Hydrophobic biomimetic membrane as well as preparation method and application thereof

Abstract

The invention provides a hydrophobic biomimetic membrane and application thereof. A layer of phospholipid membrane embedded with cholesterol is deposited on a substrate through a membrane hydration method and serves as the hydrophobic biomimetic membrane for separation and concentration of drugs such as paclitaxel, and target drug molecules selectively penetrate through the hydrophobic biomimetic membrane based on reverse dialysis. The separation membrane disclosed by the invention has the advantages of simple preparation and use methods, good separation and concentration effects and recyclability.

Description

Technical Field

[0001] The present invention relates to a bionic membrane, in particular to a hydrophobic bionic membrane, a preparation method thereof, and an application thereof in drug separation and concentration. Background Art

[0002] Paclitaxel is an efficient, low-toxic, and broad-spectrum natural anti-cancer drug, which can be isolated and purified from the bark of Taxus chinensis. However, due to the extremely low content of paclitaxel in plants and the slow growth of Taxus plants, the semi-synthesis, biosynthesis, total synthesis and other synthetic methods of paclitaxel are becoming increasingly perfect. At present, artificial synthesis has become the main source of obtaining paclitaxel [Li Xiandeng, Zhang Li. Research progress on the synthesis of paclitaxel [J]. Chemical Engineering Design Communications, 2021, 47(01): 163-164.].

[0003] For the crude paclitaxel obtained by biological purification or chemical synthesis, it usually contains paclitaxel analogs, such as 10-deacetylbaccatin III, cephalomannine, 7-epi-10-deacetylpaclitaxel, etc. The structural formulas of paclitaxel and its analogs are shown as follows. Separation and purification is a key step to improve the efficacy of anti-cancer drugs. Therefore, it is necessary to remove impurities such as paclitaxel analogs and organic solvents contained in the crude paclitaxel. At present, the chromatographic separation method of paclitaxel is relatively mature and has the advantage of high separation coefficient [Zhu Shunqin, Tan Feng, Tang Kexuan. Research progress on the extraction and purification technology of paclitaxel [J]. Chinese Journal of Biochemical Pharmaceutics, 2004, 25(01): 59-62.], but it still requires multiple separation and concentration steps and has a high cost.

[0004]

[0005] Structural formulas of paclitaxel and its analogs

[0006] In recent years, due to its advantages such as low cost and simple and easy operation of the device, the membrane separation method has been continuously tried and developed in the field of drug separation and concentration. For example, a paclitaxel separation membrane based on the principle of molecular imprinting adsorbs and intercepts paclitaxel, and selectively permeates its analog (10-deacetylbaccatin III) to achieve separation [Hui Zhang, Yuqi Li, Deyong Zheng, et al. Bio-inspired construction of cellulose-based molecular imprinting membrane with selective recognition surface for paclitaxel separation. Applied Surface Science, 2019, 466: 244-253]. However, there is currently no report on the universality of the separation membrane, that is, it has separation performance for a variety of analogs and other types of impurities, which is the research prospect in the design and application fields of paclitaxel separation membranes. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a hydrophobic biomimetic membrane, which comprises a substrate and a phospholipid membrane formed on the surface of the substrate, wherein cholesterol or its derivative is embedded in the phospholipid membrane.

[0008] According to an embodiment of the present invention, the phospholipid membrane comprises a phospholipid monolayer (or phospholipid single molecule layer) and a phospholipid bilayer (or phospholipid double molecule layer). According to an embodiment of the present invention, the phospholipid membrane is a phospholipid monolayer.

[0009] According to an embodiment of the present invention, the phospholipid membrane is deposited on the surface of the substrate.

[0010] According to an embodiment of the present invention, the phospholipid molecules forming the phospholipid membrane are selected from one or more of lecithins, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, and natural lipid extracts.

[0011] According to an embodiment of the present invention, the lecithins include one or more of the following groups:

[0012] 1,2-Dimyristoylphosphatidylcholine (DMPC), 1,2-Dipalmitoylphosphatidylcholine (DPPC), 1,2-Distearoylphosphatidylcholine (DSPC), 1,2-Dioleoylphosphatidylcholine (DOPC), 1,2-Dimyristoleoylphosphatidylcholine, 1,2-Dipalmitoleoylphosphatidylcholine, 1,2-Dipetroselinoylphosphatidylcholine, 1,2-Dielaidoylphosphatidylcholine, 1,2-Dilinoleoylphosphatidylcholine, 1,2-Dilinolenoylphosphatidylcholine, 1,2-Dieicosenoylphosphatidylcholine, 1,2-Diarachidonoylphosphatidylcholine, 1,2-Dierucoylphosphatidylcholine, 1,2-Dinervonoylphosphatidylcholine, 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-Palmitoyl-2-linoleoylphosphatidylcholine, 1-Palmitoyl-2-arachidonoylphosphatidylcholine, 1-Palmitoyl-2-docosahexaenoylphosphatidylcholine, 1-Stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-Stearoyl-2-linoleoylphosphatidylcholine, 1-Stearoyl-2-arachidonoylphosphatidylcholine, 1-Stearoyl-2-docosahexaenoylphosphatidylcholine, 1-Oleoyl-2-palmitoylphosphatidylcholine, 1-Oleoyl-2-stearoylphosphatidylcholine, 1,2-Didocosahexaenoylphosphatidylcholine.

