WO2020259079A1

WO2020259079A1 - Preparation method for and application of p-glycoprotein bioaffinity chromatography column

Info

Publication number: WO2020259079A1
Application number: PCT/CN2020/087975
Authority: WO
Inventors: 童珊珊; 陈昱初; 余江南; 徐希明
Original assignee: 江苏大学
Priority date: 2019-06-28
Filing date: 2020-04-30
Publication date: 2020-12-30
Also published as: CN110308233A

Abstract

A preparation method for and an application of a bioaffinity chromatography column rich in P-glycoprotein. The preparation method comprises: extracting a plasma membrane layer containing P-glycoprotein in a Caco-2 cell and placing same in a dialysis bag, performing dialysis to obtain a plasma membrane solution rich in P-glycoprotein, mixing the plasma membrane solution with amino-bonded silica gel uniformly and then leaving standing and performing centrifugation, adding a Tris-HCL solution to the precipitate and mixing same uniformly to obtain a P-glycoprotein plasma membrane stationary phase suspension, introducing the P-glycoprotein plasma membrane stationary phase suspension into an empty column core, and completing column packing to obtain a P-glycoprotein bioaffinity chromatography column. The invention provides a new method for screening of P-glycoprotein target drugs, and has strong specificity and stability, a long service life, and a good application prospect.

Description

Preparation method and application of P-glycoprotein bioaffinity chromatography column

Technical field

The invention belongs to the technical field of medicine and relates to a preparation method and application of a P-glycoprotein bioaffinity chromatography column.

Background technique

P-glycoprotein (P-gp) is a transmembrane glycoprotein with a molecular weight of 170KD, which has an energy-dependent "drug pump" function. It is a drug export pump that relies on ATPase and a channel for Ca ²⁺ and Cl ^- . It directly or indirectly pumps the drug out of the cell through ATP, so that the intracellular drug is pumped out of the cell, reducing the concentration of the drug in the cell and making the cell resistant.

Substances that inhibit the P-gp transport substrate are called P-gp inhibitors. P-gp inhibitors can improve the bioavailability of therapeutic drugs by blocking drug efflux. In recent years, it has been widely used clinically to improve the efficacy of chemotherapy drugs. Many drugs and reagents have been proven to be P-gp inhibitors, such as verapamil, ginsenoside Rg3, naringenin, chuanxiong, paeonol, piperine, capsaicin, etc. The existing methods for screening P-gp substrates and inhibitors in vitro are commonly two-way transport, uptake and efflux, and ATPase activity assay, but these methods have long experimental periods (generally more than 21 days in culture) and cell models can only One-time use, not suitable for the screening of large quantities of target drugs. The technical solution of the present application aims to provide a method for preliminary screening of P-glycoprotein target drugs that can be used multiple times, and each screening only takes tens of minutes or several hours.

Summary of the invention

The present invention aims to provide a P-glycoprotein bioaffinity chromatography column with the advantages of strong specificity, strong affinity adsorption, good system stability, long service life, etc., which is applied to the detection of P-glycoprotein target drugs .

In order to solve the above technical problems, the technical solutions adopted by the present invention are:

A method for preparing a P-glycoprotein bioaffinity chromatography column includes the following steps: extracting the plasma membrane layer containing P-glycoprotein in Caco-2 cells in a dialysis bag, and obtaining a P-glycoprotein-rich quality after dialysis Membrane solution, mix the plasma membrane solution with amino-bonded silica gel and then stand still and centrifuge, add the precipitate to the Tris-HCL solution and mix the P-glycoprotein membrane stationary phase suspension to obtain the P-glycoprotein membrane stationary phase suspension The suspension is injected into the blank column core, and the P-glycoprotein bioaffinity chromatography column is obtained after the column is loaded.

Further, the plasma membrane merging layer is a merging layer in which the plasma membrane layer rich in P-glycoprotein is selected by the western blot method after plasma membrane extraction of the Caco-2 cells.

The dosage ratio of the plasma membrane solution to the amino-bonded silica gel is 0.6mL:0.6-1.2g.

