Conductive phospholipid microtube material as well as preparation method and application thereof
By using a method of combining phospholipids with conductive polymers, conductive phospholipid microtubule materials were prepared, which solved the problem of poor biocompatibility of existing electrical stimulating materials, and achieved efficient neuroaxon regeneration and improved therapeutic effects.
Patent Information
- Application Number
- CN202411641213.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-04-01
AI Technical Summary
The existing electrically stimulating materials have poor biocompatibility, resulting in safety hazards and irreversible damage when used in vivo.
Using phospholipids as the substrate, the conductive phospholipid microtubule material is prepared by liposome extrusion technology and polymerization. The material is coated with a submicron-scale phospholipid vesicle matrix layer and a conductive polymer, and has excellent biocompatibility and electrical conductivity.
This material can effectively promote the growth and regeneration of neural axons, improve the biocompatibility problems of conductive materials in traditional electrical stimulation therapy, reduce patient pain and medical costs, and improve treatment effect.
Smart Images
Figure CN119733106A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of tissue engineering materials, and in particular relates to a conductive phospholipid microtubule material and a preparation method and application thereof. Background Art
[0002] Peripheral nerve injury usually leads to motor and sensory dysfunction in the corresponding area, with a high disability rate, affecting the quality of life of patients and bringing heavy medical and economic burdens to society. The repair of long-distance nerve defects has been a long-standing clinical problem. Axonal degeneration and inflammatory response of damaged nerve tissue gradually cause neuronal degeneration and even necrosis; Schwann cells and fibroblasts at the site of injury interact with each other to form connective tissue scars, which hinder axon regeneration and reach distal target organs. Therefore, how to effectively promote nerve tissue regeneration, explore nerve repair materials from the perspective of nerve regeneration mechanism and combine them with tissue engineering technology, which provides a new idea for achieving regeneration of damaged nerve tissue and functional reconstruction.
[0003] Electrical stimulation therapy is a known treatment method that can promote the regeneration of nerve tissue. Appropriate electrical stimulation can accelerate the repair of peripheral nerve damage by guiding Schwann cells to migrate to the site of injury and promoting these cells to release neurotrophic factors (such as nerve growth factor NGF and brain-derived neurotrophic factor BDNF). These trophic factors help accelerate the regeneration and functional recovery of nerve axons. However, most traditional electrical stimulation treatment methods are performed in vitro (i.e., without implanting the device in the patient's body). Specifically, in vitro electrical stimulation therapy refers to the placement of an electrical stimulation device outside the patient's body to transmit electrical stimulation to the target tissue or organ through skin or other surface contact. A small amount of electrical stimulation therapy using built-in conductive materials has the problem of poor biocompatibility of conductive materials. Usually, a secondary operation is required to remove them after completing the corresponding function, causing irreversible damage to the patient. Therefore, it is urgent to develop new electrical stimulation materials that are both highly biocompatible and conductive to better meet the needs of electrical stimulation therapy, thereby improving safety, reducing patient pain and medical costs, and improving treatment effects. Summary of the invention
[0004] 1. Technical issues to be resolved
[0005] In view of the technical problem of poor biocompatibility of electrical stimulation materials in the prior art, the present invention provides a conductive phospholipid microtubule material and a preparation method and application thereof. The phospholipid microtubule is conductive, can promote axon growth, and has good biocompatibility, thus solving the technical problem of insufficient biocompatibility of existing electrical stimulation therapy materials.
[0006] (II) Technical solution
[0007] In a first aspect, the present invention provides a conductive phospholipid microtubule material, which comprises a submicron tubular phospholipid vesicle matrix layer and a conductive polymer coated on the outer surface of the tubular phospholipid vesicle matrix layer, wherein the conductive polymer is formed by in-situ polymerization of pyrrole and / or pyrrole carboxylic acid monomers.
[0008] According to a preferred embodiment of the present invention, the tubular submicron phospholipid vesicle matrix layer is obtained by liposome extrusion using phospholipids with a phase transition temperature between 20-80°C as matrix raw materials.