[0013] According to an embodiment of the present invention, the phosphatidylethanolamine includes one or more of the following group:

[0014] 1,2-Dimyristoylphosphatidylethanolamine (DMPE), 1,2-Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Distearoylphosphatidylethanolamine (DSPE), 1,2-Dioleoylphosphatidylethanolamine (DOPE), 1-Palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-Palmitoyl-2-linoleoylphosphatidylethanolamine, 1-Palmitoyl-2-arachidonoylphosphatidylethanolamine, 1-Palmitoyl-2-docosahexaenoylphosphatidylethanolamine, 1-Stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-Stearoyl-2-linoleoylphosphatidylethanolamine, 1-Stearoyl-2-arachidonoylphosphatidylethanolamine,

[0015] 1-Stearoyl-2-docosahexaenoyl phosphatidylethanolamine, 1,2-dielaidoyl phosphatidylethanolamine, 1,2-dilinoleoyl phosphatidylethanolamine, 1,2-dilinolenoyl phosphatidylethanolamine, 1,2-diarachidonoyl phosphatidylethanolamine, 1,2-didocosahexaenoyl phosphatidylethanolamine, 1,2-dipalmitoleoyl phosphatidylethanolamine.

[0016] According to an embodiment of the present invention, the phosphatidylglycerols include one or more of the following group:

[0017] 1,2-Dimyristoyl phosphatidylglycerol (DMPG), 1,2-Dipalmitoyl phosphatidylglycerol (DPPG), 1,2-Distearoyl phosphatidylglycerol (DSPG), 1,2-Dioleoyl phosphatidylglycerol (DOPG), 1-Palmitoyl-2-oleoyl phosphatidylglycerol (POPG), 1-Palmitoyl-2-linoleoyl phosphatidylglycerol, 1-Palmitoyl-2-arachidonoyl phosphatidylglycerol, 1-Palmitoyl-2-docosahexaenoyl phosphatidylglycerol, 1-Stearoyl-2-oleoyl phosphatidylglycerol (SOPG), 1-Stearoyl-2-linoleoyl phosphatidylglycerol, 1-Stearoyl-2-arachidonoyl phosphatidylglycerol, 1-Stearoyl-2-docosahexaenoyl phosphatidylglycerol.

[0018] According to an embodiment of the present invention, the phosphatidylserines include:

[0019] 1-Palmitoyl-2-oleoyl phosphatidylserine (POPS), 1-Palmitoyl-2-linoleoyl phosphatidylserine, 1-Palmitoyl-2-arachidonoyl phosphatidylserine, 1-Palmitoyl-2-docosahexaenoyl phosphatidylserine, 1-Stearoyl-2-oleoyl phosphatidylserine (SOPS), 1-Stearoyl-2-linoleoyl phosphatidylserine, 1-Stearoyl-2-arachidonoyl phosphatidylserine, 1-Stearoyl-2-docosahexaenoyl phosphatidylserine, 1,2-Dimyristoyl phosphatidylserine (DMPS), 1,2-Dipalmitoyl phosphatidylserine (DPPS), 1,2-Distearoyl phosphatidylserine (DSPS), 1,2-Dioleoyl phosphatidylserine (DOPS), 1,2-Didocosahexaenoyl phosphatidylserine, 1,2-Dierucoyl phosphatidylserine.

[0020] According to an embodiment of the present invention, the natural lipid extract includes: egg yolk lecithin, bovine heart lecithin, brain lecithin, bovine liver lecithin, soybean lecithin.

[0021] According to an embodiment of the present invention, the cholesterol derivative is a cholesterol ester. According to an embodiment of the present invention, the cholesterol esters include cholesterol C1-20 alkyl carboxylates, cholesterol C2-20 alkenyl carboxylates, and cholesterol benzoates. For example, cholesterol palmitate, cholesterol benzoate, and cholesterol succinate monoester.

