Pre-assembly solution of conductive bionic vesicles and preparation method of conductive bionic vesicles

Abstract

The invention relates to a pre-assembly solution of conductive bionic vesicles. The pre-assembly solution comprises phospholipid, cholesterol, a conductive monomer, a dopant, water and a first polymerization trigger factor, the first polymerization trigger factor is a combination of hydrogen ions and an oxidant, a combination of hydrogen ions and a bio-enzyme, an oxidant or a bio-enzyme; the conductive monomer is selected from at least one of pyrrole, pyrrole-3-carboxylic acid and the like; the dopant is at least one of phytic acid, polystyrolsulfon acid (PSS), benzene methanesulfonic acid and p-toluenesulfonic acid; the biological enzyme in the first polymerization trigger factor is at least one of catalase CAT, glucose oxidase and horse radish peroxidase HRP; an oxidizing agent in the first polymerization trigger factor is at least one of hydrogen peroxide, ferrous iron and ferric iron. The pre-assembled solution provided by the invention can be used for synthesizing conductive biological vesicles in vivo or in vitro, can be in signal connection with a biological interface for tissue regeneration, improves the reconstruction of a bioelectrical signal or is used for monitoring physiological signals of organoid and cells and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a pre-assembly solution of a conductive bionic vesicle and a method for preparing the conductive bionic vesicle. Background Art

[0002] Conductive polymers (CPs) can provide ideal biointerface connections and have attracted widespread attention in biomedical applications. The dual conduction of electrons and ions is closely related to the transmission mode of living systems, thereby reducing the local electrochemical impedance between biomaterials and living cells. CPs exhibit high stability, stimulus-responsive properties, good biocompatibility, etc. under physiological environmental conditions, can promote peripheral nerve regeneration, and are conductive under physiological conditions. In addition, CPs can be synthesized as independent biomaterials for local delivery of electric fields.

[0003] Biomimetic vesicles are artificial vesicles designed to mimic the structure and function of biological cell membranes. Conductive biomimetic vesicles are biomimetic vesicles with conductive characteristics. These vesicles have the ability to transmit electrons, enabling them to respond to stimuli such as electric fields, currents or electromagnetic waves.

[0004] It is understood that although CP can be directly manufactured in living neural tissues through electrochemical polymerization, it is usually limited by specific tissue structures. Moreover, it is still a huge challenge to adjust the microstructure of the material at the subcellular scale to form conductive biomimetic vesicles between the conductive substrate and cells to provide a seamless biological interface. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a preassembled solution of conductive bionic vesicles and a method for preparing conductive bionic vesicles. The preassembled solution of conductive bionic vesicles can synthesize conductive biological vesicles in vivo or in vitro, which can be connected with biological interface signals for tissue regeneration, improve the reconstruction of bioelectric signals, or be used to monitor the physiological signals of organoids and cells.

[0007] (II) Technical solution

[0008] In a first aspect, the present invention provides a pre-assembled solution of a conductive biomimetic vesicle, which comprises: phospholipids, cholesterol, a conductive monomer, a dopant, water and a first polymerization triggering factor;

[0009] The first polymerization triggering factor is a combination of hydrogen ions and oxidants or a combination of hydrogen ions and biological enzymes; or the first polymerization triggering factor is an oxidant or a biological enzyme;

[0010] The conductive monomer is at least one selected from pyrrole, pyrrole-3-carboxylic acid, 2,2'-dipyrrole, pyrrole-3-sulfonic acid, aniline, p-aminodiphenylamine, thiophene, 3,4-ethylenedioxythiophene, 3,4-ethyldioxypyrrole, pyrrolethiophene copolymer and pyrrole-3,4-ethyldioxythiophene, or a copolymer of any two or more of the foregoing monomers;

[0011] The dopant is at least one of phytic acid, polystyrene sulfonic acid (PSS), toluene sulfonic acid and p-toluene sulfonic acid;

[0012] The biological enzyme in the first polymerization trigger factor is at least one of catalase CAT, glucose oxidase and horseradish peroxidase HRP; the oxidant in the first polymerization trigger factor is hydrogen peroxide (H2O2), divalent iron (Fe 2+ ) and ferric iron (Fe 3+ ) at least one of.

[0013] According to the above pre-assembly solution, it further comprises water, and the pre-assembly solution is formed by taking water as a dispersion solvent.

[0014] According to the above pre-assembled solution, the pre-assembled solution also contains functional active molecules, which are drugs, genes or proteins. The functional active molecules play a role in regulating the biological microenvironment or treating diseases; the proteins can carry oxidoreductases, transferases, hydrolases, lyases, isomerases or synthetases as needed.

