Artificial cell and preparation method thereof, and bionic tissue and preparation method thereof

Abstract

The invention discloses an artificial cell and a preparation method thereof as well as a bionic tissue and a preparation method thereof, an aqueous dispersion of a negative electricity nanometer material is sprayed into an aqueous phase receiving bath containing a cationic polyelectrolyte and a surfactant through a gas-liquid shearing microfluidic technology, and one-step construction of an artificial cell membrane and a cytoskeleton is realized. According to the method, the internal skeleton enhanced artificial cell is constructed in a water phase by a one-step method by utilizing the electrostatic interaction of the negative electricity nano material and the cationic polyelectrolyte, and the method has the characteristics of convenience in preparation and excellent stability. By introducing different functional materials, multifunctional integration of the artificial cell can be realized. Meanwhile, the adhesion inducer is added, so that a bionic tissue can be further constructed, and the bionic tissue integrates excellent mechanical properties of the artificial cells and can bear disturbance to a certain degree without disintegration.

Description

Technical Field

[0001] This invention belongs to the field of artificial cell and biomimetic tissue preparation, and relates to a method for in-situ construction of artificial cells and biomimetic tissues in an aqueous phase using gas-liquid shear microfluidics technology. Background Technology

[0002] Artificial cells, as miniature biomimetic systems that mimic the structure and function of natural cells, not only provide simplified models for exploring complex physiological processes within cells, but also demonstrate enormous application potential in research on the origin of life, cell engineering, biosensors, and drug delivery, becoming a frontier in life science research. Currently, the construction of artificial cells is mainly based on two methods: top-down and bottom-up. The top-down method removes or replaces certain components in cells using physical or chemical means to obtain the smallest units capable of performing basic life activities. This approach can preserve the biological functions of real cells to the greatest extent and has good biocompatibility, but the highly precise and complex operations involved, high costs, and potential ethical issues limit its widespread application. Therefore, current research mainly focuses on the bottom-up method, which uses the most basic chemical substances to gradually construct structures with cellular characteristics, providing extremely high design flexibility for artificial cell construction. Various artificial cell assemblies, such as liposomes, polymers, colloids, and condensates, have been constructed based on the bottom-up method, but their lower mechanical strength and stability limit the further development and application of artificial cells. To address this challenge, researchers drew inspiration from the complex network structure within cells—the cytoskeleton. The cytoskeleton not only maintains cell morphology but also provides essential mechanical strength. By constructing the cytoskeleton within artificial cells, researchers successfully improved the long-term stability of these artificial cells. However, existing artificial cytoskeleton mimics, such as DNA, proteins, metal-polyphenol backbones, and synthetic polymers, typically suffer from poor mechanical stability, complex construction methods, and high costs, making them unsuitable for large-scale production. Therefore, developing a simple and efficient method to prepare artificial cells with superior mechanical properties has become an urgent problem to be solved. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide an artificial cell and its preparation, and a biomimetic tissue and its preparation method. By using gas-liquid shear microfluidics, an aqueous dispersion of negatively charged nanomaterials is sprayed into an aqueous receiving bath containing cationic polyelectrolytes and surfactants to achieve one-step construction of artificial cell membranes and cytoskeleton. The prepared artificial cells have excellent mechanical stability and functional modification properties. Furthermore, by adding an adhesion inducer to perform interfacial adhesion on the artificial cells, a one-step biomimetic tissue with specific structure and excellent mechanical strength is constructed.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] The method for preparing artificial cells includes the following steps:

[0006] S11) An extruded phase solution A1 was prepared using negatively charged nanomaterials and ultrapure water;

[0007] S12) The receiving bath solution C1 is prepared using cationic polyelectrolytes, surfactants and ultrapure water;

[0008] S13) The extrusion phase solution A1 obtained in step S11) is pumped into the receiving bath solution C1 using a syringe pump and a coaxial needle to obtain artificial cells. The specific operation is as follows:

[0009] The extruded phase solution A1 is pumped into the inner layer of the coaxial needle using a syringe pump. When the extruded phase solution A1 forms droplets D1 at the outlet of the inner layer needle, the shearing action of the gas blown from the outer layer needle breaks the droplets D1 into uniform microdroplets. The microdroplets fall into the receiving bath solution C1 to form artificial cell microcapsules Cell-1, which are artificial cells. The gas blown from the outer layer needle is air or an inert gas, and the flow rate of the gas blown from the outer layer needle is controlled by a rotor flow meter.