[0009] According to a preferred embodiment of the present invention, the phospholipids are phospholipids with a phase transition temperature between 20-80°C, and are at least one of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE). Among them, the phosphatidylcholine (PC) can be selected as dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC). Phosphatidylethanolamine (PE) can be selected as 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine DSPE.
[0010] In a second aspect, the present invention provides a method for preparing a conductive phospholipid microtubule material, comprising:
[0011] S1, dissolving a phospholipid having a phase transition temperature between 20 and 80° C. in an organic solvent, evaporating the organic solvent, and further drying to remove the residual organic solvent; then adding peroxidase and deionized water for hydration to form a mixed solution;
[0012] S2, using liposome extrusion technology to repeatedly extrude the mixed solution prepared in S1 through a micron-sized pore to obtain a phospholipid aqueous solution that has been shaped;
[0013] S3, adding pyrrole monomer and / or pyrrole carboxylic acid monomer to the phospholipid aqueous solution of S2, mixing evenly, adding reactive oxygen species ROS to initiate polymerization reaction, and obtaining a conductive phospholipid microtubule material.
[0014] According to a preferred embodiment of the present invention, in S1, the phospholipid is a phospholipid with a phase transition temperature between 20-80° C. and is at least one of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE). Preferably, the phosphatidylcholine (PC) can be dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC).
[0015] Phospholipids with a phase transition temperature between 20-80°C can maintain a stable liquid crystal state at room temperature and body temperature, which can simplify the operation, do not require special temperature control equipment, can be operated at room temperature, and can also ensure that the liposomes can work effectively in the in vivo environment. In the liquid crystal state, the phospholipid bilayer has a high fluidity.
[0016] According to a preferred embodiment of the present invention, in S1, the organic solvent is at least one of chloroform, dichloromethane, methanol, tetrahydrofuran and diethyl ether.
[0017] According to a preferred embodiment of the present invention, in S1, the phospholipid is dissolved in an organic solvent to prepare a solution with a concentration of 1 mg / mL±0.5 mg / mL.
[0018] According to a preferred embodiment of the present invention, in S1, the peroxidase is catalase CAT and / or horseradish peroxidase HRP.
[0019] According to a preferred embodiment of the present invention, in S2, the extrusion is achieved by using at least one of a polycarbonate membrane, a polytetrafluoroethylene membrane, a cellulose acetate membrane and a nylon membrane with a micron-sized pore size, and the pore size is 1 μm to 10 μm. The extrusion process is operated at room temperature or 25-35°C.
[0020] According to a preferred embodiment of the present invention, in S1, the drying temperature for removing the residual organic solvent is 25-35°C.
[0021] According to a preferred embodiment of the present invention, in S3, the final concentration of the pyrrole monomer and / or the pyrrole carboxylic acid monomer in the reaction system is 20-100 mM.
[0022] According to a preferred embodiment of the present invention, in S3, the reactive oxygen species ROS is hydrogen peroxide, and the final concentration of hydrogen peroxide in the reaction system is 1nM-10nM; when the monomer added in S3 is a pyrrole monomer, hydrogen peroxide is added dropwise until the solution presents a dark blue-purple color (indicating the formation of polypyrrole, PPy), which indicates that the conductive phospholipid microtubule material has been obtained.
[0023] Preferably, after the conductive phospholipid microtubule material is prepared in step S3, it is sterilized by gamma ray irradiation, with a total irradiation dose of 9 Gy, in preparation for subsequent ganglion cell co-culture.
[0024] In a third aspect, the present invention provides a method for culturing ganglion cells, which uses the above-mentioned conductive phospholipid microtubule material to co-culture with ganglion cells.
[0025] In a fourth aspect, the present invention also relates to the use of the above-mentioned conductive phospholipid microtubule material in the preparation of nerve damage repair materials.
[0026] (III) Beneficial effects
[0027] The present invention uses phospholipid as a base material and combines phospholipid extrusion technology with a polymerization reaction of a conductive monomer to prepare a submicron-sized conductive phospholipid microtubule material that is covered with a conductive polymer on the outside and has excellent biocompatibility. During the preparation process, catalase CAT and / or horseradish peroxidase HRP and reactive oxygen species ROS are used to trigger the polymerization of pyrrole (or pyrrole carboxylic acid), thereby avoiding the addition of biotoxic reagents, having no toxic effect on cells, and having high biocompatibility.