When the packing is completely for detecting the effluent after packing, the total protein and total cholesterol content should not be detected.

The present invention also provides the application of the P-glycoprotein bioaffinity chromatographic column prepared according to the above method in screening P-glycoprotein target drugs. The said application is the use of a chromatographic analysis method with ammonium acetate as the mobile phase and chromatography The column is a P-glycoprotein bioaffinity chromatography column for high performance liquid chromatography analysis. When there are chromatographic peaks in the sample to be tested, it means that the sample to be tested contains P-glycoprotein target drugs, otherwise it is not; Peak retention means that the retention time of the chromatographic peak in the sample to be tested is longer than the control blank column, and the peak range is wider.

Further, the concentration of the mobile phase ammonium acetate in the chromatographic analysis is 5-15 mmol/L; the chromatographic column temperature of the high performance liquid chromatography is 37°C, and the mobile phase flow rate is 0.2-0.4 mL/min.

Compared with the prior art, the beneficial effects of the present invention are:

The invention extracts the plasma membrane rich in P-glycoprotein from Caco-2 cells, constructs a P-glycoprotein bioaffinity chromatographic column with amino-bonded silica gel, and applies the constructed P-glycoprotein bioaffinity chromatographic column to chromatography In the analysis, it was verified that it can specifically bind to P-glycoprotein-targeted drugs, and has a specific affinity adsorption function for P-glycoprotein-positive drugs verapamil, but not for drugs that are not targeted by P-glycoprotein. Adsorption. It provides a new method for the screening of P-glycoprotein target drugs and has broad application prospects. The P-glycoprotein-rich bioaffinity chromatography column prepared by the invention has undergone performance evaluation and proved to have strong specificity, stability, long service life and good application prospects.

Description of the drawings

Figure 1 is a diagram of the layered plasma membrane of Caco-2 cells after centrifugation;

Figure 2 is a diagram showing the characterization of Caco-2 plasma membrane P-glycoprotein;

Figure 3 is an immunofluorescence imaging image of Caco-2 plasma membrane. In the figure, A is a blank control of Caco-2 plasma membrane; B is an immunofluorescence imaging image of extracted P-gp protein membrane;

Figure 4 is an electron microscopic characterization diagram of P-glycoprotein membrane silica gel stationary phase. In the figure, A is blank silica gel, and B is the bioaffinity silica gel stationary phase of P-glycoprotein membrane silica gel;

Figure 5 is a comparison diagram of the adsorption performance of P-glycoprotein bioaffinity chromatography column on verapamil when the mobile phase ammonium acetate concentration is 5mmol/L, 10mmol/L and 15mmol/L respectively;

Figure 6 is a comparison diagram of the adsorption performance of P-glycoprotein bioaffinity chromatography column for verapamil when the flow rates are 0.2mL/min, 0.3mL/min and 0.4mL/min;

Figure 7 is a comparison diagram of the adsorption performance of P-glycoprotein bioaffinity chromatography column for verapamil when the column temperature is 27℃, 37℃ and 47℃;

Figure 8 is the specific evaluation diagram of P-glycoprotein bioaffinity chromatography column; in the figure, A is the chromatogram of pyrazinamide-blank column, B is the chromatogram of verapamil-blank column, and C is the chromatogram of pyrazinamide-P -Glycoprotein bioaffinity chromatography column chromatogram, D is the chromatogram of verapamil-P-glycoprotein bioaffinity chromatography column;

Figure 9 is a comparison diagram of retention time of P-glycoprotein bioaffinity chromatography column on day 0, day 30 and day 90;

Figure 10 is a comparison diagram of the retention time of the P-glycoprotein bioaffinity chromatography column at 0 hour, 48 hour, 96 hour, 120 hour and 200 hour usage time.

Detailed ways

Hereinafter, the technical solutions and advantages of the present invention will be explained and described in more detail in conjunction with the drawings and specific implementations of the specification. It should be understood that the content presented in the specification, specific embodiments, and drawings of the specification are only for more clearly describing the technical solutions and advantages of the present invention, and do not limit the protection scope of the present invention.