[0010] In the above-mentioned method for preparing artificial cells, the negatively charged nanomaterial is a water-dispersible nanomaterial with a negatively modified surface, including cellulose nanofibers; in the extrusion phase solution A1, the weight of the negatively charged nanomaterial is 0.05 to 5% of the weight of the extrusion phase solution A1.

[0011] The above-mentioned method for preparing artificial cells also includes one or more of the following water-dispersible nanomaterials: graphene oxide, MXene, Fe3O4 nanoparticles, gold nanoparticles, and silica nanoparticles.

[0012] In the above-mentioned method for preparing artificial cells, the cationic polyelectrolyte is one or more of polydimethyldiallylammonium chloride, chitosan, polyethyleneimine, polylysine, cationic polyacrylamide, and polyquaternary ammonium salt acrylate / methacrylate; in the receiving bath solution C1, the weight of the cationic polyelectrolyte is 0.05 to 5% of the weight of the receiving bath solution C1.

[0013] In the above-mentioned method for preparing artificial cells, the surfactant in the receiving bath solution C1 is an ionic surfactant and / or a nonionic surfactant, and the weight of the surfactant is 0.01% to 1% of the weight of the receiving bath solution C1. Ionic surfactants include anionic surfactants, cationic surfactants, and zwitterionic surfactants. Anionic surfactants include sulfonates, sulfates, carboxylates, phosphates, and N-acylaminocarboxylates, etc. Cationic surfactants include quaternary ammonium salts, quaternary phosphine salts, and ammonium oxides, etc. Zwitterionic surfactants include amino acids, betaines, and lecithin, etc.; nonionic surfactants include polyoxyethylene, polyols, alkanolamides, and polyethers, etc.

[0014] In the above-mentioned method for preparing artificial cells, the injection rate of the extrusion phase solution A1 is 10-30 mL / h, the gas flow rate blown out from the outer needle is 0.5-1.0 L / min, the outer needle specification is between 12G and 18G, and the inner needle specification is between 17G and 30G.

[0015] Artificial cells, wherein the artificial cells are artificial cells prepared by the above-described artificial cell preparation method.

[0016] The method for preparing biomimetic tissue includes the following steps:

[0017] S21) Extrusion phase solution B is prepared by using extrusion phase solution A2 and adhesion inducer, wherein extrusion phase solution A2 is extrusion phase solution A1 in claim 1;

[0018] S22) The extruded phase solution B obtained in step S21) is pumped into the receiving bath solution C2 using a syringe pump and a coaxial needle to obtain biomimetic tissue. The specific operation is as follows:

[0019] The extruded phase solution B is pumped into the inner layer of the coaxial needle using a syringe pump. When the extruded phase solution B forms droplets D2 at the outlet of the inner layer needle, the shearing action of the gas blown from the outer layer needle breaks the droplets D2 into uniform microdroplets. The microdroplets fall into the receiving bath solution C2 to form artificial cell microcapsules Cell-2. Under the action of an adhesion inducer, the artificial cell microcapsules Cell-2 spontaneously adhere in the receiving bath solution C2 to form biomimetic tissue. The gas blown from the outer layer needle is air or an inert gas, and the flow rate of the gas blown from the outer layer needle is controlled by a rotor flow meter. The receiving bath solution is the receiving bath solution C1 as described in claim 1.

[0020] In the above-mentioned biomimetic tissue preparation method, the extrusion phase solution B is prepared by mixing the extrusion phase solution A2 and the adhesion inducer at a volume ratio of 4:1. The adhesion inducer is a strong electrolyte solution with a concentration of 0.1–1.0 M or a hydrophobic protein aqueous solution with a concentration of 0.5–5 mg / mL. The strong electrolytes include sodium chloride, calcium chloride, and magnesium chloride, etc., and the hydrophobic proteins include horseradish peroxidase, glucose oxidase, and various membrane proteins.

