Nanodrug Carrier with P-Glycoprotein Inhibitory Function, Preparation Method and Application

Abstract

The present invention provides a nano-drug carrier with P-glycoprotein inhibitory function, which is composed of methoxypolyethylene glycol-polylactic acid copolymer (mPEG-PDLLA) covalently coupled with a P-gp inhibitor (ER) through a disulfide bond linker. In view of the deficiencies of the existing technology of nano-drug carriers for clinical treatment of drug-resistant tumors, the present invention has conducted in-depth research and proposed an effective solution: (1) adding disulfide bonds to endow the carrier with redox responsiveness inside tumor cells, thereby increasing the local concentration of drugs at the treatment site and reducing toxicity and side effects; (2) through precise ratio control, covalently coupling the P-gp inhibitor ER with mPEG-PDLLA molecules, which not only ensures the stability of the carrier but also endows it with P-gp inhibitory ability.

Description

Technical Field

[0001] The present invention belongs to the field of nano-drug carriers, and particularly relates to a nano-drug carrier with P-glycoprotein inhibitory function, a preparation method and an application thereof. Background Art

[0002] Cancer is a disease formed by abnormal cell proliferation, characterized by high incidence, high mortality, etc. It causes tens of millions of deaths every year and is one of the main causes of death globally.

[0003] Currently, chemotherapy is still one of the main means for treating cancer. Traditional chemotherapy drugs either enter the gastrointestinal tract through oral administration, are absorbed by the digestive system, enter the blood circulation, and then are transported to the tumor site in the body through the blood, or are intravenously injected directly into the blood circulation to reach all parts of the body, including the tumor site. Generally speaking, whether through oral administration or intravenous injection, due to the lack of targeting ability, drugs are non-selectively and inefficiently delivered to the lesion site, and it is impossible to avoid their toxic and side effects on surrounding healthy tissues or cells. In addition, tumor cells repeatedly exposed to chemotherapy drugs may be affected by drug selection pressure, resulting in overexpression of intracellular P-glycoprotein (P-gp); P-gp is a transmembrane transport protein with the function of pumping chemotherapy drugs out of cells. Therefore, overexpression of P-glycoprotein can reduce the accumulation of drugs in cells, reduce the toxicity of drugs to cells, and cause cancer drug resistance.

[0004] Encequidar (ER) is a specific P-gp inhibitor used for developing an oral preparation of Paclitaxel (PTX). Binding with paclitaxel and administering orally can only inhibit P-gp in gastrointestinal tissues. Although it increases the blood drug concentration of PTX, it brings great gastrointestinal side effects, and ER cannot reach the cytoplasm of tumor cells to inhibit P-gp here. Therefore, its curative effect on drug-resistant cell carcinoma is limited. Therefore, achieving specific inhibition of P-gp in tumor cytoplasm by ER, enhancing the uptake of active substances, and reducing side effects at the same time is an effective method to reduce tumor drug resistance and prolong the survival time of patients; the chemical structure of ER is as follows:

[0005]

[0006]

[0007] Nanodrug delivery technology is an important technological advancement in the medical field in recent years. It has a wide range of applications and aims to improve the efficacy of drugs and reduce side effects. This technology uses nanoscale carriers to precisely deliver drugs into target tissues or cells, thereby achieving targeted drug therapy. The development of this technology stems from the limitations and deficiencies of traditional drug delivery systems, such as low bioavailability of drugs, non-specific distribution, and increased side effects. Nanodrug delivery technology has become a new drug delivery system that has attracted much attention by optimizing the release characteristics of drugs and enhancing the stability of drugs.

[0008] Both methoxypolyethylene glycol (mPEG) and poly (D, L-lactic acid) (PDLLA) are materials with good biocompatibility, having little toxicity and immunogenicity to the body. Therefore, they can reduce the adverse reactions of the drug delivery system to the body. At the same time, they have good stability, can protect drugs from degradation and inactivation, and prolong the plasma half-life of drugs. Therefore, in the field of nanodrug carriers, mPEG-PDLLA copolymers have broad application prospects and development potential. However, mPEG-PDLLA as an anti-tumor drug carrier material also has many deficiencies: (1) Limited drug loading capacity. The drug loading capacity of the mPEG-PDLLA carrier may be limited, which may affect the efficacy and treatment effect of drugs; (2) Non-selective drug release. It is unable to rapidly release drugs in tumor tissues or tumor cells; (3) Treatment resistance. Although the nanodrugs of the mPEG-PDLLA carrier can enter cells through endocytosis and temporarily bypass the P-gp efflux effect, the released anti-tumor drugs will still be pumped out of the cells by the P-gp pump, resulting in drug resistance.