The instruments and reagents used in the present invention are all common commercially available products, which are all available in the market.

Example 1:

Cultivation of Caco-2 cells: Caco-2 cells are cultured in a high-glycemic medium containing 10% fetal bovine serum, 1% double antibodies and 1% non-essential amino acids. The cell mass is counted as 20 25cm ² cell flasks. Collect the cells. Add the plasma membrane extract to the collected Caco-2 cells. The components of the plasma membrane extract are: 1mL lysate (1% Triton-100, 10mmol/LTris, 150mmol/LNaCl, 2.5mmol/LEDTA), 10μL100mmol/LPMSF And 75μL aprotinin; lightly beat the cells and grind them at 4°C. After grinding, let them stand for 15min. Transfer the cell crushing liquid to a special centrifuge tube, and then slowly add it along the wall in a trickle water with a volume ratio of 2: 2:1 80% sucrose solution, 30% sucrose solution, 5% sucrose solution, the sucrose layer has an interface, stand for 20min at 4℃, centrifuge at 4℃, 200000g for 16h and take it out; Figure 1 is the result after centrifugation Plasma membrane layering of Caco-2 cells is shown in Figure 1. The centrifuged Caco-2 cells are divided into 7 layers of macroscopic flocculent layers from top to bottom, of which the bottom is the sedimentation layer. Westernblot characterization of each flocculent layer with P-glycoprotein as the characterizing protein, identification and extraction of the plasma membrane layer rich in P-glycoprotein; the collected plasma membrane rich in P-glycoprotein combined layer at 3000 Da The dialysis bag was dialyzed in PBS with pH 7.4 for 48 hours to obtain a plasma membrane solution rich in P-glycoprotein, which was stored in a refrigerator at -80°C until use.

Figure 2 is a diagram showing the characterization of P-glycoprotein in the plasma membrane of Caco-2 cells; in the figure, numbers 1-6 indicate layer 1 to layer 6 in turn, and number 7 indicates sedimentation layer; as shown in Figure 2, The results show that the 3-6 layers are rich in P-glycoprotein, while the precipitation layer P-glycoprotein has almost no expression. It can be seen that the collected and extracted plasma membrane layer is the 3-6 layer flocculent layer; with P-gp The monoclonal antibody is the primary antibody, and the fluorescently-labeled goat anti-rabbit is the secondary antibody. Indirect immunofluorescence experiments were performed to further verify the extracted P-gp-rich plasma membrane combined layer. Figure 3 is an immunofluorescence imaging image of the plasma membrane of Caco-2 cells, where A is a blank control of the plasma membrane of Caco-2 cells; B is an immunofluorescence imaging image of the combined plasma membrane of the extracted P-gp; as shown in Figure 3. The plasma membrane of the blank control and the extracted P-gp were combined and observed under a fluorescence microscope. The extracts were observed to emit specific fluorescence, which further verified the combined plasma membrane of the collected P-gp (layers 3-6). ) It is rich in P-glycoprotein and can fully extract P-glycoprotein.

Take 0.6 mL of the plasma membrane solution rich in P-glycoprotein and 0.6 g of amino-bonded silica gel at 4°C and shake for 48 hours, leave it to stand for centrifugation, and mix the precipitate with 100 mM Tris-HCL solution to obtain rich P-sugar The protein membrane stationary phase suspension is set aside.

Identification of P-glycoprotein-rich membrane stationary phase suspension: Take 0.1-1 mL of the prepared plasma membrane stationary phase mixture and place it on a cover glass, and dry it naturally at room temperature. Take the dry powder and directly fix it on the conductive glue, and perform gold spraying and scanning electron microscopy for characterization. Figure 4 is an electron microscopic characterization diagram of a silica gel stationary phase rich in P-glycoprotein membrane. In the figure, A is blank silica gel, and B is a bioaffinity silica gel stationary phase rich in P-glycoprotein membrane silica gel; as shown in Figure 4, Compared with the blank silica gel, the plasma membrane rich in P-glycoprotein wrapped silica gel and did not fall off, indicating that the P-glycoprotein was successfully attached to the surface of the silica gel, and the preparation of the silica stationary phase suspension of the P-glycoprotein rich membrane was completed.