[0015] According to the above-mentioned pre-assembly solution, the phospholipid is 1,2-dioleoylphosphatidylcholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA) or phospholipid-polyethylene glycol-thiol (DOPE-PEG-SH) or one of other specific phospholipid functional group derivatives.

[0016] According to the above pre-assembly solution, the mass ratio of phospholipid to cholesterol is 1-10:1-10, and the mass concentrations of phospholipid and cholesterol in the pre-assembly solution are 0.1-5 mg / mL and 0.2-2 mg / mL, respectively.

[0017] According to the above pre-assembly solution, the total concentration of the conductive monomer in the pre-assembly solution is 1-20 mM; the concentration ratio of the conductive monomer to the dopant is 1:0.1-8;

[0018] According to the above pre-assembly solution, when the first polymerization triggering factor contains a biological enzyme, the concentration of the biological enzyme in the pre-assembly solution is 0.1-10 mg / mL;

[0019] When the first polymerization triggering factor contains an oxidant, the concentration of the oxidant in the pre-assembly solution is 1-50 mM; the concentration ratio of the conductive monomer to the oxidant is 1:0.1-5.

[0020] In a second aspect, the present invention provides a method for preparing a pre-assembled solution of conductive biomimetic vesicles, comprising the following steps:

[0021] (1) dissolving phospholipids and cholesterol in an organic solvent A to form a first solution, and mixing by ultrasonication; after the ultrasonic treatment, removing the solvent by rotary evaporation, and drying under vacuum to remove the remaining solvent, thereby obtaining preassembled liposomes of phospholipids and cholesterol;

[0022] (2) dissolving the conductive monomer in solvent B to form a second solution;

[0023] (3) adding the second solution, the dopant acid solution, and the aqueous solution of the first polymerization trigger factor to the preassembled liposomes of step (1), mixing, and obtaining a precursor solution;

[0024] (4) The precursor solution is subjected to a pre-assembly treatment of liposome vesicles, and the particle size of the liposome vesicles is uniformly screened by a particle size control method to obtain a pre-assembly solution.

[0025] According to the above preparation method, organic solvent A is at least one of ethanol, ether, methanol, dichloromethane and chloroform (preferably chloroform); in the first solution, the mass ratio of phospholipid to cholesterol is 1-10:1-10; the mass volume ratio of phospholipid to organic solvent A is 1-10:1-10; in the third solution, the concentration ratio of conductive monomer to dopant is 1:0.1-8; solvent B is at least one of water, ethanol, ether, methanol, dichloromethane and chloroform.

[0026] The mass to volume ratios are all expressed in standard units, such as mass in g, corresponding to volume in L, or mass in mg, corresponding to volume in mL.

[0027] According to the above preparation method, in step (1), the ultrasonic time is ≤60min; the vacuum drying time is ≤48h; the rotary evaporation temperature is ≤70°C; and the vacuum drying temperature is ≤70°C. The ultrasonic time may be ≤10min, 10-20min, 20-30min, 30-40min, 40-50min or 50-60min. Preferably, the ultrasonic time is 10min. The vacuum drying time may be <10h, 10-15h, 15-20h, 20-24h, 24-30h or 30-48h. Preferably, the vacuum drying time is 24h. The rotary evaporation temperature may be <40°C, 40-60°C, 60-70°C. Preferably, the rotary evaporation temperature is 41°C. The drying temperature may be ≤25°C, 25-50°C, 50-70°C. Preferably, the drying temperature is 25°C.

[0028] Preferably, the conductive monomer is at least one of pyrrole, pyrrole-3-carboxylic acid, 2,2'-dipyrrole, pyrrole-3-sulfonic acid, aniline, p-aminodiphenylamine, thiophene and 3,4-ethylenedioxythiophene; in a preferred embodiment, the conductive monomer is pyrrole or pyrrole-3-carboxylic acid.

[0029] Preferably, the phospholipid is at least one of 1,2-dioleoylphosphatidylcholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA) or phospholipid-polyethylene glycol-thiol (DOPE-PEG-SH) or other specific phospholipid functional group derivatives. In a preferred embodiment, the phospholipid is DPPC.

[0030] Preferably, the dopant acid solution is at least one of phytic acid or polystyrene sulfonic acid (PSS) or toluene sulfonic acid or p-toluene sulfonic acid. In a preferred embodiment, the dopant acid solution is a phytic acid solution.

[0031] Preferably, the mass ratio of phospholipid to cholesterol in the first solution is 9:1-2, and the mass volume ratio of phospholipid to organic solvent A is 9:2-4. That is, preferably, 9 mg of phospholipid and 1 or 2 mg of cholesterol are dissolved in 2 mL of chloroform, or in a mixed solution of 2 mL of chloroform and 2 mL of methanol.