[0021] Bionic tissue, wherein the bionic tissue is a bionic tissue prepared using the above-described bionic tissue preparation method.

[0022] The technical solution of the present invention achieves the following beneficial technical effects:

[0023] 1. This invention develops a low-cost, high-volume method for preparing artificial cell microcapsules with excellent mechanical properties. Compared with traditional artificial cell systems, this system has advantages such as convenient preparation, simple composition, and easy functionalization.

[0024] 2. This method can achieve one-step formation of artificial cell membranes and cytoskeleton, greatly simplifying the construction method of artificial cytoskeleton. Furthermore, artificial cells constructed by this method have excellent stability and can exist stably in aquatic environments for a long time.

[0025] 3. By adjusting the composition of the extrusion liquid, various functional materials can be introduced to achieve functional modification of artificial cell microcapsules. By adding adhesion inducers, a one-step biomimetic tissue can be constructed. This biomimetic tissue integrates the excellent mechanical properties of microcapsules, has good stability in an aqueous environment, and can withstand a certain degree of disturbance without disintegrating. Attached Figure Description

[0026] Figure 1-a The image shows a scanning electron microscope image of the artificial cell microcapsules prepared in Example 1 after lyophilization.

[0027] Figure 1-b The image shows a scanning electron microscope image of the artificial cell microcapsules prepared in Example 2 after lyophilization.

[0028] Figure 2-a Laser scanning confocal images of the artificial cell microcapsules prepared in Example 1;

[0029] Figure 2-b This is a laser scanning confocal image of the artificial cell microcapsules prepared in Example 2;

[0030] Figure 3 An optical photograph showing the directional movement of the artificial cell microcapsules prepared in Example 3 by attracting them with a magnet;

[0031] Figure 4 Optical photographs of biomimetic tissues constructed using artificial cell microcapsules;

[0032] Figure 5 This is a scanning electron microscope image of the biomimetic tissue prepared in Example 4. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. Obviously, the described embodiments are only some preferred embodiments of the present invention, and the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels.

[0034] Example 1

[0035] Prepare an aqueous dispersion of approximately 1 wt% cellulose nanofibers (CNF), and after ultrasonic defoaming, use it as the extrusion phase solution A1. Prepare an aqueous solution containing 0.5 wt% polydimethyldiallylammonium chloride (PDDA) and 0.1 wt% Triton X-100 as the receiving bath solution C1. Draw up the extrusion phase solution A1 with a syringe, place the syringe on the syringe pump, and connect the outlet to the inner needle of the coaxial needle using a PVC tube. Connect the lower inlet of the rotor flowmeter to the nitrogen cylinder with a silicone tube, and the upper inlet to the outer needle of the coaxial needle. Adjust the syringe pump flow rate to 30 mL / h, and adjust the rotor flowmeter to maintain the nitrogen flow rate at approximately 1.0 L / min. Fix the coaxial needle directly above the receiving bath solution C1 and keep it vertical. First, start the syringe pump to spray droplets. After the extruded liquid delivery stabilizes, begin collecting until all the extruded liquid is sprayed into the receiving bath solution C1. At this point, the microcapsules in the receiving bath solution C1 sink to the bottom of the beaker under the influence of gravity. Slowly pour out most of the receiving bath solution, add ultrapure water to wash, and repeat three times to obtain stable artificial cell microcapsules Cell-1, which are artificial cells.

[0036] Example 2

[0037] The specific implementation steps are the same as in Example 1, wherein the extruded phase solution A1 is a mixed solution prepared by mixing 1 wt% CNF aqueous dispersion and 2 mg / mL graphene oxide (GO) aqueous dispersion in a volume ratio of 4:1.

[0038] Example 3

[0039] The specific implementation steps are the same as in Example 1, wherein the extrusion phase solution A1 is a mixed solution prepared by 1 wt% CNF aqueous dispersion and 5 mg / mL iron(Fe3O4) nanoparticle aqueous dispersion in a volume ratio of 4:1.