[0009] Therefore, developing an mPEG-PDLLA nanodrug carrier that can improve the drug loading capacity, achieve controlled release of drugs, has P-gp inhibitory function and can encapsulate hydrophobic drugs is a very promising research direction in this technical field. Summary of the Invention

[0010] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a nanodrug carrier with P-glycoprotein inhibitory function, a preparation method and applications thereof.

[0011] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0012] In the first aspect, the present invention provides a nanodrug carrier with P-glycoprotein inhibitory function, which is composed of a methoxypolyethylene glycol-poly (D, L-lactic acid) copolymer (mPEG-PDLLA) covalently coupled with a P-gp inhibitor (ER) through a disulfide bond Linker, and the structural formula is shown as follows:

[0013]

[0014] The nano-drug carrier (SH181) contains the disulfide bond Linker and the P-gp inhibitor (ER) in its molecule, and has good redox responsiveness and P-gp inhibitory ability inside tumor cells compared with the mPEG-PDLLA copolymer.

[0015] In a second aspect, the present invention provides a method for preparing a nano-drug carrier (SH181) with P-glycoprotein inhibitory function, including:

[0016] Covalently coupling the mPEG-PDLLA copolymer with the P-gp inhibitor (ER) through the disulfide bond Linker, centrifuging to remove impurities, and drying to obtain the nano-drug carrier (SH181) with P-glycoprotein inhibitory function. Specifically, it includes the following steps:

[0017] (1) Using tetrahydrofuran as a solvent, first couple the P-gp inhibitor (ER) and the disulfide bond Linker;

[0018] (2) Using anhydrous dichloromethane as a solvent, couple the product obtained in step (1) with the mPEG-PDLLA copolymer under the action of a catalyst;

[0019] (3) Dissolve the product obtained in step (2) in deionized water, vortex and oscillate, and perform ultrasonic emulsification to obtain an emulsion;

[0020] (4) Ultracentrifuge the emulsion at low temperature, transfer the supernatant to an ultrafiltration centrifugal tube, centrifuge, wash, and freeze-dry to obtain the product.

[0021] Further, the feeding ratio of the P-gp inhibitor (ER) and the disulfide bond Linker in step (1) is 1:2.

[0022] Further, the number-average molecular weight of the mPEG-PDLLA in step (2) is 4 kD.

[0023] Further, the catalyst in step (2) is 4-dimethylaminopyridine and dicyclohexylcarbodiimide.

[0024] Further, the ultrasonic power in step (3) is 8 W, and the ultrasonic time is 15 s.

[0025] Further, the low-temperature ultracentrifugation in step (4) refers to centrifuging at 4 °C and 12,000 rpm for 15 min, and the minimum molecular weight cut-off of the ultrafiltration centrifugal tube is 3.5 kD.

[0026] In a third aspect, the present invention provides an application of a nano-drug carrier (SH181) with P-glycoprotein inhibitory function, including its application in the preparation of anti-drug-resistant tumor nano-drugs.

[0027] Specifically, a nano-drug carrier (SH181) with P-glycoprotein inhibitory function is used to encapsulate poorly soluble anti-tumor drugs to prepare anti-drug-resistant tumor nano-drugs.

[0028] Furthermore, the poorly soluble anti-tumor drugs include paclitaxel (Paclitaxel, PTX), docetaxel (Docetaxel, DTX), and cabazitaxel (Cabazitaxel, CTX).

[0029] Furthermore, taking the encapsulation of the paclitaxel as an example, the encapsulation method includes preparing corresponding nano-micelles:

[0030] (1) Dissolve an appropriate amount of the nano-drug carrier and the paclitaxel (mass ratio of nano-drug carrier: paclitaxel = 10:1) in an appropriate volume of dimethyl sulfoxide, vortex and oscillate to mix them evenly, and fully dissolve.

