Immobilized artificial membrane chromatography stationary phase as well as preparation method and application thereof

Abstract

The invention discloses an immobilized artificial membrane chromatography (IAMC) stationary phase as well as a preparation method and application thereof, and belongs to the technical field of bionic chromatography stationary phases. According to the method, a matrix containing an initiating group is used as an initiator, under the action of a catalyst, a phospholipid monomer is fixed on the surface of the matrix by utilizing an active free radical polymerization method, and the stable IAMC stationary phase is formed. According to the method, the phospholipid can be efficiently, simply and conveniently grafted on the surface of the silica gel matrix, and the prepared stationary phase has good acid-base stability and bionic property. The IAMC stationary phase provided by the invention has a wide application prospect, can greatly improve the application potential of a chromatographic technology in the fields of life science, environmental protection, drug research and development and the like, and has relatively high economic and social values.

Description

Technical Field

[0001] This invention relates to the field of biomimetic chromatographic stationary phase technology, and more specifically to an immobilized artificial membrane chromatographic stationary phase, its preparation method, and its application. Background Technology

[0002] Biomimetic phospholipid membrane chromatography, also known as immobilized artificial membrane chromatography (IAMC), is a novel chromatographic technique that simulates the environment of biological membranes. It immobilizes phospholipids or phospholipid analogs on the surface of different matrices through physical adsorption or covalent bonding to construct an artificial membrane environment with similar structure and function to biological membranes. By analyzing the retention behavior of compounds on this membrane, the membrane permeability, absorption, and distribution characteristics of the compounds can be evaluated. Currently, commercially available IAMC stationary phases are mainly of the IAM.PC type (IAM.PC.MG, IAM.PC.DD, and IAM.PC.DD2), which use covalent bonding to immobilize phosphatidylcholine-like substances on the surface of a silica matrix to simulate a biological membrane environment. These are widely used in drug screening, drug-membrane interaction analysis, and environmental toxicity prediction. Although commercially available IAMC stationary phases provide relatively realistic membrane environment simulations, they still face challenges such as high production costs, limited phospholipid variety, and complex preparation processes, requiring further improvements to enhance their performance and expand their application range.

[0003] Our research group has previously designed various phospholipid monomers (including single-chain and double-chain phospholipids) and successfully prepared a variety of polymer-based monolithic phospholipid columns, covering both single-phospholipid and mixed-phospholipid monolithic columns. These chromatographic columns have significant application value in drug-membrane interaction studies and the prediction of drug-induced phospholipid deposition risk. Summary of the Invention

[0004] The purpose of this invention is to provide an immobilized artificial membrane chromatography (IAMC) stationary phase, its preparation method, and its applications. This method modifies the surface of a silica matrix using an organic polymer coating, and then grafts phospholipid functional groups onto the matrix via active free radical polymerization, thereby constructing a novel IAMC stationary phase. This preparation method is simple, improves the phospholipid diversity and acid-base stability of the stationary phase, and achieves biomembrane-like properties, potentially solving the problems of cumbersome preparation, limited phospholipid types, and poor stability in existing technologies. The method of grafting phospholipids onto the silica surface developed in this study successfully solves the key technical bottlenecks of commercial IAMC stationary phases: phospholipid grafting is achieved through a controllable and efficient "surface-initiated grafting" method, significantly reducing the complexity of traditional preparation processes; this method can efficiently graft multiple phospholipid structures, greatly expanding the diversity of the stationary phase; and in terms of acid-base stability, surface modification technology improves the acid-base stability of the stationary phase.

[0005] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0006] An immobilized artificial membrane chromatography stationary phase, characterized in that the structure of the immobilized artificial membrane chromatography stationary phase is as shown in Formula I:

[0007]

[0008] Where X is the matrix material, Z is an atom or group that can be transferred by free radicals, and R1 is selected from hydrogen atoms, straight-chain or branched hydrocarbon groups;

[0009] R2 is selected from -OR or -NHR, where R is selected from straight-chain or branched hydrocarbon groups;

[0010] R3 is selected from a hydrogen atom or a unit of negative charge;

[0011] R4 is selected from hydrogen atom, 2-(trimethylammonium)ethyl, 2-aminoethyl, 2-carboxy-2-aminoethyl, glyceryl, inositoloxy or derivatives thereof;

[0012] Degree of aggregation n>1.