[0040] Example 4

[0041] The specific implementation steps are the same as in Example 1, except that the extrusion phase solution B is a mixed solution prepared by mixing 1 wt% CNF aqueous dispersion, 2 mg / mL GO aqueous dispersion, and 1 M NaCl aqueous solution in a volume ratio of 3:1:1. This enables the adhesion between artificial cell microcapsules to prepare biomimetic tissues.

[0042] Performance testing

[0043] (1) Scanning electron microscope image (SEM)

[0044] First, the artificial cell microcapsules Cell-1 prepared in Examples 1 and 2 were freeze-dried using freeze-drying technology. Then, the morphology of the freeze-dried artificial cell microcapsules Cell-1 was analyzed using scanning electron microscopy. Figure 1-a The morphology of CNF / PDDA lyophilized artificial cell microcapsules Cell-1 was shown. Figure 1-b The morphology of CNF-GO / PDDA lyophilized artificial cell microcapsules Cell-1 is shown. Figures 1-a to 1-b It can be seen that the prepared artificial cell microcapsules Cell-1 all exhibit a dense membrane layer and an internal three-dimensional network structure.

[0045] (2) Laser Scanning Confocal Microscope (LSCM)

[0046] The structure and composition of the artificial cell microcapsules Cell-1 prepared in Examples 1 and 2 were analyzed using laser scanning confocal microscopy. During the preparation of the artificial cell microcapsules Cell-1 used for LSCM characterization, each component was mixed with 10 wt% fluorescently labeled nanomaterials. CNF was labeled with FITC (fluorescein isothiocyanate, green fluorescence), and GO was labeled with Pyrene (pyrene, blue fluorescence). Figure 2-a Fluorescence images of the CNF / PDDA artificial cell microcapsule Cell-1 are shown. Figure 2-b Fluorescence images of the CNF-GO / PDDA artificial cell microcapsule Cell-1 are shown. Figure 2-a It is known that a large amount of CNF is distributed on the dense membrane layer and participates in the composition of the artificial cell membrane, while some CNF that does not participate in assembly is distributed inside the microcapsule as contents, responsible for maintaining the morphology of the artificial cell. Figure 2-b It can be seen that the distribution of CNF in CNF-GO / PDDA artificial cell microcapsule Cell-1 is similar to that in CNF / PDDA artificial cell microcapsule Cell-1, while GO is mainly concentrated on the dense membrane layer and is responsible for regulating the mechanical properties and permeability of the dense membrane layer.

[0047] (3) Macro photos

[0048] The magnetic response and motion behavior of the magnetic artificial cells prepared in Example 3 were photographed using a camera. The results are shown below. Figure 3 .Depend on Figure 3 It is known that magnetic artificial cells can move in a directional manner under the attraction of a magnet.

[0049] The preparation process of the biomimetic tissue obtained in Example 4 was photographed using a camera, and the results are shown below. Figure 4 The structure of the prepared biomimetic tissue was analyzed using scanning electron microscopy, and the results are shown in [Figure number missing]. Figure 5 .Depend on Figure 4 It can be seen that as the artificial cells generated by gas-liquid shearing fall into the receiving bath solution, the artificial cells continuously accumulate and adhere at the bottom, gradually forming a tissue-like structure with a certain structural strength. Furthermore, this biomimetic tissue remains stable without disintegrating under slight external disturbances (such as shaking of the receiving bath solution containing the biomimetic tissue, or slight prodding or stirring of the biomimetic tissue by other instruments). Figure 5 It is known that there are adhesion structures between artificial cells in biomimetic tissues, such as... Figure 5 The part enclosed by the dotted line is the reason why the adhesion structure between artificial cells can maintain the structural and morphological stability of the biomimetic tissue under slight external disturbances.