[0031] (2) Gradually add the mixed solution obtained in step (1) dropwise to an appropriate amount of deionized water, and continuously stir at 1000 rpm at room temperature for 20 min.

[0032] (3) Transfer the solution obtained in step (2) to a dialysis bag with a molecular weight cut-off of 3.5 kD, and dialyze at room temperature for 8 h.

[0033] (4) Filter the liquid obtained in step (3) through a membrane (0.22 μm), aliquot, and freeze-dry to obtain paclitaxel nano-micelles (SH181@PTX NPs).

[0034] The advantages of the present invention are as follows:

[0035] In view of the deficiencies of the existing technology of nano-drug carriers for treating drug-resistant tumors in clinical treatment, the present invention has conducted in-depth research and proposed effective solutions: (1) Disulfide bonds are added to endow the carrier with redox responsiveness inside tumor cells, thereby increasing the local concentration of the drug at the treatment site and reducing toxicity and side effects; (2) By precise ratio control, the P-gp inhibitor ER is covalently coupled with the mPEG-PDLLA molecule, which not only ensures the stability of the carrier but also endows it with P-gp inhibitory ability.

[0036] Compared with ordinary mPEG-PDLLA nano drug carriers, the product of the present invention has obvious redox responsiveness in tumor cells, is relatively stable in a 10% serum and phosphate buffer (PBS, pH 7.4) simulating the human blood circulation environment, and will accelerate the release of drugs in a phosphate buffer (PBS, pH 7.4) and exogenous glutathione (GSH, 5 mM) simulating the inside of human tumor cells. In addition, through precise ratio control, the structure of this product contains the P-gp inhibitor ER component, has obvious P-gp inhibitory function, and the formed drug-loaded nanoparticles have uniform particle size and good dispersion coefficient. This novel drug carrier can be used to encapsulate a variety of poorly soluble anti-tumor drugs. The paclitaxel nano micelles prepared using the SH181 can have a drug loading capacity of up to 5.1%; it has obvious advantages in the treatment of drug-resistant tumors. Description of the Drawings

[0037] In order to more clearly and explicitly illustrate the technical solutions in the present invention, the following provides the drawings in each embodiment. All the drawings are limited to the embodiments in the present invention.

[0038] Figure 1 1H NMR spectrum of the nano drug carrier (SH181) with P-glycoprotein inhibitory function in an embodiment of the present invention;

[0039] Figure 2 Ultraviolet absorption spectrum of the nano drug carrier (SH181) with P-glycoprotein inhibitory function in an embodiment of the present invention;

[0040] Figure 3 Hemolysis rate effect diagram of the nano drug carrier (SH181) with P-glycoprotein inhibitory function in an embodiment of the present invention;

[0041] Figure 4 Cell safety experiment effect diagram of the nano drug carrier (SH181) with P-glycoprotein inhibitory function in an embodiment of the present invention;

[0042] Figure 5 Redox responsiveness experiment effect diagram of the nano drug carrier (SH181) with P-glycoprotein inhibitory function in an embodiment of the present invention;

[0043] Figure 6 P-gp inhibition diagram of the nano drug carrier (SH181) with P-glycoprotein inhibitory function in an embodiment of the present invention;

[0044] Figure 7 Flow cytometry diagram of the uptake of the nano drug carrier (SH181) with P-glycoprotein inhibitory function by human drug-resistant triple-negative breast cancer cells in an embodiment of the present invention;

[0045] Figure 8 Schematic diagram of the structure of paclitaxel nanomicelles (SH181@PTX NPs) in an embodiment of the present invention;

[0046] Figure 9 Particle size distribution diagram of paclitaxel nanomicelles (SH181@PTX NPs) in an embodiment of the present invention;

[0047] Figure 10 Zeta potential diagram of paclitaxel nanomicelles (SH181@PTX NPs) in an embodiment of the present invention;

[0048] Figure 11 Transmission electron microscope image of paclitaxel nanomicelles (SH181@PTX NPs) in an embodiment of the present invention;

[0049] Figure 12 Statistical chart of the cytotoxicity of paclitaxel nanomicelles (SH181@PTX NPs) against human drug-resistant triple-negative breast cancer cells in an embodiment of the present invention;

[0050] Figure 13 Flow cytometry diagram of the effect of paclitaxel nanomicelles (SH181@PTX NPs) on apoptosis of human drug-resistant triple-negative breast cancer cells in an embodiment of the present invention;

[0051] Figure 14 Quantitative statistical chart of the effect of paclitaxel nanomicelles (SH181@PTX NPs) on apoptosis of human drug-resistant triple-negative breast cancer cells in an embodiment of the present invention. Detailed implementation manners

[0052] The present invention will be further described in detail below with reference to the embodiments and the drawings, but the implementation manners of the present invention are not limited thereto.