Prepare the chromatographic empty column core, column sleeve, and corresponding frit. Before installing the column, rinse with double distilled water, then install the column core in the direction indicated by the arrow on the column sleeve, and install a sieve plate at the lower end. Pack the column with a pressure of 50MPa, slowly inject the prepared P-glycoprotein membrane stationary phase suspension into the column core, use PBS as the packing liquid, and use an HPLC pump at a flow rate of 2mL/min to accelerate the filling to the front of the column. After the pressure rises slowly, open the column cover and the upper sieve, and then slowly inject the stationary phase suspension into the core again. Repeatedly, until the column pressure is stable, receive the effluent from the column to determine the total protein and cholesterol content to determine Whether the packing is complete; the blank silica gel column packing method is the same as the above, the difference is that the pure silica gel that is not bound to the plasma membrane is injected into the column core; the determination of the protein and cholesterol content in the plasma membrane before and after packing indicates that the packing is complete and the effect is good. Table 1 shows the total protein and total cholesterol content of the P-glycoprotein bioaffinity chromatographic column before and after packing; from Table 1, it can be seen that the P-glycoprotein bioaffinity chromatographic column is completely packed.

Table 1. Total protein and total cholesterol content before and after loading

Note: "-" means not detected.

Example 2:

Add 1mL lysate (1% Triton-100, 10mmol/LTris, 150mmol/L NaCl, 2.5mmol/LEDTA), 10μL 100mmol/LPMSF and 75μL aprotinin to the collected Caco-2 cells, and gently mix the cells. Grind at 4°C, leave it for 15 minutes after grinding, transfer the cell crushing liquid to a special centrifuge tube, and slowly add the 80% sucrose solution and 30% sucrose solution with a volume ratio of 2:2:1 in a trickle water along the wall. , 5% sucrose solution, the interface of the sucrose layer appears. Let stand for 20 min at 4°C, and collect and extract the plasma membrane layer after centrifugation at 4°C under 200,000g centrifugal conditions for 16 hours. The plasma membrane layer takes the 3-6 flocculent layer; The obtained plasma membrane layer was dialyzed in a 3000Da dialysis bag in PBS with pH 7.4 for 36 hours to obtain a plasma membrane solution rich in P-glycoprotein, which was stored in the refrigerator at -80°C for later use; The plasma membrane solution of the protein and 1.2g of amino-bonded silica gel were shaken at 4°C for 48 hours, and then left to stand and centrifuge. The precipitate was mixed with 100mM Tris-HCL solution to obtain a P-glycoprotein-rich membrane stationary phase suspension. The P-glycoprotein membrane stationary phase suspension was injected into the blank column core, and the P-glycoprotein bioaffinity column was obtained after verifying that the column was loaded.

Example 3:

Determine the retention time of the P-glycoprotein bioaffinity column to the P-glycoprotein positive drug verapamil when the mobile phase concentration is 5mmol/L, 10mmol/L and 15mmol/L ammonium acetate, and investigate the flow at different concentrations The influence of relative adsorption performance. Chromatographic conditions: P-glycoprotein bioaffinity chromatography column 30mmx4.6mm; flow rate: 0.3mL/min; column temperature: 37°C; injection volume: 20μL; injection concentration: 200μg/mL; UV detector; detection wavelength: 228nm. Figure 5 is a comparison diagram of the adsorption performance of verapamil on the P-glycoprotein bioaffinity column when the mobile phase ammonium acetate concentration is 5mmol/L, 10mmol/L and 15mmol/L, respectively; Figure 5 shows the ammonium acetate concentration At 5mmol/L, 10mmol/L and 15mmol/L, the peak time is 100min, 65min and 50min respectively. It can be seen that as the concentration of ammonium acetate in the mobile phase increases, the peak time of retained components is earlier.