[0028] In step S1, adding peroxidase and deionized water for hydration can improve the uniformity of the distribution of peroxidase on the phospholipid vesicles, thereby improving the uniformity of polypyrrole coating on the outside of the phospholipid vesicles and improving the uniformity of the distribution of the conductive material outside the conductive phospholipid microtubule material.
[0029] The conductive phospholipid microtubule material prepared by the present invention can form a series circuit with the biological electric field in the body to provide electrical stimulation to the damaged nerve tissue, which can effectively improve the problems of poor biocompatibility and difficulty in biological metabolism of conductive materials in traditional electrical stimulation therapy.
[0030] Experiments have shown that when the conductive phospholipid microtubule material is co-cultured with ganglion cells, the ganglion cells are in good condition, and as the co-culture time increases, the regeneration rate of ganglion axons is significantly accelerated. This shows that the conductive phospholipid microtubule material can effectively promote the growth of ganglion axons and is beneficial to the repair of long-distance peripheral nerve injuries.
[0031] The conductive phospholipid microtubule material provided by the present invention has a carrying space with a high aspect ratio, and its tube diameter can reach the submicron level, and can be widely used in different fields such as cell biology, membrane biophysics, biochemistry, bionics, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 This is a cryo-scanning electron microscope image of the conductive phospholipid microtubule material prepared in Example 1 at 4K magnification.
[0033] Figure 2 This is a cryo-scanning electron microscope image of the conductive phospholipid microtubule material prepared in Example 1 at 40K magnification.
[0034] Figure 3 This is an infrared spectrum analysis diagram of the conductivity of the conductive phospholipid microtubule material prepared in Example 1.
[0035] Figure 4 These are pictures of immunofluorescence staining performed with NF200 / DAPI counterstaining solution observed under a laser confocal microscope when the fluorescently labeled conductive phospholipid microtubule material was co-cultured with rat dorsal root ganglion cells for 1 day (a), 2 days (b), and 3 days (c) in Example 2. DETAILED DESCRIPTION
[0036] 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.
[0037] Example 1
[0038] This embodiment provides a method for preparing a conductive phospholipid microtubule material, and the steps are as follows:
[0039] (1) Weigh 250 mg of dipalmitoylphosphatidylcholine (DPPC) and dissolve it in chloroform to obtain a phospholipid solution with a concentration of 1 mg / mL. Place the phospholipid solution in a round-bottom flask.
[0040] (2) Using a rotary evaporator, evaporate the chloroform in the solution in step (1) at room temperature, and then transfer the solution into a vacuum drying oven at 25-35° C. to further remove residual chloroform.
[0041] (3) Add horseradish peroxidase HRP and deionized water into the round-bottom flask to form a mixed solution.
[0042] (4) The mixed solution prepared in step (3) is extruded back and forth using a polycarbonate membrane with a pore size of 3 μm to shape the phospholipids to obtain a shaped phospholipid aqueous solution.
[0043] (5) Pyrrole monomer is added to the phospholipid aqueous solution, and after being mixed evenly, the concentration of pyrrole monomer in the reaction system is 25 mM, and 1 nM hydrogen peroxide solution is added dropwise to initiate the polymerization reaction of pyrrole monomer until the mixed solution turns dark blue-purple (indicating that the pyrrole monomer is polymerized to produce polypyrrole, and the polypyrrole is polymerized in situ and coated on the outside of the phospholipid), thereby preparing a conductive phospholipid microtubule material.
[0044] like Figure 1 As shown, the prepared conductive phospholipid microtubule material was observed under a cryo-scanning electron microscope at 4000 and 40000 times magnification, respectively. Figure 1 and Figure 2 Pictures shown.