[0022] According to an embodiment of the present invention, the substrate is a porous support layer.

[0023] According to an embodiment of the present invention, the porous support layer is selected from one or more of a polymer membrane with a porous structure and an inorganic membrane with a porous structure.

[0024] According to an embodiment of the present invention, the polymer membrane with a porous structure is selected from one or more of a cellulose membrane, a polyamide membrane, a polyacrylonitrile membrane, a polycarbonate membrane, a polysulfone membrane, a polyolefin membrane, a polyvinyl alcohol membrane, a polystyrene membrane, a polymethacrylate membrane, an ethylene-vinyl acetate copolymer membrane, and a polyether block copolymer membrane.

[0025] According to an embodiment of the present invention, the cellulose membrane includes a regenerated cellulose membrane, a cellulose acetate membrane, a cuprammonium cellulose membrane, etc.

[0026] According to an embodiment of the present invention, the polymer membrane is a dialysis membrane, such as a regenerated cellulose dialysis membrane.

[0027] According to an embodiment of the present invention, the inorganic membrane with a porous structure includes a ceramic membrane, etc.

[0028] The present invention also provides a method for preparing the hydrophobic biomimetic membrane, and the preparation method includes the following steps:

[0029] (1) Dissolve phospholipid molecules in a solvent to obtain a phospholipid solution;

[0030] (2) Remove the solvent from the phospholipid solution obtained in step (1) to obtain self-assembled phospholipid vesicles;

[0031] (3) Mix a cholesterol solution with the phospholipid vesicles obtained in step (2) to embed cholesterol in the phospholipid vesicles to obtain a reconstituted solution;

[0032] (4) Contact the reconstituted solution obtained in step (3) with the substrate to form a phospholipid membrane embedded with cholesterol on the surface of the substrate.

[0033] According to an embodiment of the present invention, the solvent for dissolving phospholipids in step (1) is selected from C1-4 alkyl alcohol solvents, C5-9 alkanes, halogenated C1-4 alkanes, and ethers. According to an embodiment of the present invention, the C1-4 alkyl alcohol solvents include methanol, ethanol, n-propanol, isopropanol, etc., the C5-9 alkanes include n-pentane, n-hexane, n-heptane, n-octane, etc., the halogenated C1-4 alkanes include dichloromethane, chloroform, etc., and the ethers include diethyl ether. According to an embodiment of the present invention, the solvent for dissolving phospholipids in step (1) is selected from one or more of methanol, chloroform, n-hexane, and diethyl ether, preferably methanol.

[0034] According to an embodiment of the present invention, the concentration of phospholipids in the phospholipid solution in step (1) is 0.5-8 mg / mL, such as 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, preferably 1.0-4.0 mg / mL, more preferably 1.5-3.0 mg / mL, and even more preferably 2.0 mg / mL.

[0035] According to an embodiment of the present invention, in step (2), the phospholipid vesicles formed by self-assembly are obtained by vacuum evaporating the solvent (such as rotary evaporation).

[0036] According to an embodiment of the present invention, there is a white film-like substance adhering to the container wall in step (2).

[0037] According to an embodiment of the present invention, the solvent in the cholesterol solution in step (3) is selected from C1-4 alkyl alcohol solvents, C5-9 alkanes, halogenated C1-4 alkanes, and ethers. According to an embodiment of the present invention, the C1-4 alkyl alcohol solvents include methanol, ethanol, n-propanol, isopropanol, etc., the C5-9 alkanes include n-pentane, n-hexane, n-heptane, n-octane, etc., the halogenated C1-4 alkanes include dichloromethane, chloroform, etc., and the ethers include diethyl ether. According to an embodiment of the present invention, the solvent for dissolving phospholipids in step (1) is selected from one or more of methanol, chloroform, n-hexane, and diethyl ether, preferably methanol.

[0038] According to an embodiment of the present invention, the molar ratio of cholesterol to phospholipids in step (1) in the cholesterol solution in step (3) is 1:2-1:15, such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, preferably 1:4-1:12, more preferably 1:5-1:10, and even more preferably 1:6.

[0039] According to an embodiment of the present invention, a phosphate buffer solution (PBS solution) also needs to be added in step (3).

[0040] According to an embodiment of the present invention, the mixing temperature (i.e., the reconstitution temperature) in step (3) is controlled at 10 - 50 °C, such as 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C; preferably, the mixing temperature is 20 °C - 35 °C; preferably, the mixing temperature is room temperature.

[0041] According to an embodiment of the present invention, mixing in step (3) is carried out by ultrasonic oscillation.

[0042] According to an embodiment of the present invention, the mixing time (i.e., the reconstitution time) in step (3) is 5 - 60 min, such as 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, preferably 20 - 40 min, preferably 30 min.

