dialyzed in ultrapure water (dialysis bag molecular weight cutoff 35 kDa). The dialyzate was filtered and freeze-dried to obtain a white solid, which was stored at -20°C.

[0046] (3) Preparation of phenylboronic acid functionalized protein vesicles: Prepare 10 mg mL of phenylboronic acid functionalized base solution synthesized in (2). -1 , use a pipette to draw 20 μL of the building block solution into a 1.5 mL centrifuge tube, then add 8 μL of phosphate buffer (pH 8.0, 50 mM), mix well, add 400 μL of isooctyl alcohol and shake vigorously for 30 seconds to obtain emulsion vesicles ( Figure 2 ), and the film structure after drying can be observed by optical microscope ( Figure 3 If the aqueous phase is required to be transferred, add 0.5 mg of cross-linking agent (NHS-PEG 16 -NHS), and after the cross-linking reaction overnight, the mixture was transferred to the aqueous phase by gradient dialysis with 75%, 50%, and 25% ethanol, respectively. Figure 4 The throughput process of the protein vesicle under a micropipette can be observed under an optical microscope, demonstrating the flexibility of the protein vesicle membrane.

[0047] 2. Preparation of polysaccharide aggregate droplets

[0048] (1) Synthesis of carboxylated amylose: Weigh amylose (12 kDa, 150 mg) and sodium hydroxide (360 mg) and dissolve them in 3 mL of water under heating at 70°C, then add chloroacetic acid (270 mg) and keep the temperature at 70°C for 2 hours. After the reaction, adjust the pH to neutral with acetic acid, precipitate in 40 mL of ice ethanol, and then dissolve in ultrapure water. The solution is dialyzed in ultrapure water (dialysis bag molecular weight cutoff is 3.5 kDa), and the dialyzate is filtered and freeze-dried to obtain a white solid, which is then stored at -20°C. Figure 5 The carboxylation H NMR spectrum of amylose can prove the success of the modification process.

[0049] (2) Preparation of polysaccharide aggregate droplets: Carboxylated amylose and polydimethyl ammonium chloride were prepared in PBS (pH = 7.4) buffer at a concentration of 2 mg mL -1 120 μL of polydimethyl ammonium chloride solution was slowly added to 90 μL of stirred carboxylated amylose solution to make the final concentration of positively charged moieties and negatively charged moieties 4:3. The solution became turbid after mixing, and its morphology could be clearly seen under a microscope ( Figure 6 ).

[0050] 3. Preparation of artificial tissue containing intercellular matrix (condensate phase)

[0051] The aggregate droplets were constructed by the basic carboxylated amylose and polydiallyl dimethyl ammonium chloride (2 mg mL -1 ) in a ratio of 3:4, and 200 μL was added to the protein vesicles (0.5 mg mL -1 , 100 μL), wherein the dispersion solution adopts a neutral solution environment of PBS buffer solution (pH=7.4, 10 mM) to ensure the supramolecular interaction between phenylboronic acid and sugar groups, and the centrifuge tube is slightly shaken with fingers to ensure that the two are mixed evenly, and then allowed to stand to form an artificial tissue containing interstitial cells ( Figure 7 ).

[0052] 4. Dormant enzyme reaction activation effect test

[0053] like Figure 8 As shown in the figure, the artificial tissue containing intercellular matrix is ​​separated from water and is called condensate phase, and the activation process of dormant enzyme reaction is shown in the figure. Fig. 9Specifically, lactate dehydrogenase (LDH) was selected for testing, and protein vesicles (100 μL) and aggregate droplets (200 μL) were added to a 200 μL LDH solution and allowed to stand at room temperature for assembly (the final concentration of LDH was 0.15 μg mL -1 ), forming an aggregate phase to enrich LDH, and then adding NADH (final concentration 0.5mM) and sodium pyruvate (final concentration 5mM) to test whether the enzyme reaction is activated. Fig.10 The large decrease in NADH in the condensed phase can be used to conclude that the enzyme reaction is activated.