[0050] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for preparing artificial cells, characterized in that, Includes the following steps: S11) An extruded phase solution A1 was prepared using negatively charged nanomaterials and ultrapure water; S12) The receiving bath solution C1 is prepared using cationic polyelectrolytes, surfactants and ultrapure water; S13) The extrusion phase solution A1 obtained in step S11) is pumped into the receiving bath solution C1 using a syringe pump and a coaxial needle to obtain artificial cells. The specific operation is as follows: The extruded phase solution A1 is pumped into the inner layer of the coaxial needle using a syringe pump. When the extruded phase solution A1 forms droplets D1 at the outlet of the inner layer needle, the shearing action of the gas blown from the outer layer needle breaks the droplets D1 into uniform microdroplets. The microdroplets fall into the receiving bath solution C1 to form artificial cell microcapsules Cell-1, which are artificial cells. The gas blown from the outer layer needle is air or an inert gas, and the flow rate of the gas blown from the outer layer needle is controlled by a rotor flow meter.

2. The method for preparing artificial cells according to claim 1, characterized in that, The negatively charged nanomaterials are water-dispersible nanomaterials with negatively charged surfaces, including cellulose nanofibers; in the extruded phase solution A1, the weight of the negatively charged nanomaterials is 0.05 to 5% of the weight of the extruded phase solution A1.

3. The method for preparing artificial cells according to claim 2, characterized in that, Water-dispersible nanomaterials also include one or more of graphene oxide, MXene, Fe3O4 nanoparticles, gold nanoparticles, and silica nanoparticles.

4. The method for preparing artificial cells according to claim 1, characterized in that, The cationic polyelectrolyte is one or more of polydimethyldiallylammonium chloride, chitosan, polyethyleneimine, polylysine, cationic polyacrylamide, and polyquaternary ammonium salt acrylate / methacrylate; in the receiving bath solution C1, the weight of the cationic polyelectrolyte is 0.05 to 5% of the weight of the receiving bath solution C1.

5. The method for preparing artificial cells according to claim 1, characterized in that, In the receiving bath solution C1, the surfactant is an ionic surfactant and / or a nonionic surfactant, and the weight of the surfactant is 0.01 to 1% of the weight of the receiving bath solution C1.

6. The method for preparing artificial cells according to claim 1, characterized in that, The injection rate of the extruded phase solution A1 is 10-30 mL / h, the gas flow rate blown from the outer needle is 0.5-1.0 L / min, the outer needle specification is between 12G and 18G, and the inner needle specification is between 17G and 30G.

7. An artificial cell, characterized in that, The artificial cell is an artificial cell prepared using the artificial cell preparation method described in claim 1.

8. A method for preparing biomimetic tissue, characterized in that, Includes the following steps: S21) Extrusion phase solution B is prepared by using extrusion phase solution A2 and adhesion inducer, wherein extrusion phase solution A2 is extrusion phase solution A1 in claim 1; S22) The extruded phase solution B obtained in step S21) is pumped into the receiving bath solution C2 using a syringe pump and a coaxial needle to obtain biomimetic tissue. The specific operation is as follows: The extruded phase solution B is pumped into the inner layer of the coaxial needle using a syringe pump. When the extruded phase solution B forms droplets D2 at the outlet of the inner layer needle, the shearing action of the gas blown from the outer layer needle breaks the droplets D2 into uniform microdroplets. The microdroplets fall into the receiving bath solution C2 to form artificial cell microcapsules Cell-2. Under the action of an adhesion inducer, the artificial cell microcapsules Cell-2 spontaneously adhere in the receiving bath solution C2 to form biomimetic tissue. The gas blown from the outer layer needle is air or an inert gas, and the flow rate of the gas blown from the outer layer needle is controlled by a rotor flow meter. The receiving bath solution is the receiving bath solution C1 as described in claim 1.

9. The biomimetic tissue preparation method according to claim 8, characterized in that, The extrusion phase solution B is prepared by mixing the extrusion phase solution A2 with the adhesion inducer at a volume ratio of 4:

1. The adhesion inducer is a strong electrolyte solution with a concentration of 0.1 to 1.0 M or a hydrophobic protein pure aqueous solution with a concentration of 0.5 to 5 mg / mL.

10. A biomimetic tissue, characterized in that, The biomimetic tissue is a biomimetic tissue prepared using the biomimetic tissue preparation method described in claim 8.

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