[0043] According to an embodiment of the present invention, the phospholipid membrane embedded with cholesterol is deposited on the surface of the substrate in step (4).

[0044] According to an embodiment of the present invention, the substrate is immersed in the reconstitution solution in step (4).

[0045] According to an embodiment of the present invention, the temperature at which the phospholipid membrane embedded with cholesterol is deposited on the surface of the substrate in step (4) is 10 - 80 °C, such as 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, preferably 30 - 70 °C, further preferably 40 °C - 60 °C, further preferably 50 °C.

[0046] The present invention also provides a method for separating and concentrating a drug, including contacting a solution of the drug with the hydrophobic biomimetic membrane of the present invention.

[0047] According to an embodiment of the present invention, the drug is selected from one of paclitaxel, camptothecin, and artemisinin.

[0048] According to an embodiment of the present invention, the target drug molecule selectively permeates through the hydrophobic biomimetic membrane of the present invention based on reverse dialysis.

[0049] According to an embodiment of the present invention, the separation and concentration method includes the following steps:

[0050] (1) Dissolve the crude paclitaxel in a solvent to obtain a crude paclitaxel solution.

[0051] (2) Add a good solvent for paclitaxel to a dialysis bag with a cholesterol-embedded phospholipid membrane deposited on its surface, immerse it in the crude paclitaxel solution, and perform reverse dialysis; wherein, the dialysis bag with a cholesterol-embedded phospholipid membrane deposited on its surface is made of the hydrophobic biomimetic membrane of the present invention.

[0052] According to an embodiment of the present invention, the solvent described in step (1) is selected from one or a mixed solution of water and methanol.

[0053] According to an embodiment of the present invention, a PBS solution is also added in step (1).

[0054] According to an embodiment of the present invention, the good solvent for paclitaxel in step (2) is methanol.

[0055] The present invention also provides the application of the hydrophobic biomimetic membrane in the separation and concentration of paclitaxel.

[0056] The present invention also provides a device, and the device contains the hydrophobic biomimetic membrane described in the present invention.

[0057] Beneficial effects

[0058] (1) The raw materials of the hydrophobic biomimetic membrane of the present invention are cheap and easily available, the preparation method is simple, and the substrate can select a commercially available dialysis membrane (such as a regenerated cellulose dialysis membrane). By using the thin film hydration method, a phospholipid molecular layer (such as a phospholipid monolayer) embedded with cholesterol is deposited on the outer surface of the dialysis membrane. Only four simple operations of "dissolution - solvent evaporation - redissolution - solvent evaporation" are required, with high repeatability, large-scale preparation possible, and short time consumption.

[0059] (2) The use method of the hydrophobic biomimetic membrane of the present invention is simple. Based on the reverse dialysis method, an aqueous solution of a crude drug containing various analog impurities is placed in a container. By utilizing the difference in the hydrophobic interaction of the separation membrane with the drug (such as paclitaxel) and its analogs, the drug molecules (such as paclitaxel) can selectively enter the dialysis bag and dissolve in an organic good solvent, thereby achieving separation and concentration.

[0060] (3) The separation and purification effect is good, the purity of the separated product is relatively high, and the drug purity can be significantly improved. For example, only one treatment can increase the purity of the crude paclitaxel from 30.1% to 97.4%, a three-fold increase.

[0061] (4) The hydrophobic biomimetic membrane of the present invention has universality, can achieve the interception of various analog impurities, and realize the separation and concentration of the crude drug (such as the crude paclitaxel).

[0062] (5) The hydrophobic biomimetic membrane of the present invention can be recycled and is more environmentally friendly.

[0063] Definitions and explanations:

[0064] C1 - 20 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and C20; C1 - 4 is selected from C1, C2, C3, and C4; C5 - 9 is selected from C5, C6, C7, C8, and C9.

[0065] As used herein, the term "halo" means fluorine, chlorine, bromine, and iodine.

[0066] As used herein, the term "alkyl" is understood to mean a saturated aliphatic hydrocarbon group having a straight or branched chain. For example, "C1-20 alkyl" means straight and branched chain alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. "C 1-4 alkyl" means straight and branched chain alkyl groups having 1, 2, 3, or 4 carbon atoms. Examples of said alkyl groups are methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, isopentyl, n-dodecyl, n-tetradecyl, n-hexadecyl, and n-octadecyl.

[0067] In the term "cholesterol C1-20 alkyl carboxylate" as used herein, the number of carbon atoms in "C1-20 alkyl carboxylate" includes the carbon atom of the carboxyl group. For example, octadecyl carboxylate is stearate, and hexadecyl carboxylate is palmitate.