[0045] Depend on Figure 2 It can be clearly seen that a large number of submicron-scale tube-like structures are formed in the solution. The conductive phospholipid microtubule material presents a composite structure of vesicles and microtubules. The diameter of the microtubules is at the submicron level, and has a high aspect ratio carrying space. The conductive phospholipid microtubule material is obtained by repeatedly extruding and shaping phospholipids into tube-like phospholipid vesicles with micron-scale apertures. Then, pyrrole monomers undergo free radical polymerization under the catalysis of hydrogen peroxide to form conductive polypyrrole. The polypyrrole uses phospholipids as a supporting structure and is coated on the outside of the tube-like phospholipid vesicles to form a conductive polypyrrole. Figure 1 or Figure 2The conductive phospholipid microtubule material shown. It can be seen that the conductive phospholipid microtubule material prepared by the optimization method has good morphology, high yield, and the diameter of the microtubule is at the submicron level, which can be widely used in different fields such as cell biology, membrane biophysics, biochemistry, and bionics.
[0046] Furthermore, the prepared conductive nanovesicle-to-tuble composites were tested for conductivity. The test method is as follows:
[0047] The conductive phospholipid microtubes are coated with polypyrrole, which is a conductive polymer with excellent conductive properties. In the infrared spectroscopy test method, the KBr tablet method is used, that is, a proper amount of CNVTCs powder and KBr crystal are mixed in an agate mortar and ground into extremely fine powder, then put into a mold and cold-pressed into thin sheets by a tablet press for testing. The scanning range is 4000cm -1 Up to 400cm -1 Infrared spectroscopy analysis can reveal the chemical environment and relative position of each atom in the polypyrrole molecule. The characteristic vibration of the pyrrole ring is at 1560cm -1 、1480cm -1 、1310cm -1 and 1190cm -1 Appeared at 1560cm -1 、1480cm -1 They correspond to the C=C and CC stretching vibrations of the pyrrole ring respectively. Test results Figure 3 As shown, it is proved that CNVTCs are conductive.
[0048] Example 2
[0049] In this example, the conductive phospholipid microtubules were co-cultured with SD rat dorsal root ganglion cells to analyze the biological properties of the conductive phospholipid microtubules. The experimental steps of this example include:
[0050] 1. A conductive phospholipid microtubule material was prepared according to the method of Example 1. However, in order to facilitate visual observation, in this embodiment, the dipalmitoylphosphatidylcholine (DPPC) in Example 1 was replaced with a mixed phospholipid of phospholipid DSPE (1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine) and DPPC (dipalmitoylphosphatidylcholine) labeled with a fluorescent group (FITC). Specifically, in step (1), 5 mg DSPE-FITC and 200 mg DPPC were weighed and dissolved in chloroform / methanol (1:1, v / v) to obtain a phospholipid solution with a concentration of 1 mg / mL. The remaining steps were carried out according to the method of Example 1, and finally a conductive phospholipid microtubule material labeled with a fluorescent group (FITC) was obtained. The conductive phospholipid microtubule material was sterilized at a 9 Gy irradiation dose.
[0051] 2. Preparation of rat dorsal root ganglion cells
[0052] The cells used in this embodiment are SD rat dorsal root ganglion cells, and the specific steps are: select 5-day-old rat suckling mice, put them in 75% ethanol for disinfection after being killed, gradually dissect out the sciatic nerve and spine, separate the muscles on both sides of the spine, cut along the lamina and destroy the intervertebral foramen, pick the DRG (dorsal root ganglion) at the intersection of the sciatic nerve and the spinal cord, and plant them in a culture dish pre-coated with matrix glue after being acted on by type I collagenase and trypsin solution, and replace them with Neurobasal complete medium for cultivation after they adhere to the wall. The extracted and cultured DRG neuron group and neuron growth are normal, which provides a good foundation for further research on neuron axon growth.
[0053] 3. Use the above-mentioned conductive phospholipid microtubule material labeled with a fluorescent group (FITC) and SD rat dorsal root ganglion cells for co-culture. The co-culture conditions are as follows:
[0054] Primary dorsal root ganglion cells of SD rats were extracted in advance and plated. Sterilized CNVTCs were mixed with complete culture medium of dorsal ganglion cells at a volume ratio of 1:100, and co-culture of DRG cells and CNVTCs cells was performed at a culture temperature of 37°C and 5% CO2.