[0013] Preferably, the matrix material of X is selected from silicon dioxide, aluminum oxide, zirconium oxide, or titanium oxide; the atoms or groups of Z are selected from halogen atoms, dithioester groups, or trithiocarbonate groups.

[0014] Preferably, the structure of the immobilized artificial membrane chromatography stationary phase is as shown in Formula II:

[0015]

[0016] Preferably, the preparation method includes the following steps: first, the matrix material is treated; then, a reagent with free radical transferable atoms or groups is coupled to the surface of the treated matrix material to form an initiator; then, phospholipid monomers are provided to the surface of the initiator; and the reaction is carried out under the conditions of a catalyst and a reducing agent to obtain the immobilized artificial membrane chromatography stationary phase.

[0017] Preferably, the procedure specifically includes the following steps:

[0018] S1. The matrix material is acidified using a mixture of concentrated hydrochloric acid and methanol to obtain an activated matrix material; the activated matrix material is coupled with a silanizing agent using toluene as a reaction solvent to obtain an aminated matrix material.

[0019] S2. Add the aminated matrix material, triethylamine, and a reagent with free radical transferable atoms or groups to anhydrous tetrahydrofuran and react at room temperature for 1–24 h to obtain the acylated matrix material.

[0020] S3. Mix the phospholipid monomer, acyl bromide matrix material, catalyst and reducing agent and react for 1 to 60 hours to obtain the immobilized artificial membrane chromatography stationary phase.

[0021] Preferably, in S1, the ratio of concentrated hydrochloric acid to methanol is 10% to 60%, and the molar concentration of concentrated hydrochloric acid is 6 to 12 mol / L; the mass-to-volume ratio of the matrix material to the mixed solution of concentrated hydrochloric acid and methanol is 1 to 5 g: 20 to 200 mL; the mass-to-volume ratio of the activated matrix material, silanizing reagent, and toluene is 0.1 to 5 g: 0.1 to 8 mL: 10 to 200 mL.

[0022] In S2, the mass-to-volume ratio of the aminated matrix material, triethylamine, and reagents with free radical transferable atoms or groups is 0.1–5 g : 0.1–6 mL : 0.1–8 mL;

[0023] In S3, the molar ratio of the phospholipid monomer to the catalyst ranges from 100:1 to 10000:1; the acylation matrix material is 0.1% to 30% of the mass of the phospholipid monomer; the molar ratio of the catalyst to the reducing agent is 1:0.5 to 1:10; the amount of the phospholipid monomer is more than 400 mg; the reaction temperature is 50 to 60 °C, and the reaction time is 16 to 30 h.

[0024] Preferably, in S1, the matrix material is silicon dioxide, aluminum oxide, zirconium oxide, or titanium oxide, with a particle size of 1.7–100 μm;

[0025] The silanizing agent is 3-aminopropyltriethoxysilane;

[0026] In S2, the reagent for the free radical transferable atom or group is 2-bromoisobutyryl bromide.

[0027] Preferably, in S3, the phospholipid monomer is phosphatidylcholine or phosphatidylethanolamine or phosphatidic acid or phosphatidylserine or phosphatidylinositol or phosphatidylglycerol or derivatives thereof or combinations thereof, including single-chain, double-chain or multi-chain with different chain lengths;

[0028] The catalyst is a complex formed by a transition metal salt and a ligand; the transition metal salt includes copper salt, iron salt, rhodium salt, rhenium salt, vanadium salt, zinc salt, gold salt, silver salt, molybdenum salt, manganese salt, chromium salt, tungsten salt, cobalt salt, ruthenium salt, or combinations thereof; the ligand is selected from bipyridine and its derivatives, tris(2-pyridyl)phosphine and its derivatives, dithiocarboxylic acid esters, or combinations thereof; the molar ratio of the transition metal salt to the ligand is 1:1 to 1:4;

[0029] The reducing agent is selected from ascorbic acid or sodium ascorbate or tin(II) 2-ethylhexanoate or combinations thereof.