[0068] As used herein, the term "alkane" is understood to mean a saturated aliphatic hydrocarbon having a straight or branched chain. For example, "C5-9 alkane" means straight and branched chain alkanes having 5, 6, 7, 8, or 9 carbon atoms. Examples of said alkanes are n-pentane, n-hexane, n-heptane, n-octane, n-nonane, etc.

[0069] As used herein, the term "alkenyl" refers to a straight or branched chain aliphatic hydrocarbon group having one or more carbon-carbon double bonds. For example, the term "C2-20 alkenyl" as used herein refers to an alkenyl group having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and one, two, or three carbon-carbon double bonds, such as vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl, etc.

[0070] In the term "cholesterol C2-20 alkenyl carboxylate" as used herein, the number of carbon atoms in "C2-20 alkenyl carboxylate" includes the carbon atom of the carboxyl group. For example, 9-octadecenyl carboxylate is oleate. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Schematic diagram of a hydrophobic biomimetic membrane structure deposited with a phospholipid monolayer film prepared in Example 1 of the present invention.

[0072] Figure 2Scanning tunneling microscope (STM) surface topography and cross-sectional height difference diagrams of the hydrophobic biomimetic membrane deposited with a phospholipid monolayer film prepared in Example 1 of the present invention.

[0073] Figure 3 Schematic diagram of the separation and concentration device of Example 2 of the present invention.

[0074] Figure 4 Chromatograms of the paclitaxel samples before and after separation using the hydrophobic biomimetic membrane of Example 2. Among them, the chromatogram in the upper part is the chromatogram of the separated paclitaxel sample, and the chromatogram in the lower part is the chromatogram of the crude paclitaxel before separation. Detailed implementation manners

[0075] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only illustrative explanations of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0076] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0077] Preparation of the hydrophobic biomimetic membrane deposited with a phospholipid monolayer film in Example 1

[0078] The phospholipid molecule used is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The preparation method includes the following steps:

[0079] (1) Prepare a DOPC solution with a concentration of 20 mg / mL using methanol as the solvent;

[0080] (2) Take 2.5 mL of the DOPC solution (20 mg / mL) and 7.5 mL of methanol into a round-bottom flask, and ultrasonicate for 3 min at room temperature to mix evenly;

[0081] (3) Evaporate the solvent methanol to dryness under vacuum using a rotary evaporator. It can be observed that a white film-like substance adheres to the round-bottom flask, which is the self-assembled phospholipid vesicle;

[0082] (4) Add 0.2 mL of a methanol solution of cholesterol (2 g / 100 g) and 20 mL of PBS solution (10 mM, pH = 7.4) to the round-bottom flask for reconstitution, and ultrasonically oscillate for 30 min at room temperature to embed cholesterol into the phospholipid vesicle. The reconstituted solution is milky white;

[0083] (5) After pre-treating a 10-cm commercially available regenerated cellulose dialysis membrane (MW 1000D, MD 25 mm) (boiling in water for 10 min and then washing with distilled water), clamp both ends and lay it flat in a petri dish. Add the above-mentioned reconstituted solution to immerse the dialysis membrane. Place it in an oven and heat at 50 °C to slowly and evenly deposit the cholesterol-embedded phospholipid membrane on the outer surface of the dialysis membrane. Take it out after 30 min, wash it with distilled water and set aside to obtain a regenerated cellulose dialysis membrane with a cholesterol-embedded phospholipid membrane deposited on its surface.

[0084] The structural schematic diagram of the hydrophobic biomimetic membrane with a phospholipid monolayer membrane deposited in Example 1 is as Figure 1 shown. From Figure 1 it can be seen that a 1,2-dioleoyl-sn-glycero-3-phosphocholine monolayer membrane is deposited on the surface of the regenerated cellulose dialysis membrane, and cholesterol molecules are embedded in the phospholipid membrane.

[0085] The thickness of the deposited phospholipid membrane was measured by scanning tunneling microscopy (STM), and the results are as Figure 2 shown. From Figure 2 the above (two-dimensional image), it can be seen that the deposited phospholipid membrane is dense and uniform; at the same time, from Figure 2 the below (one-dimensional spectrum line), the thickness of the deposited layer can be read as 2.6 nm, which is close to the length of a phospholipid molecule. Comparing with the thickness of the phospholipid bilayer membrane which is generally 5 - 6 nm, it can be determined that a phospholipid monolayer membrane is formed in Example 1.