[0032] Preferably, the concentration ratio of the conductive monomer to the dopant is 1:0.1-8, for example, 1:0.1-1, 1:0.1-2, 1:0.1-3, 1:0.1-4, 1:0.1-5, 1:0.1-6, 1:0.1-7, 1:0.1-8, preferably 1:1.

[0033] Preferably, the concentration ratio of the conductive monomer to the oxidant is 1:0.1-5, for example, 1:0.1-1, 1:0.1-2, 1:0.1-3, 1:0.1-4 or 1:0.1-5, preferably 1:2.

[0034] According to the above preparation method, the liposome vesicle assembly method in step (4) is one of solvent injection method, thin film hydration method, reverse evaporation method, double emulsion method or microfluidic method.

[0035] According to the above preparation method, the particle size control method in step (4) is a method of using ultrasound, shearing, homogenization and extrusion at a temperature of ≤80°C; the particle size control device is one of a micro-hand-push extruder, a tank-type liposome extruder and a gas-pressurized extruder. The particle size control temperature can be <40°C, 40-60°C, 60-70°C, 70-80°C. Preferably, the particle size control temperature is 41°C.

[0036] In a third aspect, the present invention provides a method for preparing conductive bionic vesicles in vitro, comprising: adding a second polymerization trigger factor to a pre-assembled solution, specifically: when the first polymerization trigger factor in the pre-assembled solution of the conductive bionic vesicle does not include an oxidant, the second polymerization trigger factor includes an oxidant; when the first polymerization trigger factor in the pre-assembled solution of the conductive bionic vesicle does not include a biological enzyme, the second polymerization trigger factor includes a biological enzyme; and based on the above principle, when the first polymerization trigger factor in the pre-assembled solution of the conductive bionic vesicle does not include H + When the second polymerization trigger factor also contains H + ; After polymerization reaction, conductive bionic vesicles are obtained.

[0037] According to the above-mentioned method for preparing conductive biomimetic vesicles in vitro, when the second polymerization trigger factor comprises an oxidant, the oxidant is H2O2, and the concentration of H2O2 is 30-100mM, and the concentration of H2O2 can be 30-50mM, 50-80mM, or 80-100mM. More preferably, the concentration of H2O2 is 100mM.

[0038] When preparing conductive bionic vesicles in vitro, for example, after mixing the pre-assembled solution with the neuronal cells, an appropriate amount of the second polymerization trigger factor is added, and the conductive bionic vesicles are generated by stirring at room temperature for culturing rat dorsal root ganglion cells (DRG) and promoting axonal growth. During the culture period, the concentration of neuronal cells is 0.01-1mM, and the concentration of conductive bionic vesicles is 0.01-0.05mM, 0.05-0.1mM, 0.1-0.5mM, 0.5-1mM, preferably 1mM. For another example, the neurovascular adventitia collagen scaffold is immersed in the pre-assembled solution, and after adding an appropriate amount of the second polymerization trigger factor, it is shaken on a shaker for a period of time, so that the vascular scaffold is fully in contact with the solution and attached to react, so that the conductive bionic vesicles grow on the neurovascular adventitia collagen scaffold, and the effect of neurovascular adventitia reconstruction is achieved. The shaking time of the shaking table can be 12-24h, the shaking time of the shaking table can be 12-16h, 16-20h, 20-24h, preferably 24h.

[0039] When preparing conductive bionic vesicles in vivo, the preassembled solution containing the first polymerization trigger factor (the first polymerization trigger factor is a combination of hydrogen ions and an oxidant) can be directly injected into the injured part of the animal body, and the second polymerization trigger factor (CAT, glucose oxidase or HRP, etc.) generated by the microenvironment of the injured part of the animal is used to provide oxidative polymerization conditions, promote the in situ polymerization of the preassembled solution and generate conductive bionic vesicles at the injured part of the animal body. The conductive bionic vesicles can be used for in situ polymerization of neuronal cells and neural tissues to achieve seamless connection, stimulate the proliferation of neuronal cells and the growth and repair of nerve axons, modify and assemble the interface of engineered blood vessels, promote the reconstruction of the outer membrane of nerve vessels, etc., and the antioxidant enzymes and chemical factors carried by the conductive bionic vesicles can also be used to remove ROS in the damaged area.

[0040] It should be noted that when the conductive biomimetic vesicles are generated by in situ polymerization in vivo, the pre-assembly solution must contain H + ions, but when conducting biomimetic vesicles were prepared by in vitro polymerization, H + It can be added before synthesizing conductive biomimetic vesicles, so the pre-assembly solution does not contain H + ion.