[0030] More preferably, the phospholipid monomer is 2-methacryloyloxydodecylphosphocholine, 2-methacryloyloxydodecylphosphoglycerol, 2-methacryloyloxydodecylphosphocarboxylic acid, 2-methacryloyloxydodecylphosphoethanolamine, 2-methacryloyloxydodecylphosphophosphate, 1-decyl-2-(11-methacrylamidoundecyl)-sn-glycerol-3-phosphoethanolamine, 1-decyl-2-(11-methacrylamidoundecyl)-sn-glycerol-3-phosphate, 1-decyl-2-(11-methacrylamidoundecyl)-sn-glycerol-3-phosphate, At least one of the following: (1-acrylamidoundecanoyl)-sn-glycerol-3-phosphate glycerol-cardiolipin, 1-dodecanoyl-2-(12-methacryloyloxydodecanoyl)-sn-glycerol-3-phosphate choline, 1-dodecanoyl-2-(12-methacryloyloxydodecanoyl)-sn-glycerol-3-phosphate, 1-dodecanoyl-2-(12-methacryloyloxydodecanoyl)-sn-glycerol-3-cardiolipin, and 1-dodecanoyl-2-(12-methacryloyloxydodecanoyl)-sn-glycerol-3-phosphate.

[0031] The present invention relates to the application of an immobilized artificial membrane chromatography stationary phase.

[0032] Preferably, the immobilized artificial membrane chromatography immobilizer is used in the study of drug-cell membrane interactions, and in environmental and food applications. Compared with the prior art, the present invention has the following advantages:

[0033] (1) The existing IAMC stationary phase preparation process is complicated, while the present invention uses a simple and efficient silica matrix phospholipid grafting technology, which makes the preparation process simpler.

[0034] (2) In the prior art, the phospholipids in IAMC columns are of a single type. This invention can prepare a variety of phospholipid membrane stationary phases to more accurately simulate the biomembrane environment.

[0035] (3) Protected by the polymer coating, the grafted silica matrix exhibits excellent acid and base stability over a wide pH range (2-10). Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the preparation process of the IAMC stationary phase prepared in Example 1.

[0037] Figure 2 Here is a scanning electron microscope image of the IAMC stationary phase prepared in Example 1, wherein... Figure 2 a is an electron microscope image of bare silicone. Figure 2 b is an electron microscope image of the IAMC stationary phase.

[0038] Figure 3 The image shows the EDS energy dispersive spectroscopy (EDS) spectrum of the IAMC stationary phase prepared in Example 1.

[0039] Figure 4 Separation diagram of several β-blocker drugs in reverse-phase mode using the IAMC packed column prepared in Example 1.

[0040] Figure 5 The image shows the separation of benzophenone compounds by the IAMC packed column prepared in Example 1 in reverse phase mode.

[0041] Figure 6 The image shows the separation of polycyclic aromatic hydrocarbons using the IAMC packed column prepared in Example 1 in reverse phase mode.

[0042] Figure 7 CHI IAMC packed column prepared in Example 1 7.4(MDPC) The CHI IAMC values ​​measured by commercial IAMC column IAMC.PC.DD2 7.4(PC.DD2) Correlation analysis of values.

[0043] Figure 8 Qualitative comparison of the IAMC packed column prepared in Example 1 in an in vitro early warning model of drug-induced phospholipid deposition risk. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise stated, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0045] Example 1: Preparation of IAMC stationary phase

[0046] (1) Preparation of phospholipid monomers

[0047] a. 25 g of 1,12-dodecanediol, 250 mL of anhydrous tetrahydrofuran, and 4 mL of dry pyridine were added to a 500 mL two-necked flask and stirred at 35 °C until clear. Then, 5 mL of methacrylamide chloride was diluted with 20 mL of tetrahydrofuran and added dropwise through a constant pressure funnel over 1 hour. The reaction was then carried out at 25 °C for 24 hours. The resulting pyridine salt was removed by filtration, and after filtration and drying, 200 mL of dichloromethane was added and stirred overnight at 25 °C. The mixture was filtered again and dried, and purified by column chromatography to obtain a colorless oil.

[0048] b. Take 3 mL of the oily substance obtained in step (1) into a 50 mL single-necked flask, add 20 mL of phosphorus oxychloride, react for 16 h, then pour directly onto 200 mL of ice. After the ice melts, react for another 1 h. Extract three times with 60 mL of ethyl acetate and collect the upper organic phase. Then dry with anhydrous magnesium sulfate and evaporate to dryness for later use.