Example 4:

Determine the retention time of the P-glycoprotein bioaffinity column to the P-glycoprotein positive drug verapamil at flow rates of 0.2mL/min, 0.3mL/min and 0.4mL/min, respectively, and investigate the effect of different flow rates on the chromatographic The effect of column adsorption performance. Chromatographic conditions: P-glycoprotein bioaffinity column 30mmx4.6mm; mobile phase 10mmol/L ammonium acetate (PH7.2-7.4); column temperature 37℃; sample volume 20μL; sample concentration 200μg/mL; UV detection Detector; detection wavelength 228nm. Figure 6 is a comparison diagram of the adsorption performance of P-glycoprotein bioaffinity column for verapamil when the flow rates are 0.2mL/min, 0.3mL/min, and 0.4mL/min. As can be seen from Figure 6, the flow rate is 0.2mL /min peak time is 175min, when the flow rate is 0.3mL/min, the peak time is 61.5min, the flow rate is 0.4mL/min, and the peak time is 46min. Within the flow rate range of 0.2mL/min～0.4mL/min, the retention effect is better. it is good.

Example 5:

The retention time of the P-glycoprotein bioaffinity column for the P-glycoprotein positive drug verapamil at the column temperature of 27℃, 37℃ and 47℃ was determined, respectively, and the adsorption performance of the column at different column temperatures was investigated. influences. Chromatographic conditions: P-glycoprotein bioaffinity column 30mmx4.6mm; mobile phase 10mmol/L ammonium acetate (pH7.2-7.4); flow rate 0.3mL/min; injection volume 20μL; injection concentration 200μg/mL; UV Detector; detection wavelength 228nm. Figure 7 is a comparison diagram of the adsorption performance of P-glycoprotein bioaffinity column for verapamil when the column temperature is 27°C, 37°C and 47°C; it can be seen from Figure 7 that the peak time is as the temperature rises in advance. The peak time at 27°C is longer, and the peak time at 47°C is the earliest. Considering that the efflux of P-glycoprotein occurs under the physiological environment of the human body, it is necessary to ensure the affinity screening of biochromatography as much as possible. The microenvironment is closer to the real situation, and 37℃ is selected as the column temperature for analysis.

Example 6:

Because pyrazinamide and P-glycoprotein have no significant effect on the function and expression. Therefore, pyrazinamide was selected as the P-glycoprotein negative drug. Using retention time as an indicator, the retention of P-glycoprotein negative drug pyrazinamide and P-glycoprotein positive drug verapamil on the blank column and P-glycoprotein bioaffinity chromatography column were investigated respectively. Chromatographic conditions: P-glycoprotein bioaffinity chromatography column 30mmx4.6mm; mobile phase 10mmol/L ammonium acetate; flow rate 0.3mL/min; column temperature 37℃; sample volume 20μL; sample concentration 200μg/mL; UV detection The detection wavelength of pyrazinamide is 268nm; the detection wavelength of verapamil is 228nm. Figure 8 is the specific evaluation diagram of P-glycoprotein bioaffinity chromatography column; in the figure, A is the chromatogram of pyrazinamide-blank column, B is the chromatogram of verapamil-blank column, and C is the chromatogram of pyrazinamide-P -Glycoprotein bioaffinity column chromatogram, D is the chromatogram of verapamil-P-glycoprotein bioaffinity column. It can be seen from Figure 7 that the blank column does not retain the P-glycoprotein positive drug verapamil and the P-glycoprotein negative drug pyrazinamide, while the P-glycoprotein bioaffinity chromatography column does not retain the P-glycoprotein negative drug pyrazine. The azine amide has no retention, the retention time for the P-glycoprotein positive verapamil is 61.5 min, the retention range is 50-120 min, and there is strong retention. It can be seen that the P-glycoprotein bioaffinity chromatography column constructed by the present invention The specificity is good and the reproducibility is good. The drug verapamil acting on the P-glycoprotein target has a strong binding ability to the receptor on the Caco-2 plasma membrane. This bioaffinity chromatography model can be applied to In vitro screening of P-glycoprotein substrates and inhibitors.