[0041] (III) Beneficial effects

[0042] The conductive bionic vesicles of the present invention are obtained by polymerizing conductive monomers on the template using liposomes as templates in the presence of chemical triggering polymerization factors and enzymes. The conductive bionic vesicles can not only be phagocytosed by immune macrophages, but also reach specific locations for cell-level transduction through targeted delivery, thereby achieving biological interface signal connection with tissue regeneration, improving the reconstruction of bioelectric signals or assembling into flexible electrodes to monitor the physiological signals of organoids and cells, etc. The conductive bionic vesicles can be combined with advanced nano-detection technology to fit tissues as soft interfaces, construct electrochemical sensors, in-situ electrodes, soft electrodes, etc., which provide a platform for monitoring physiological and electrochemical signals; in addition, some therapeutic strategies guided by conductive bionic vesicles can also be applied to immune regulation, inflammation regulation, neural regeneration, angiogenesis regulation, tissue repair and brain-gut axis regulation, etc.

[0043] The pre-assembly solution of the conductive bionic vesicle of the present invention can carry an appropriate amount of conductive monomers, enzyme amounts and enzyme types and chemical factors according to the requirements of the microenvironment in the animal body. It can be synthesized in vitro to obtain conductive bionic vesicles, or injected into the damaged area in the body, and assembled with the damaged tissue in situ under the oxidative polymerization conditions provided by the damaged area. The conductive bionic vesicle has good biocompatibility and biodegradability, and can be seamlessly connected with neuronal cells and neural tissue in situ polymerization to achieve the effect of stimulating the proliferation of neuronal cells and the growth of nerve axons, or used for interface modification and assembly of engineered blood vessels to promote the reconstruction of vascular adventitia. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The toxicity of different concentrations of Py or PyCA monomers on DRG neuronal cells.

[0045] Figure 2 SEM images of conductive biomimetic vesicles PPy CMV and PPyCA CMV assembled in vitro.

[0046] Figure 3 Fluorescence microscopy bright-field images of the conductive biomimetic vesicles PPy CMV and PPyCA CMV promoting the axon growth of DRG neuronal cells in vitro co-culture.

[0047] Figure 4 The reconstruction effect of co-culture of conductive bionic vesicles PPy CMV and PPyCA CMV on the adventitia of neurons.

[0048] Figure 5 Schematic diagram of the platform for monitoring tissue electrochemical signals using conductive bionic vesicles for soft interface modification of gold electrodes. DETAILED DESCRIPTION

[0049] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0050] In living animals, areas of tissue damage are often accompanied by oxidative stress. Increased production of reactive oxygen species (ROS) leads to tissue degeneration and remodeling. In this process, microvesicles (MVs) produced by cells in the tissue microenvironment are closely related to ROS. This relationship occurs because MVs can produce ROS or scavenge the inflammatory response caused by ROS. MVs can carry different antioxidant enzymes (such as catalase (CAT)) to scavenge ROS. CAT is not abundant under normal conditions, but it is overexpressed in damaged areas and plays an important role in tolerance to oxidative stress. Hydrogen peroxide (H2O2) can be trapped at the active site of CAT, where it oxidizes heme iron to produce π-cationic porphyrin radicals. π-cationic porphyrin radicals can initiate the polymerization reaction of monomers. Therefore, the microenvironment of the damaged area of ​​the animal body combined with chemical factors can provide the synthesis conditions for oxidative polymerization to produce conductive bionic vesicles. This provides favorable conditions for the in situ polymerization to generate conductive bionic vesicles, so that the grown conductive bionic vesicles can be seamlessly fitted with nerve repair and engineering vascular modification.

[0051] Therefore, if the in situ generation of conductive biomimetic vesicles is to be triggered in vivo, the pre-assembly solution of conductive biomimetic vesicles should contain a certain amount of oxidants and H + , the oxidant is, for example, H2O2, so that after the pre-assembled solution contacts the CAT enzyme (glucose oxidase or HRP) in the damaged area in vivo, hydrogen peroxide (H2O2) can be trapped at the active site of CAT, oxidizing the heme iron at this site to generate π-cationic porphyrin free radicals, and using the cation free radicals to initiate the polymerization reaction of the monomers, so that the conductive monomers in the pre-assembled solution are polymerized on the liposomes (phospholipid-cholesterol pre-assembled liposomes), and the conductive bionic vesicles are synthesized in situ. If the conductive bionic vesicles are to be synthesized in vitro, a certain amount of CAT (glucose oxidase or HRP) can be added to the pre-assembled solution of the conductive bionic vesicles, and a certain amount of hydrogen peroxide (H2O2) can be added in the in vitro synthesis environment to generate cation free radicals to trigger the oxidative polymerization reaction to synthesize the conductive bionic vesicles; or a certain amount of H2O2 can be added to the pre-assembled solution, and a certain amount of CAT can be artificially added in the in vitro synthesis environment to trigger the oxidative polymerization reaction to synthesize the conductive bionic vesicles.