[0086] Example 2 Separation and Concentration of Paclitaxel

[0087] (1) Weigh 0.5 g of crude paclitaxel containing analog impurities into a beaker, add methanol dropwise and sonicate until it just dissolves, and then dilute it to 200 mL with PBS solution (10 mM, pH = 7.4);

[0088] (2) Make the regenerated cellulose dialysis membrane with a cholesterol-embedded phospholipid membrane deposited on its surface prepared in Example 1 into a dialysis bag with a cholesterol-embedded phospholipid membrane deposited on its surface. Add methanol (3 mL), a good solvent for paclitaxel, to the above dialysis bag with a cholesterol-embedded phospholipid membrane deposited on its surface, clamp both ends, immerse it in the crude paclitaxel solution, add a magnetic stir bar to the beaker and place it on a magnetic stirrer, and perform reverse dialysis at room temperature;

[0089] (3) Monitor the dialysis equilibrium by monitoring the intensity of the ultraviolet absorption spectra (absorption peak: 227 nm) of the crude solution and the product solution in the dialysis bag. The dialysis equilibrium is reached in 3 h, and after reaching the equilibrium, collect the concentrated solution in the dialysis bag.

[0090] The concentrated solution was rotary-evaporated to remove the solvent, and the purity of paclitaxel was determined according to the high performance liquid chromatography method in Chinese Pharmacopoeia (General Rule 0512: chromatographic column: octadecylsilyl silica gel column; mobile phase: acetonitrile: water (40:60); flow rate: 1.5 mL / min; column temperature: 30 °C; detection wavelength: 227 nm; retention time: 34 minutes). The purity of paclitaxel in the separated product reached 97.4% as determined by chromatography. The purity of the crude paclitaxel used (the total amount of paclitaxel / paclitaxel and its analogs, mol%) was 30.1% as determined by chromatography. The purity of the separated product was increased by 3 times compared with the crude product.

[0091] The schematic diagram of the separation and concentration device in Example 2 is as Figure 3 shown. As Figure 3 can be seen, the aqueous solution of crude paclitaxel containing various analog impurities was placed in a beaker. By utilizing the difference in the hydrophobic interaction of paclitaxel and its analogs with the separation membrane, paclitaxel selectively entered the dialysis bag and dissolved in the organic good solvent, thus realizing separation and concentration.

[0092] The chromatograms of the paclitaxel samples before and after separation using the hydrophobic biomimetic membrane in Example 2 are as Figure 4 shown. Figure 4 The upper chromatogram in Figure 4 is the chromatogram of the separated paclitaxel sample, and the lower chromatogram in Figure 4 is the chromatogram of the crude paclitaxel before separation. As

[0093] can be seen, after separation and concentration, the purity of paclitaxel was significantly improved. From the chromatogram of the crude paclitaxel before separation, it can be obtained that the contents (mol%) of paclitaxel and its analogs 10-deacetylbaccatin III, cephalomannine, 7-epi-10-deacetylpaclitaxel in the crude paclitaxel were 30.1%, 25.9%, 22.7% and 21.3% respectively, and the retention times were 34.025, 30.434, 36.218, 37.879 min respectively. From the chromatogram of the separated paclitaxel sample, it can be obtained that the purity of paclitaxel after separation and concentration reached 97.4%.

[0093] The above has given an exemplary description of the implementation mode of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above implementation mode. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the claims of this application.

Claims

1. A hydrophobic biomimetic membrane, characterized in that: The hydrophobic biomimetic membrane comprises a substrate and a phospholipid membrane formed on the surface of the substrate, wherein cholesterol or its derivatives are embedded in the phospholipid membrane.