[0049] c. 2g of the product from step (2) above, 0.84g of choline chloride, 2,4,6-triisopropylbenzenesulfonyl chloride, and 50mL of pyridine were added to a 100mL single-necked flask and stirred at room temperature for 24h. After direct rotary evaporation to dryness, the product was purified by column chromatography, using V as the eluent. 甲醇 :V 二氯甲烷 After removing impurities at a ratio of 1:2, use V 甲醇 :V 水 The solution was purified at a ratio of 10:1, and the solvent was removed to obtain a white solid, 2-methacryloyloxydodecyl choline phosphoryl phosphate (MDPC). 1 HNMR(300MHz,Methanol-d4)δ6.0-6.1(m,1H),5.61-5.63(m,1H),4.23-4.26(m,2H),4.16(t,2H),3.89(m,2H), 3.62-3.67(m,3H),3.24(s,9H),1.93-1.96(m,3H),1.66-1.72(m,1=2H),1.58-1.65(m,2H),1.26-1.44(m,16H).

[0050] (2) Preparation of acyl brominated silica gel

[0051] a. Preparation of activated silica gel: Take 3g of silica gel (5μm, After adding the silica gel to a 250 mL three-necked flask, add 50 mL of methanol and 50 mL of concentrated hydrochloric acid (12 mol / L), and react in an oil bath at 70 °C for 4 h to obtain activated silica gel. After the reaction is complete, wash with methanol and water until neutral, and then dry in a vacuum drying oven at 120 °C for later use.

[0052] b. Preparation of aminated silica gel: 1.5 g of activated silica gel, 4.5 mL of 3-aminopropyltriethoxysilane (APTES), and 50 mL of dry toluene were added to a 100 mL three-necked flask and reacted at 110 °C for 24 h. Afterward, the mixture was washed three times with toluene and acetone and then dried in a vacuum oven at 120 °C to obtain aminated silica gel.

[0053] c. Preparation of acylated silica gel: Take 3g of aminoized silica gel, 50mL of tetrahydrofuran and 2.5mL of triethylamine into a 100mL three-necked flask and stir at 0℃ for 15min. Then add 4.5mL of 2-bromoisobutyryl bromide into the reaction system within 1h. After stirring at room temperature for 24h, wash with tetrahydrofuran, water and acetone until neutral to obtain acylated silica gel.

[0054] (3) Polymerization of phospholipid monomers

[0055] like Figure 1As shown, a method for preparing an IAMC stationary phase includes the following steps: 400 mg of phospholipid monomer, 100 mg of acylated silica gel, 18 mg of cuprous chloride, 28 mg of 2,2'-bipyridine, 30 mg of ascorbic acid, and 10 mL of anhydrous methanol are added to a sealed container and reacted at 55 °C for 24 h. After washing three times with saturated disodium ethylenediaminetetraacetate solution, distilled water, and methanol respectively, the mixture is dried at 50 °C to obtain the IAMC stationary phase.

[0056] Example 2

[0057] A method for preparing an IAMC stationary phase differs from Example 1 in that: 400 mg of phospholipid monomer, 100 mg of acylated silica gel, 18 mg of cuprous chloride, 28 mg of 2,2'-bipyridine, 30 mg of ascorbic acid, and 10 mL of anhydrous methanol are added to a sealed container and reacted at 55 °C for 16 h. After washing three times with saturated disodium ethylenediaminetetraacetate solution, distilled water, and methanol respectively, the IAMC stationary phase is obtained by drying at 50 °C.

[0058] Example 3

[0059] A method for preparing an IAMC stationary phase differs from Example 1 in that: 400 mg of phospholipid monomer, 100 mg of acylated silica gel, 18 mg of cuprous chloride, 28 mg of 2,2'-bipyridine, 30 mg of ascorbic acid, and 10 mL of anhydrous methanol are added to a sealed container and reacted at 55°C for 8 h. After washing three times with saturated disodium ethylenediaminetetraacetate solution, distilled water, and methanol respectively, the IAMC stationary phase is dried at 50°C to obtain the aforementioned IAMC stationary phase.

[0060] Example 4

[0061] A method for preparing an IAMC stationary phase differs from Example 1 in that: 600 mg of phospholipid monomer, 100 mg of acylated silica gel, 18 mg of cuprous chloride, 28 mg of 2,2'-bipyridine, 30 mg of ascorbic acid, and 10 mL of anhydrous methanol are added to a sealed container and reacted at 55 °C for 24 h. After washing three times with saturated disodium ethylenediaminetetraacetate solution, distilled water, and methanol respectively, the IAMC stationary phase is dried at 50 °C to obtain the aforementioned IAMC stationary phase.