[0052] To this end, the present invention provides a pre-assembled solution of a conductive biomimetic vesicle, which includes: phospholipids, cholesterol, a conductive monomer, a dopant and a first polymerization trigger; the first polymerization trigger is a combination of hydrogen ions and an oxidant or a combination of hydrogen ions and a biological enzyme; or the first polymerization trigger is an oxidant or a biological enzyme; the conductive monomer is selected from at least one of pyrrole, pyrrole-3-carboxylic acid, 2,2'-dipyrrole, pyrrole-3-sulfonic acid, aniline, p-aminodiphenylamine, thiophene, 3,4-ethylenedioxythiophene, 3,4-ethyldioxypyrrole, pyrrolethiophene copolymer and pyrrole-3,4-ethyldioxythiophene, or a copolymer composed of any two or more of the aforementioned monomers; the dopant is at least one of phytic acid, polystyrene sulfonic acid (PSS), toluenesulfonic acid and p-toluenesulfonic acid; the biological enzyme in the first polymerization trigger is at least one of catalase CAT, glucose oxidase and horseradish peroxidase HRP; the oxidant in the first polymerization trigger is at least one of hydrogen peroxide, divalent iron and trivalent iron. The pre-assembly solution is formed by using water as a dispersing solvent.

[0053] Among them, dopants can improve some properties of polymers, such as: improving conductivity (can make biopolymers better biosensors, biobatteries or biocompatible electronic component materials), enhancing the mechanical strength and toughness of polymers (making them more durable and suitable for biomedical applications that need to withstand certain mechanical stresses, such as tissue engineering scaffolds or wearable medical devices), improving biocompatibility and degradability (reducing immune responses or promoting biodegradation of materials), improving polymer performance (such as higher water solubility, better optical transparency or specific hydrophilic / hydrophobic properties), promoting cell adhesion (doping with PSS can improve the surface properties of biopolymers, making them more conducive to cell adhesion, proliferation and differentiation, thereby promoting tissue regeneration), etc.

[0054] The pre-assembly solution also contains functional active molecules, which are drugs, genes or proteins. Proteins that can be carried are, for example, transferases, hydrolases, lyases, isomerases and synthetases.

[0055] Among them, the phospholipid is 1,2-dioleoylphosphatidylcholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA) or phospholipid-polyethylene glycol-thiol (DOPE-PEG-SH) or one of other specific phospholipid functional group derivatives.

[0056] Wherein, the mass ratio of phospholipid to cholesterol is 1-10:1-10, and the mass concentrations of phospholipid and cholesterol in the pre-assembly solution are 0.1-5 mg / mL and 0.2-2 mg / mL, respectively. The mass concentration of phospholipid is preferably 1-2 mg / mL, 2-3 mg / mL, 3-4 mg / mL or 4-5 mg / mL. The mass concentration of cholesterol is preferably 0.2-0.5 mg / mL or 0.5-1 mg / mL.

[0057] Wherein, the total concentration of the conductive monomer in the pre-assembly solution is 1-10mM; the concentration ratio of the conductive monomer to the dopant is 1:0.1-8; preferably 1:2-5, more preferably 1:2. When the first polymerization trigger factor contains a biological enzyme, the concentration of the biological enzyme in the pre-assembly solution is 0.1-5mg / mL, preferably 0.2-0.5mg / mL or 0.5-1mg / mL. When the first polymerization trigger factor contains an oxidant, the concentration of the oxidant in the pre-assembly solution is 0.1-50mM, preferably 5-10mM; the concentration ratio of the conductive monomer to the oxidant is 1:0.1-5.

[0058] The steps for preparing the pre-assembly solution are as follows:

[0059] (1) Dissolving phospholipids and cholesterol in an organic solvent A to form a first solution, and mixing by ultrasonication; after the ultrasonication, removing the solvent by rotary evaporation, and drying under vacuum to remove the remaining solvent, thereby obtaining preassembled liposomes of phospholipids and cholesterol. The organic solvent A is at least one of ethanol, ether, methanol, dichloromethane and chloroform (preferably chloroform); in the first solution, the mass ratio of phospholipids to cholesterol is 1-10:1-10; and the mass volume ratio of phospholipids to organic solvent A is 1-10:1-10.

[0060] (2) Dissolve the conductive monomer in solvent B to form a second solution.

[0061] (3) adding the second solution, the dopant acid solution, and the aqueous solution of the first polymerization trigger factor to the preassembled liposomes of step (1), mixing, and obtaining a precursor solution;

[0062] (4) The precursor solution is subjected to a pre-assembly treatment of liposome vesicles, and the particle size of the liposome vesicles is uniformly screened by a particle size control method to obtain a pre-assembly solution.