2. The hydrophobic biomimetic membrane according to claim 1, characterized in that: The phospholipid membrane includes a phospholipid monolayer membrane and a phospholipid bilayer membrane; preferably, the phospholipid membrane is a phospholipid monolayer membrane; and / or The phospholipid membrane is deposited on the surface of the substrate; and / or The phospholipid molecules forming the phospholipid membrane are selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, and natural lipid extracts; Preferably, the lecithins include one or more of the following groups: 1,2-dimyristoylphosphatidylcholine (DMPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dioleoylphosphatidylcholine (DOPC), 1,2-dimyristoleoylphosphatidylcholine, 1,2-dipalmitoylphosphatidylcholine, 1,2-petroselinoylphosphatidylcholine, 1,2-elaidoylphosphatidylcholine, 1,2-dilinoleoylphosphatidylcholine, 1,2-linolenoylphosphatidylcholine, 1,2-eicosenoylphosphatidylcholine, 1,2-diachidonoylphosphatidylcholine, 1,2- Erucoyl phosphatidylcholine, 1,2-di-tetradecenoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine, 1-palmitoyl-2-arachidonoyl phosphatidylcholine, 1-palmitoyl-2-docosahexaenoyl phosphatidylcholine, 1-hard Acyl-2-oleoylphosphatidylcholine (SOPC), 1-stearoyl-2-linoleoylphosphatidylcholine, 1-stearoyl-2-arachidonoylphosphatidylcholine, 1-stearoyl-2-docosahexaenoylphosphatidylcholine, 1-oleoyl-2-palmitoylphosphatidylcholine, 1-oleoyl-2-stearoylphosphatidylcholine, 1,2-docosahexaenoylphosphatidylcholine. Preferably, the phosphatidylethanolamine comprises one or more of the following groups: 1,2-dimyristoylphosphatidylethanolamine (DMPE), 1,2-dipalmitoylphosphatidylethanolamine (DPPE), 1,2-distearoylphosphatidylethanolamine (DSPE), 1,2-dioleoylphosphatidylethanolamine (DOPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-palmitoyl-2-linoleoylphosphatidylethanolamine, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine, 1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine, 1-stearoyl-2 -oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-linoleoylphosphatidylethanolamine, 1-stearoyl-2-arachidonoylphosphatidylethanolamine, 1-stearoyl-2-docosahexaenoylphosphatidylethanolamine, 1,2-di-re-oleoylphosphatidylethanolamine, 1,2-dilinoleoylphosphatidylethanolamine, 1,2-dilinolenoylphosphatidylethanolamine, 1,2-diarachidonoylphosphatidylethanolamine, 1,2-docosahexaenoylphosphatidylethanolamine, 1,2-dipalmitoleoylphosphatidylethanolamine; Preferably, the phosphatidylglycerols include one or more of the following groups: 1,2-dimyristoylphosphatidylglycerol (DMPG), 1,2-dipalmitoylphosphatidylglycerol (DPPG), 1,2-distearoylphosphatidylglycerol (DSPG), 1,2-dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), 1-palmitoyl-2-linoleoylphosphatidylglycerol, 1-palmitoyl-2-arachidonoylphosphatidylglycerol, 1-palmitoyl-2-docosahexaenoylphosphatidylglycerol, 1-stearoyl-2-oleoylphosphatidylglycerol (SOPG), 1-stearoyl-2-linoleoylphosphatidylglycerol, 1-stearoyl-2-arachidonoylphosphatidylglycerol, 1-stearoyl-2-docosahexaenoylphosphatidylglycerol; Preferably, the phosphatidylserines include one or more of the following groups: 1-palmitoyl-2-oleoylphosphatidylserine (POPS), 1-palmitoyl-2-linoleoylphosphatidylserine, 1-palmitoyl-2-arachidonoylphosphatidylserine, 1-palmitoyl-2-docosahexaenoylphosphatidylserine, 1-stearoyl-2-oleoylphosphatidylserine (SOPS), 1-stearoyl-2-linoleoylphosphatidylserine, 1-stearoyl-2-arachidonoylphosphatidylserine Phosphatidylserine, 1-stearoyl-2-docosahexaenoylphosphatidylserine, 1,2-dimyristoylphosphatidylserine (DMPS), 1,2-dipalmitoylphosphatidylserine (DPPS), 1,2-distearoylphosphatidylserine (DSPS), 1,2-dioleoylphosphatidylserine (DOPS), 1,2-didocosahexaenoylphosphatidylserine, 1,2-dierucoylphosphatidylserine; Preferably, the natural lipid extract comprises: egg yolk lecithin, beef heart lecithin, brain lecithin, beef liver lecithin, and soybean lecithin. Preferably, the phospholipid molecule is selected from one or more of 1,2-dioleoyl-sN-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sN-glycero-3-phosphocholine (DPPC); and / or The cholesterol derivative is a cholesterol ester; and / or The substrate is a porous supporting layer; preferably, the porous supporting layer is selected from one or more of a polymer membrane with a porous structure and an inorganic membrane with a porous structure; preferably, the polymer membrane with a porous structure is selected from one or more of a cellulose membrane, a polyamide membrane, a polyacrylonitrile membrane, a polycarbonate membrane, a polysulfone membrane, a polyolefin membrane, a polyvinyl alcohol membrane, a polystyrene membrane, a polymethacrylate membrane, an ethylene-vinyl acetate copolymer membrane, and a polyether block copolymer membrane; preferably, the cellulose membrane includes a regenerated cellulose membrane, a cellulose acetate membrane, and a cuprammonium cellulose membrane; preferably, the polymer membrane is a dialysis membrane, such as a regenerated cellulose dialysis membrane; preferably, the inorganic membrane with a porous structure is selected from a ceramic membrane.