[0055] 4. Prepare three groups in total according to the above method. In one group, the cells / composite materials were immunofluorescently stained with NF200 / DAPI counterstaining solution after 1 day of culture and observed under a laser confocal microscope. The results are as follows: Figure 4 As shown in (a); the second group used NF200 / DAPI counterstaining solution to perform immunofluorescence staining on the cells / composite materials after 2 days of culture and observed them under a laser confocal microscope. The observation results are shown in Figure 4As shown in (b); the last group of cells / composite materials were immunofluorescently stained with NF200 / DAPI counterstaining solution after 3 days of culture and observed under a laser confocal microscope. The results are shown in Figure 4 as shown in (c).
[0056] Depend on Figure 4 It can be seen that after co-culture with CNVTCs (conductive phospholipid microtubule material), the rat dorsal root ganglion cells are in good condition, and the regeneration rate of neurites is accelerated as the co-culture time is prolonged. This proves that CNVTCs prepared by liposome extrusion technology combined with conductive monomer polymerization modification have no toxic effect on nerve cells, are conducive to cell growth, and are expected to be used for the repair of long-distance nerve damage.
[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A conductive phospholipid microtubule material, characterized in that: It includes: A submicron-scale tube-like phospholipid vesicle matrix layer and a conductive polymer coated on the outer surface of the tube-like phospholipid vesicle matrix layer. The conductive polymer is formed by in-situ polymerization of pyrrole and / or pyrrole carboxylic acid monomers.
2. The conductive phospholipid microtubule material according to claim 1, characterized in that: The tubular submicron phospholipid vesicle matrix layer is obtained by using phospholipid with a phase transition temperature between 20 and 80° C. as a matrix raw material and by adopting a liposome extrusion method.
3. The conductive phospholipid microtubule material according to claim 1, characterized in that: The phospholipid is a phospholipid with a phase transition temperature between 20-80° C. and is at least one of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine; the phosphatidylcholine is dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; and the phosphatidylethanolamine is 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine.
4. A method for preparing a conductive phospholipid microtubule material, characterized in that: It includes: S1, dissolving a phospholipid having a phase transition temperature between 20 and 80° C. in an organic solvent, evaporating the organic solvent, and further drying to remove the residual organic solvent; then adding peroxidase and deionized water for hydration to form a mixed solution; S2, using liposome extrusion technology to repeatedly extrude the mixed solution prepared in S1 through a micron-sized pore to obtain a phospholipid aqueous solution that has been shaped; S3, adding pyrrole monomer and / or pyrrole carboxylic acid monomer to the phospholipid aqueous solution of S2, mixing evenly, adding reactive oxygen species ROS to initiate polymerization reaction, and obtaining a conductive phospholipid microtubule material.
5. The preparation method according to claim 4, characterized in that: In S1, the organic solvent is at least one of chloroform, dichloromethane, methanol, tetrahydrofuran and diethyl ether; the phospholipid is dissolved in the organic solvent to prepare a solution with a concentration of 1 mg / mL±0.5 mg / mL.
6. The preparation method according to claim 4, characterized in that: In S1, the peroxidase is catalase CAT and / or horseradish peroxidase HRP.
7. The preparation method according to claim 4, characterized in that: In S2, the extrusion is achieved by using at least one of a polycarbonate membrane, a polytetrafluoroethylene membrane, a cellulose acetate membrane and a nylon membrane with a pore size of micrometer level, and the pore size is 1 μm to 10 μm.
8. The preparation method according to claim 4, characterized in that: The drying temperature for removing the residual organic solvent is 25-35°C.
9. The preparation method according to claim 4, characterized in that: In S3, the final concentration of pyrrole monomer and / or pyrrole carboxylic acid monomer in the reaction system is 20-100 mM; the reactive oxygen species ROS is hydrogen peroxide, and the final concentration of hydrogen peroxide in the reaction system is 1 nM-10 nM; when the monomer added in S3 is a pyrrole monomer, hydrogen peroxide is added dropwise until the solution turns dark blue-purple, which indicates that the conductive phospholipid microtubule material has been obtained.
10. Use of the conductive phospholipid microtubule material according to any one of claims 1 to 3 or the conductive phospholipid microtubule material prepared by the preparation method according to any one of claims 4 to 9 in preparing nerve damage repair materials.