[0063] Furthermore, the volume of organic solvent A is 1-10 mL, the mass of phospholipids is 1-10 mg, and the mass of cholesterol is 1-10 mg. The volume of solvent B is 1-10 mL. Solvent B is at least one of water, ethanol, ether, methanol, dichloromethane and chloroform, preferably water. The concentration ratio of the conductive monomer to the dopant is 1:0.1-8. When the first polymerization trigger factor is an oxidant, the concentration ratio of the conductive monomer to the oxidant is 1:0.1-5. In step (1), the ultrasonic time is ≤60 min; the vacuum drying time is ≤48 h; the rotary evaporation temperature is ≤70 ° C; the vacuum drying temperature is ≤70 ° C.

[0064] In a preferred embodiment, the dopant acid solution is a phytic acid solution.

[0065] Preferably, the mass ratio of phospholipid to cholesterol in the first solution is 9:1-2, and the mass volume ratio of phospholipid to organic solvent A is 9:2-4. That is, preferably, 9 mg of phospholipid and 1 or 2 mg of cholesterol are dissolved in 2 mL of chloroform, or in a mixed solution of 2 mL of chloroform and 2 mL of methanol.

[0066] The liposome vesicle assembly method in step (4) is one of solvent injection method, thin film hydration method, reverse evaporation method, emulsion method or microfluidic method, the diameter control method is a method of using ultrasound, shearing, homogenization and extrusion at a temperature of ≤80°C; the particle size control device is one of a micro-hand-push extruder, a tank-type liposome extruder and a gas-pressurized extruder. Among them, the particle size control temperature can be <40°C, 40-60°C, 60-70°C, 70-80°C. Preferably, the particle size control temperature is 41°C.

[0067] The preassembled solution prepared by the above method can synthesize conductive bionic vesicles under in vitro polymerization triggering conditions, or can be injected into the damaged area in an animal body to synthesize conductive bionic vesicles under the triggering conditions provided by the damaged area.

[0068] The following is a description of the preferred embodiments of the present invention.

[0069] Example 1

[0070] This example investigates the biocompatibility of pyrrole monomer (Py) and pyrrole carboxylic acid (pyrrole-3-carboxylic acid) monomer (PyCA), and the investigation method is as follows: primary DRG neuron cells were cultured for 5 days and then incubated with Py or PyCA monomer (0.5mM to 10mM, PBS solution) for 24h. After washing three times with PBS solution (pH 7.4), the cells were stained with a Live / Dead staining kit and observed under a fluorescence microscope.

[0071] The experimental results are as follows Figure 1As shown, no decrease in cell viability was observed with Py or PyCA at Py or PyCA monomer concentrations up to 1 mM (e.g. Figure 1 A, B), indicating that at low concentrations (1 mM), Py or PyCA were non-toxic to DRG neuronal cells; when the PyCA monomer concentration was as high as 5 mM, only a slight reduction in cell viability was observed, and at high concentrations (2-5 mM), PyCA showed significantly better biocompatibility than Py (see Figure 1 C, D).

[0072] Example 2

[0073] In this embodiment, conductive bionic vesicles are assembled in vitro, and the method is as follows:

[0074] (1) Weigh 9 mg of phospholipid DPPC and 2 mg of cholesterol and dissolve them in 2 mL of chloroform and 2 mL of methanol solution. Dissolve them completely by ultrasonication, remove the solvent by rotary evaporation, and dry them in a vacuum oven at 25°C overnight to remove the remaining solvent until they are completely dry, so that the phospholipid and cholesterol are preassembled to obtain preassembled liposomes.

[0075] (2) Pyrrole monomer (Py) and pyrrole carboxylic acid (pyrrole-3-carboxylic acid) monomer (PyCA) were dissolved in water to obtain a conductive monomer solution with a concentration of 50 mM. Phytic acid was dissolved in water to obtain a phytic acid solution with a concentration of 100 mM. CAT enzyme was dissolved in water to obtain a CAT enzyme solution with a concentration of 0.2 mg / mL.

[0076] (3) Add the conductive monomer solution, phytic acid solution, and CAT enzyme solution to the preassembled liposomes in step (1) at a volume ratio of 1:1:2 to make the final solution volume reach 5 mL.

[0077] (4) Hydrate in an aqueous solution at 41°C for 1 h, and repeatedly extrude and screen using a 3 μm PC membrane at 41°C to form a pre-assembled solution.