3. The method for preparing the hydrophobic biomimetic membrane according to claim 1 or 2, comprising the following steps: (1) dissolving phospholipid molecules in a solvent to obtain a phospholipid solution; (2) removing the solvent from the phospholipid solution obtained in step (1) to obtain self-assembled phospholipid vesicles; (3) mixing the cholesterol solution with the phospholipid vesicles obtained in step (2) to embed the cholesterol into the phospholipid vesicles to obtain a reconstituted solution; (4) The reconstituted solution obtained in step (3) is brought into contact with a substrate to form a phospholipid membrane embedded with cholesterol on the surface of the substrate.

4. The method for preparing the hydrophobic biomimetic membrane according to claim 3, characterized in that: One or more of the following conditions are met: (1) The solvent for dissolving phospholipids in step (1) is selected from one or more of C1-4 alkyl alcohol solvents, C5-9 alkanes, halogenated C1-4 alkanes and ethers; preferably, the C1-4 alkyl alcohol solvents include methanol, ethanol, n-propanol, isopropanol, the C5-9 alkanes include n-pentane, n-hexane, n-heptane, n-octane, the halogenated C1-4 alkanes include dichloromethane, chloroform, and the ethers include diethyl ether; preferably, the solvent for dissolving phospholipids in step (1) is selected from one or more of methanol, chloroform, n-hexane, and diethyl ether; preferably methanol; and / or The concentration of phospholipids in the phospholipid solution in step (1) is 0.5 to 8 mg / mL, preferably 1.0 to 4.0 mg / mL, more preferably 1.5 to 3.0 mg / mL, and more preferably 2.0 mg / mL; (2) in step (2), the solvent is evaporated in vacuum to obtain self-assembled phospholipid vesicles; (3) The solvent in the cholesterol solution in step (3) is selected from one or more of C1-4 alkyl alcohol solvents, C5-9 alkanes, halogenated C1-4 alkanes and ethers; preferably, the C1-4 alkyl alcohol solvents include methanol, ethanol, n-propanol, isopropanol, the C5-9 alkanes include n-pentane, n-hexane, n-heptane, n-octane, the halogenated C1-4 alkanes include dichloromethane and chloroform, and the ethers include diethyl ether; preferably, the solvent for dissolving phospholipids in step (1) is selected from one or more of methanol, chloroform, n-hexane and diethyl ether, preferably methanol; and / or The molar ratio of cholesterol in the cholesterol solution in step (3) to the phospholipid in step (1) is 1:2 to 1:15, preferably 1:4 to 1:12, more preferably 1:5 to 1:10, more preferably 1:6; and / or In step (3), a phosphate buffer solution (PBS solution) is also required; and / or In step (3), the mixing temperature is controlled at 10 to 50° C.; preferably, the mixing temperature is room temperature; and / or In step (3), mixing is performed by ultrasonic vibration; and / or The mixing time in step (3) is 5-60 min; (4) the phospholipid membrane embedded with cholesterol in step (4) is deposited on the surface of the substrate; and / or In step (4), the substrate is immersed in a reconstitution solution; and / or In step (4), the temperature at which the cholesterol-embedded phospholipid membrane is deposited on the substrate surface is 10 to 80°C.

5. A method for separating and concentrating drugs, characterized in that: The method comprises contacting a solution of the drug with the hydrophobic biomimetic membrane according to claim 1 or 2.

6. The method for separating and concentrating drugs according to claim 6, characterized in that: The drug is selected from one of paclitaxel, camptothecin and artemisinin, preferably paclitaxel; and / or The target drug molecules selectively permeate the hydrophobic biomimetic membrane based on reverse dialysis.

7. The method according to claim 5 or 6, characterized in that: The steps include: (1) dissolving crude paclitaxel in a solvent to obtain a crude paclitaxel solution; (2) adding a good solvent for paclitaxel to a dialysis bag having a cholesterol-embedded phospholipid membrane deposited on the surface, immersing the bag in a crude paclitaxel solution, and performing reverse dialysis; wherein the dialysis bag having a cholesterol-embedded phospholipid membrane deposited on the surface is made of the hydrophobic biomimetic membrane according to claim 1 or 2; Preferably, the solvent in step (1) is selected from water and methanol or a mixed solution of the two; Preferably, a PBS solution is also added in step (1). Preferably, the good solvent for paclitaxel in step (2) is methanol.

8. Use of the hydrophobic biomimetic membrane according to claim 1 or 2 in the separation and concentration of paclitaxel.

9. A device, characterized in that: The device contains the hydrophobic biomimetic membrane according to claim 1 or 2.

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