[0062] Example 5

[0063] A method for preparing an IAMC stationary phase differs from Example 1 in that: 200 mg of phospholipid monomer, 100 mg of acylated silica gel, 18 mg of cuprous chloride, 28 mg of 2,2'-bipyridine, 30 mg of ascorbic acid, and 10 mL of anhydrous methanol are added to a sealed container and reacted at 55 °C for 24 h. After washing three times with saturated disodium ethylenediaminetetraacetate solution, distilled water, and methanol respectively, the IAMC stationary phase is dried at 50 °C to obtain the aforementioned IAMC stationary phase.

[0064] Result detection

[0065] (1) SEM and EDS characterization of the IAMC stationary phase

[0066] The morphology of the IAMC stationary phase prepared in Example 1 was characterized using scanning electron microscopy (SEM), and the results are as follows: Figure 2 As shown. Among them, Figure 2 a is an SEM image of bare silicone. Figure 2 b is an IAMC fixed-phase SEM image. From Figure 2 It can be clearly observed that the modified silicone surface exhibits obvious modification marks. To further verify whether phospholipid modification exists on the silicone surface, energy-dispersive spectroscopy (EDS) was used to analyze the sample, and the results are as follows. Figure 3 As shown. From Figure 3 The EDS spectrum showed a distribution of different elements, with phosphorus clearly visible on the surface of the silica spheres. This result indicates that phospholipid monomers containing carbon-carbon double bonds can be successfully modified onto a silica matrix using living radical polymerization.

[0067] The elemental analysis results of C, H, O, N, Si, Br, and P in each embodiment are shown in Table 1 below:

[0068] Table 1

[0069]

[0070] As can be seen from Examples 1 to 3 above, a reaction time of 16 to 24 hours is more conducive to the modification of phospholipid functional monomers onto silica gel.

[0071] As can be seen from Examples 1, 4 and 5 above, using an amount of phospholipid monomer of 400 mg or more is more conducive to the modification of phospholipid monomer on silica gel.

[0072] (2) Separation test of several drugs using IAMC packed column in reverse phase mode:

[0073] The IAMC packed column prepared in Example 1 was used to separate β-blockers, benzophenones, and polycyclic aromatic hydrocarbons. The specific separation results are as follows:

[0074] a. Isolation of β-receptor blockers

[0075] Using isothermal conditions, a series of β-receptor blockers were tested, and the separation results are as follows: Figure 4 .

[0076] Test conditions:

[0077] Mobile phase: 40% acetonitrile and 60% 50mM ammonium acetate solution (pH 7.4)

[0078] Samples: Atenolol, Acetaminophen, Esmolol, Talinolol, Alpralol, Propranolol; Flow rate: 800 nL / min

[0079] Detection wavelength: 210nm

[0080] Column: IAMC packed column prepared in Example 1, 150 mm × 0.1 mm ID.

[0081] b. Separation of phenyl ketone compounds

[0082] A series of benzophenone compounds were tested under isocratic conditions, and the separation results are as follows: Figure 5 .

[0083] Test conditions:

[0084] Mobile phase: 40% acetonitrile and 60% 50mM ammonium acetate solution (pH 7.4)

[0085] Samples: Acetophenone, Phenylacetone, Butylene-methylphenidate, Acetophenone, Hexanone, Phenylacetone, Octanone

[0086] Flow rate: 700 nL / min

[0087] Detection wavelength: 254nm

[0088] Column: IAMC packed column prepared in Example 1, 150 mm × 0.1 mm ID.

[0089] c. Separation of polycyclic aromatic hydrocarbons

[0090] A series of polycyclic aromatic hydrocarbons were tested under isocratic conditions, and the separation results are as follows: Figure 6 :

[0091] Test conditions:

[0092] Mobile phase: 40% acetonitrile and 60% 50mM ammonium acetate solution (pH 7.4)

[0093] Samples: Naphthalene, Fluorene, Phenanthrene, Pyrene, Benzo[a]anthracene

[0094] Flow rate: 800 nL / min

[0095] Detection wavelength: 210nm

[0096] Column: IAMC packed column prepared in Example 1, 150 mm × 0.1 mm ID.

[0097] from Figures 4 to 6 It can be seen that the IAMC packed column prepared in this invention has good separation ability for small molecule compounds.