[0078] (5) Add 100 mM hydrogen peroxide solution to the pre-assembly solution, react for 24 h, and observe the morphology under an electron microscope. Figure 2 The two types of conductive biomimetic vesicles shown are marked as the Control group without adding any polymerized monomers; according to the different conductive monomers, the conductive biomimetic vesicles are marked as PPy CMV and PPyCA CMV.

[0079] Example 3

[0080] The DRG neuronal cells were cultured with the conductive bionic vesicles prepared in Example 2, with a neuronal cell concentration of 1 mM and a conductive bionic vesicle concentration of 1 mM, and cultured in a 37°C constant temperature incubator (containing 5% CO2) for 24 hours. During the experiment, the cultured neuronal cells without any conductive vesicles were recorded as the Control group, and the experimental group was cultured by adding PPy CMV and PPyCA CMV when the cultured neuronal cells adhered to the wall.

[0081] The cell growth was observed under bright field fluorescence microscope. Figure 3 As shown in the figure, PPy CMV and PPyCA CMV can promote the growth of axons compared with the control group. Figure 3 It can be seen that culturing DRG neurons with PPy CMV and PPyCA CMV can promote the growth of axons, while the growth of axons in DRG neurons without conductive vesicles is relatively slow. Compared with PPy CMV, culturing with PPyCA CMV has a better effect on promoting the growth of axons.

[0082] Example 4

[0083] A preassembly solution was prepared according to the method of Example 2, and the neurovascular adventitia collagen scaffold was immersed in the preassembly solution, and 100 mM H2O2 solution was added, and the mixture was shaken on a shaker at 100 times per minute for 24 hours. The reaction temperature was 37°C, and the mixture was slowly rinsed twice with PBS. The neurovascular adventitia collagen scaffold was taken out, and the growth of conductive vesicles was observed under an electron microscope. During the experiment, the vascular scaffold cultured without any conductive vesicles was set as the Control group, and the experimental group was the vascular scaffold cultured with the conductive vesicles PPyCMV and PPyCA CMV.

[0084] Observation results such as Figure 4 As shown, compared with the control group, the surface of the neurovascular adventitia collagen scaffold cultured with PPy CMV and PPyCA CMV became rougher, indicating that loosely structured collagen fibers were formed on the neurovascular adventitia collagen scaffold, achieving the effect of neurovascular adventitia reconstruction. Among them, compared with PPy CMV, PPyCA CMV had a more obvious effect on the reconstruction of the neurovascular adventitia.

[0085] Example 5

[0086] The pre-assembly solution was prepared according to the method of Example 2, and the "9 mg phospholipid DPPC" in step (1) of Example 2 was replaced with an equal mass of DOPE-PEG-SH phospholipid to obtain a pre-assembly solution. A homemade electrode, a gold electrode with a width and spacing of 100 μm on the substrate, was added to the pre-assembly solution, and a 100 mM H2O2 solution was added, and the polymerization reaction was carried out at room temperature for 72 hours. The excess solution not coupled to the gold electrode was washed off with distilled water, and the monitoring electrode with a soft electrode interface was obtained after drying.

[0087] SD rats were used to establish the sciatic nerve injury model. The model establishment method was as follows: 3 adult male SD rats (weight distribution ranged from 200-230g) were anesthetized, the sciatic nerve of the left leg was exposed, and fine forceps were used to squeeze for 30s to cause sciatic nerve injury. ROS was detected at the sciatic nerve injury site, and the successful establishment of the sciatic nerve injury model could be confirmed by detecting the ROS level.

[0088] The pre-assembled solution was injected into the injured area of ​​the sciatic nerve of the model rat using a syringe. 2 The pre-assembled solution was injected into the injured part of the sciatic nerve of the model rat by an injection needle, and the conductive polymer was polymerized in situ. Then, the sample was cut into appropriate sizes and placed between the prepared monitoring electrodes with soft electrode interface. Figure 5 The monitoring platform was built in the manner shown in the figure. The experimental results showed that the electrochemical signals of the tissue could indeed be monitored through the monitoring electrode with a soft electrode interface, which indicated that the soft electrode interface was conductive, and the pre-assembled solution was injected into the sciatic nerve injury site of the model rat, and conductive vesicles could be synthesized in situ under the induction of the injury environment.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pre-assembled solution of conductive biomimetic vesicles, characterized in that: It includes: Phospholipids, cholesterol, conductive monomers, dopants, water and a first polymerization trigger; The first polymerization triggering factor is a combination of hydrogen ions and oxidants or a combination of hydrogen ions and biological enzymes; or the first polymerization triggering factor is an oxidant or a biological enzyme; The conductive monomer is at least one selected from pyrrole, pyrrole-3-carboxylic acid, 2,2'-dipyrrole, pyrrole-3-sulfonic acid, aniline, p-aminodiphenylamine, thiophene, 3,4-ethylenedioxythiophene, 3,4-ethyldioxypyrrole, pyrrolethiophene copolymer and pyrrole-3,4-ethyldioxythiophene, or a copolymer of any two or more of the foregoing monomers; The dopant is at least one of phytic acid, polystyrene sulfonic acid (PSS), toluene sulfonic acid and p-toluene sulfonic acid; The biological enzyme in the first polymerization triggering factor is at least one of catalase CAT, glucose oxidase and horseradish peroxidase HRP; The oxidant in the first polymerization triggering factor is at least one of hydrogen peroxide, divalent iron and trivalent iron.