[0054] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0055] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing artificial tissue containing intercellular matrix, characterized in that: The method is: Step 1: Phenylboronic acid functionalization of protein vesicles: (1) preparing a phenylboronic acid functionalized polymer: dissolving 3-acrylamidophenylboronic acid and N-isopropylacrylamide in anhydrous acetonitrile at a molar ratio of 1:30-60, deoxygenating by bubbling for 30-90 min, and heating and copolymerizing at 60-80° C. for 6-9 h after deoxygenation to prepare a polymer containing a phenylboronic acid group; (2) Preparation of phenylboronic acid functionalized moieties: incubating amino-modified bovine serum albumin with a polymer containing phenylboronic acid groups in a phosphate buffer solution at pH 6 to 8 overnight, dialyzing and freeze-drying for use; (3) Preparation of phenylboronic acid functionalized protein vesicles: The phenylboronic acid functionalized unit was prepared at 5-10 mg mL -1 The aqueous solution is mixed and then the oil phase (isooctanol) is added. After vigorous shaking, the aqueous phase is transferred for later use; Step 2: Preparation of polysaccharide aggregate droplets: (1) Synthesis of carboxylated amylose: amylose and sodium hydroxide are dissolved in water at 60-90°C, followed by addition of chloroacetic acid for 1.5-3.0 hours, followed by lyophilization after dialyzation; (2) Preparation of polysaccharide aggregate droplets: Polydimethylammonium chloride and carboxylated amylose were prepared at 1-4 mg mL -1 The aqueous solution is shaken in a ratio of 1 to 8:2 to form a white emulsion; Step 3: Preparation of artificial tissue containing interstitial cells: The phenylboronic acid functionalized protein vesicle solution obtained in step one and the polysaccharide aggregate droplets obtained in step two are mixed in a volume ratio of 1 to 6:2, and the pH of the system is adjusted to 6 to 8 using a phosphate buffer solution. The system is allowed to stand for 30 to 60 minutes at room temperature to obtain an artificial tissue containing intercellular matrix.

2. The method for preparing an artificial tissue containing intercellular matrix and its application according to claim 1, characterized in that: In step 1 (2), the mass ratio of the amino-modified bovine serum albumin to the polymer containing phenylboronic acid groups is 2:1-4, and the molecular weight cut-off of the dialysis bag used for dialysis is 30-50 kDa.

3. The method for preparing an artificial tissue containing intercellular matrix and its application according to claim 1, characterized in that: In step 1 (3), the preparation of phenylboronic acid functionalized protein vesicles is specifically as follows: using a pipette to draw the building block solution into a centrifuge tube, then adding phosphate buffer, controlling the pH to 6-8, mixing evenly, adding isooctyl alcohol oil phase and shaking vigorously for 30-120s to obtain emulsion vesicles. In addition, if there is a requirement to transfer the aqueous phase, add a final concentration of 0.02-0.1 mg μL before preparation. -1 After the cross-linking reaction was allowed to proceed overnight, the cross-linked reagent was transferred to the aqueous phase by gradient dialysis using different concentrations of ethanol.

4. The method for preparing an artificial tissue containing intercellular matrix and its application according to claim 1, characterized in that: In step 1 (3), the ratio of water to oil is 1:20-40.

5. The method for preparing an artificial tissue containing intercellular matrix and its application according to claim 1, characterized in that: In step 2 (1), the ratio of amylose, sodium hydroxide, water and chloroacetic acid is 100-200 mg: 300-500 mg: 3-10 mL: 200-400 mg; and the molecular weight cut-off of the dialysis bag used for dialysis is 3.0-5.0 kDa.

6. The method for preparing an artificial tissue containing intercellular matrix and its application according to claim 1, characterized in that: In step 2 (1), the molecular weight of the amylose is 10 to 25 kDa.

7. The method for preparing an artificial tissue containing intercellular matrix and its application according to claim 1, characterized in that: In step 2 (2), the pH of the mixed solution is controlled in the range of 6 to 8, which can be controlled by using a phosphate buffer.

8. The method for preparing an artificial tissue containing intercellular matrix and its application according to claim 1, characterized in that: In step 3, the concentration of the phenylboronic acid functionalized protein vesicle solution is 0.1-2.0 mg mL -1 .

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