[0098] (3) Acid-base stability test:

[0099] To evaluate the acid-base stability of the IAMC packed column in Example 1, the pH of the test solution was adjusted to 2 and 10 using 1M hydrochloric acid and sodium hydroxide solution, respectively, with thiourea and anisole as test substances. During the experiment, acetonitrile / water (40:60, V / V) was used as the mobile phase, and the retention times of thiourea and anisole were measured every 12 hours for a total of 72 hours. The results showed that under extreme conditions of pH 2 and pH 10, the relative standard deviation (RSD) of the retention factor k of anisole was <5%, indicating that the IAMC stationary phase did not undergo significant structural changes or loss of functional groups in strongly acidic and strongly alkaline environments, demonstrating good acid-base stability. To further verify this advantage, a literature review revealed that commercial chromatographic columns showed significant performance degradation (RSD) after running at a flow rate of 1.0 mL / min for 5 days in neutral PBS buffer (pH 7.4). m-硝基苯胺 >5%). Comparative analysis confirmed that the IAMC stationary phase prepared in Example 1 exhibited significantly better stability than conventional commercial chromatographic columns under strong acid (pH 2) and strong base (pH 10) conditions.

[0100] (3) Hydrophobicity index (CHI) of IAMC packed column chromatography 7.4 Examination of value correlation:

[0101] To evaluate the predictive performance of IAMC-packed columns on drug transmembrane transport capabilities, 62 drugs were randomly selected as test samples. The retention times of each drug were determined using the IAMC-packed column prepared in Example 1, and the column was then converted to CHIIAMC. 7.4(MDPC) Values ​​were obtained. Simultaneously, the same drug samples were measured in parallel using a commercially available IAMC column, IAMC.PC.DD2, to obtain the corresponding CHI IAMC values. 7.4(PC.DD2) The values ​​are compared and analyzed. For example... Figure 7 As shown, the CHI IAMC measured by the two chromatographic columns 7.4 The values ​​showed a significant correlation trend, with a Spearman correlation coefficient of 0.834. This result indicates that the IAMC stationary phase developed in this study possesses drug-membrane interaction prediction capabilities similar to commercially available products, and has broad application prospects, particularly in the fields of drug screening and transport behavior research.

[0102] (4) Application of IAMC packed columns in in vitro early warning model of drug-induced phospholipid deposition risk.

[0103] Drugs inhibit phospholipase activity in lysosomes, leading to the accumulation of phospholipids within the lysosome, forming lamellar structures, and accompanied by progressive organ damage and dysfunction. This has become a significant concern in drug safety. Understanding this mechanism, the mechanisms of action of related drugs, and risk control strategies can effectively improve safety prediction in new drug development and risk management in clinical drug use.

[0104] To investigate this issue, 41 drugs that have been reported to have the risk of phospholipid deposition were randomly selected, and their CHI IAM7.4 values ​​in IAMC columns were measured and compared with the predicted values ​​of in vitro models. Figure 8 The qualitative comparison results of the early warning model are presented. The prediction threshold is set at 43; values ​​greater than 43 indicate positive drugs, and values ​​less than 46 indicate negative drugs. Figure 8 It was observed that phenacetin and 6-hydroxydopamine showed false negatives, while clofibrate, flecainide, and rifampin showed false positives. Nevertheless, the model's predictive accuracy (0.89) was still higher than other predictive models such as Ploemen (76%), Tomizawa (80%), and Zhou et al. (87%), indicating that this model still possesses good predictive performance in the in vitro early warning system for drug-induced phospholipid deposition risk, providing valuable reference for safety assessment in the drug development process.

[0105] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An immobilized artificial membrane chromatography stationary phase, characterized in that, The structure of the immobilized artificial membrane chromatography stationary phase is shown in Formula I: Where X is the matrix material, Z is an atom or group that can be transferred by free radicals, and R1 is selected from hydrogen atoms, straight-chain or branched hydrocarbon groups; R2 is selected from -OR or -NHR, where R is selected from straight-chain or branched hydrocarbon groups; R3 is selected from a hydrogen atom or a unit of negative charge; R4 is selected from hydrogen atom, 2-(trimethylammonium)ethyl, 2-aminoethyl, 2-carboxy-2-aminoethyl, glyceryl, inositoloxy or derivatives thereof; The degree of aggregation n>1.