2. The pre-assembled solution according to claim 1, characterized in that: The pre-assembly solution also contains functionally active molecules, which are drugs, genes or proteins.

3. The pre-assembled solution according to claim 1, characterized in that: The phospholipid is one of 1,2-dioleoylphosphatidylcholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA) or phospholipid-polyethylene glycol-thiol (DOPE-PEG-SH) or other phospholipid functional group derivatives.

4. The pre-assembled solution according to claim 1, characterized in that: The mass ratio of phospholipid to cholesterol is 1-10:1-10, and the mass concentrations of phospholipid and cholesterol in the pre-assembly solution are 0.1-5 mg / mL and 0.2-2 mg / mL, respectively.

5. The pre-assembly solution according to claim 1, characterized in that: The total concentration of the conductive monomer in the pre-assembly solution is 1-20 mM; the concentration ratio of the conductive monomer to the dopant is 1:0.1-8; when the first polymerization trigger factor contains a biological enzyme, the concentration of the biological enzyme in the pre-assembly solution is 0.1-10 mg / mL; when the first polymerization trigger factor contains an oxidant, the concentration of the oxidant in the pre-assembly solution is 1-50 mM; the concentration ratio of the conductive monomer to the oxidant is 1:0.1-5.

6. A method for preparing a pre-assembled solution of conductive biomimetic vesicles according to any one of claims 1 to 5, characterized in that: Here are the steps: (1) dissolving phospholipids and cholesterol in an organic solvent A to form a first solution, and mixing by ultrasonication; after the ultrasonic treatment, removing the solvent by rotary evaporation, and drying under vacuum to remove the remaining solvent, thereby obtaining preassembled liposomes of phospholipids and cholesterol; (2) dissolving the conductive monomer in solvent B to form a second solution; (3) adding the second solution, the dopant acid solution, and the aqueous solution of the first polymerization trigger factor to the preassembled liposomes of step (1), mixing, and obtaining a precursor solution; (4) The precursor solution is subjected to a pre-assembly treatment of liposome vesicles, and the particle size of the liposome vesicles is uniformly screened by a particle size control method to obtain a pre-assembly solution.

7. The preparation method according to claim 6, characterized in that: In step (1), the organic solvent A is at least one of ethanol, ether, methanol, dichloromethane and chloroform; in the first solution, the mass ratio of phospholipid to cholesterol is 1-10:1-10; the mass volume ratio of phospholipid to organic solvent A is 1-10:1-10; In step (1), the ultrasonic time is ≤60 min; the vacuum drying time is ≤48 h; the rotary evaporation temperature is ≤70° C.; and the vacuum drying temperature is ≤70° C.; Solvent B is at least one of water, ethanol, ether, methanol, dichloromethane and chloroform; In the precursor solution, the concentration ratio of the conductive monomer to the dopant is 1:0.1-8.

8. The preparation method according to claim 6, characterized in that: In step (4), the pre-assembly treatment method is one of solvent injection method, thin film hydration method, reverse evaporation method, emulsion method or microfluidic method; the particle size control method is a method using ultrasound, shearing, homogenization and extrusion at a temperature of ≤80°C; the particle size control equipment is one of a micro-hand-push extruder, a tank-type liposome extruder and a gas-pressurized extruder.

9. A method for preparing conductive bionic vesicles in vitro, characterized in that: include: A second polymerization triggering factor is added to the pre-assembled solution of the conductive biomimetic vesicle according to any one of claims 1 to 5, specifically: when the first polymerization triggering factor in the pre-assembled solution of the conductive biomimetic vesicle does not include an oxidant, the second polymerization triggering factor includes an oxidant; when the first polymerization triggering factor in the pre-assembled solution of the conductive biomimetic vesicle does not include a biological enzyme, the second polymerization triggering factor includes a biological enzyme; and based on the above principle, when the first polymerization triggering factor in the pre-assembled solution of the conductive biomimetic vesicle does not include H + When the second polymerization trigger factor also contains H + ; After polymerization reaction, conductive bionic vesicles are obtained.

Images