Example 7:

The retention time of the P-glycoprotein bioaffinity column on the P-glycoprotein positive drug verapamil on the 0th day, 30th day and 90th day stored in a refrigerator at 4°C was measured respectively. Chromatographic conditions: P-glycoprotein bioaffinity chromatography column 30mmx4.6mm; mobile phase 10mmol/L ammonium acetate; flow rate 0.3mL/min; column temperature 37℃; sample volume 20μL; sample concentration 200μg/mL; UV detection Detector; detection wavelength 228nm.

Figure 9 is a comparison diagram of retention time of P-glycoprotein bioaffinity chromatography column on day 0, day 30, and day 90; as shown in Figure 9, verapamil on day 0, day 30, and day 90 The retention time of P-glycoprotein is 66.6min, 69.2min and 61.5min respectively, RSD=5.96% (n=3), there is no significant difference in chromatographic retention behavior, indicating that the P-glycoprotein bioaffinity column is stored in a refrigerator at 4℃ after preparation. In days, its chromatographic retention activity is still good, which shows that the bioaffinity chromatographic column of the present invention has better stability.

Example 8:

Determine the retention time of the P-glycoprotein bioaffinity column with the use time of 0 hour, 48 hours, 96 hours, 120 hours and 200 hours to the P-glycoprotein positive drug verapamil, and investigate the effect of the use time on the chromatography The effect of column adsorption performance. Chromatographic conditions: P-glycoprotein bioaffinity chromatography column 30mmx4.6mm; mobile phase 10mmol/L ammonium acetate; flow rate 0.3mL/min; column temperature 37℃; sample volume 20μL; sample concentration 200μg/mL; UV detection The detection wavelength is 228nm.

Figure 10 is a comparison diagram of the retention time of the P-glycoprotein bioaffinity chromatographic column when the continuous use time is 0 hour, 48 hours, 96 hours, 120 hours and 200 hours; it can be seen from Figure 10 that the column is used for 200 hours after continuous use. The retention time of the positive drug verapamil did not change significantly, indicating that there was no significant change in the retention of the positive drug verapamil after 200 hours of continuous use.

The description of the above embodiments is only used to help understand the method and core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

A method for preparing a P-glycoprotein bioaffinity chromatography column, which is characterized in that it comprises the steps of: extracting the plasma membrane layer containing P-glycoprotein in Caco-2 cells in a dialysis bag, and obtaining P-rich P- Plasma membrane solution of glycoprotein, mix the plasma membrane solution with amino-bonded silica gel, and then stand and centrifuge. The precipitate is added to the Tris-HCL solution and mixed to obtain the P-glycoprotein membrane stationary phase suspension. The protein membrane stationary phase suspension is injected into the blank column core, and the P-glycoprotein bioaffinity column is obtained after the column is loaded.
The method for preparing a chromatographic column according to claim 1, wherein the plasma membrane layer is a combined plasma membrane layer rich in P-glycoprotein.
The method for preparing a chromatographic column according to claim 2, wherein the combined plasma membrane layer is a plasma membrane layer rich in P-glycoprotein selected by a western blot method after plasma membrane extraction of Caco-2 cells The merged layer.
The method for preparing a chromatographic column according to claim 1, wherein the dosage ratio of the plasma membrane solution to the amino-bonded silica gel is 0.6 mL: 0.6-1.2 g.
The method for preparing a chromatographic column according to claim 1, characterized in that the packing of the column is to detect the content of total protein and total cholesterol when the effluent after packing is not detected.
The application of the P-glycoprotein bioaffinity chromatography column prepared by the preparation method according to any one of claims 1 to 5 in screening P-glycoprotein target drugs, characterized in that the application is based on ammonium acetate As the mobile phase, the chromatographic column is a P-glycoprotein bioaffinity chromatographic column for HPLC analysis. When there are chromatographic peaks in the sample to be tested, it means that the sample to be tested contains P-glycoprotein target drugs, otherwise there is no .
The application according to claim 6, characterized in that the concentration of the mobile phase ammonium acetate is 5-15 mmol/L.
The application according to claim 5, wherein the chromatographic column temperature of the high performance liquid chromatography is 37°C, and the flow rate of the mobile phase is 0.2 to 0.4 mL/min.