2. The immobilized artificial membrane chromatography stationary phase according to claim 1, characterized in that, The matrix material of X is selected from silicon dioxide, aluminum oxide, zirconium oxide, or titanium oxide; the atoms or groups of Z are selected from halogen atoms, dithioester groups, or trithiocarbonate groups.

3. The immobilized artificial membrane chromatography stationary phase according to claim 1, characterized in that, The structure of the immobilized artificial membrane chromatography stationary phase is shown in Formula II:

4. A method for preparing an immobilized artificial membrane chromatography stationary phase as described in any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: first, the matrix material is treated; then, a reagent with free radical transferable atoms or groups is coupled to the surface of the treated matrix material to form an initiator; then, phospholipid monomers are provided to the surface of the initiator; and the reaction is carried out under the conditions of a catalyst and a reducing agent to obtain the immobilized artificial membrane chromatography stationary phase.

5. The preparation method according to claim 4, characterized in that, Specifically, the following steps are included: S1. The matrix material is acidified using a mixture of concentrated hydrochloric acid and methanol to obtain an activated matrix material; the activated matrix material is coupled with a silanizing agent using toluene as a reaction solvent to obtain an aminated matrix material. S2. Add the aminated matrix material, triethylamine, and a reagent with free radical transferable atoms or groups to anhydrous tetrahydrofuran and react at room temperature for 1–24 h to obtain the acylated matrix material. S3. Mix the phospholipid monomer, acyl bromide matrix material, catalyst and reducing agent and react for 1 to 60 hours to obtain the immobilized artificial membrane chromatography stationary phase.

6. The preparation method according to claim 5, characterized in that, In S1, the ratio of concentrated hydrochloric acid to methanol is 10% to 60%, and the molar concentration of concentrated hydrochloric acid is 6 to 12 mol / L; the mass-to-volume ratio of the matrix material to the mixed solution of concentrated hydrochloric acid and methanol is 1 to 5 g: 20 to 200 mL; the mass-to-volume ratio of the activated matrix material, silanizing reagent, and toluene is 0.1 to 5 g: 0.1 to 8 mL: 10 to 200 mL. In S2, the mass-to-volume ratio of the aminated matrix material, triethylamine, and reagents with free radical transferable atoms or groups is 0.1–5 g : 0.1–6 mL : 0.1–8 mL; In S3, the molar ratio of the phospholipid monomer to the catalyst ranges from 100:1 to 10000:1; the acylation matrix material is 0.1% to 30% of the mass of the phospholipid monomer; the molar ratio of the catalyst to the reducing agent is 1:0.5 to 1:10; the amount of the phospholipid monomer is more than 400 mg; the reaction temperature is 50 to 60 °C, and the reaction time is 16 to 30 h.

7. The preparation method according to claim 6, characterized in that, In S1, the matrix material is silicon dioxide, aluminum oxide, zirconium oxide, or titanium oxide, with a particle size of 1.7–100 μm; The silanizing agent is 3-aminopropyltriethoxysilane; In S2, the reagent for the free radical transferable atom or group is 2-bromoisobutyryl bromide.

8. The preparation method according to claim 7, characterized in that, In S3, the phospholipid monomer is phosphatidylcholine or phosphatidylethanolamine or phosphatidic acid or phosphatidylserine or phosphatidylinositol or phosphatidylglycerol or their derivatives or combinations thereof, including single-chain, double-chain or multi-chain with different chain lengths; The catalyst is a complex formed by a transition metal salt and a ligand; the transition metal salt includes copper salt, iron salt, rhodium salt, rhenium salt, vanadium salt, zinc salt, gold salt, silver salt, molybdenum salt, manganese salt, chromium salt, tungsten salt, cobalt salt, ruthenium salt, or combinations thereof; the ligand is selected from bipyridine and its derivatives, tris(2-pyridyl)phosphine and its derivatives, dithiocarboxylic acid esters, or combinations thereof; the molar ratio of the transition metal salt to the ligand is 1:1 to 1:4; The reducing agent is selected from ascorbic acid or sodium ascorbate or tin(II) 2-ethylhexanoate or combinations thereof.

9. The application of the immobilized artificial membrane chromatography stationary phase as described in claims 1 to 3.

10. The application according to claim 9, characterized in that, The immobilized artificial membrane chromatography is used in the study of drug-cell membrane interactions, as well as in the environmental and food fields.

Images