[0053] The technical solution provided by this application utilizes the structural modification of the mPEG-PDLLA copolymer to introduce disulfide bonds (S-S), and connects a P-gp inhibitor at its hydrophobic end to obtain a novel P-gp inhibitory functionalized drug carrier. By self-assembling into nanoparticles in water, based on hydrophobic interactions, efficient loading of lipophilic small molecule anti-tumor drugs is achieved. The preparation method is simple and convenient, with good repeatability. The nano-micelles formed by the nano-drug carrier with P-glycoprotein inhibitory function in this application have uniform and stable particle sizes, with an average particle size range of 20-30 nm, a drug loading capacity of 5.1%, and an encapsulation efficiency of 90%, and have low toxicity and side effects. In addition, the nano-micelles formed by the nano-drug carrier with P-glycoprotein inhibitory function in this application can passively accumulate in tumor tissues based on the enhanced permeability and retention effect (EPR), achieving deep penetration into drug-resistant tumor tissues. In addition, this product has redox responsiveness and P-gp inhibitory function in tumor cells, can inhibit the efflux of anti-tumor drugs by drug-resistant tumor cells, effectively improve the anti-tumor effect, and has broad application prospects in drug carriers for treating drug-resistant tumors.

[0054] To make this application easier to understand, it will be shown in detail in the form of examples below. Those skilled in the art should understand that the examples are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0055] The instruments and reagents used in the examples are as follows:

[0056] 1. Instruments

[0057] Nuclear magnetic resonance spectrometer, magnetic stirrer, centrifuge, Malvern laser particle size analyzer, microplate reader, ultraviolet spectrometer, flow cytometer, freeze dryer, high performance liquid chromatograph, pure water instrument, transmission electron microscope.

[0058] 2. Reagents

[0059] mPEG 2k -PDLLA 2k , paclitaxel (PTX), PBS, fetal bovine serum Gibco10099141, CCK-8 cell proliferation detection kit, L929 cells, MDA-MB-231 / T cells, Annexin V-FITC / PI cell apoptosis detection kit.

[0060] 3.5 kD dialysis bag, centrifuge tube, cell culture plate.

[0061] Example 1 :

[0062] In this example, a nano-drug carrier (SH181) with P-glycoprotein inhibitory function was prepared. The specific method includes the following steps:

[0063]

[0064] ER (300 mg, 0.435 mmol) was suspended in tetrahydrofuran (20 mL), and sodium hydride (52.2 mg, 1.305 mmol) was added thereto. After stirring for a while, the disulfide bond Linker (SS-Linker) (444.54 mg, 0.87 mmol) was weighed and added thereto. Stir at room temperature for 3 h, evaporate the solvent to obtain a crude product; dissolve it with anhydrous dichloromethane, and successively add mPEG 2k -PDLLA 2k (1.74 g, 0.435 mmol), 4-dimethylaminopyridine (5.3 mg, 0.0435 mmol) and dicyclohexylcarbodiimide (89 mg, 0.435 mmol), and carry out a coupling reaction by stirring at room temperature for 12 h. Filter, evaporate the solvent to dryness, add 16 mL of deionized water, ultrasonically emulsify (power 8 W) for 15 s, centrifuge at 12000 rpm at 4 °C for 15 min, transfer the supernatant to a 3.5 kD ultrafiltration tube for centrifugation, redisperse it in deionized water, filter through a membrane (0.22 μm), and freeze-dry to obtain SH181.

[0065] As Figure 1 and Figure 2 shown, the results were verified by NMR and ultraviolet absorption analysis.

[0066] Example 2 :

[0067] In this example, the blood compatibility of the nano-drug carrier (SH181) with P-glycoprotein inhibitory function prepared in Example 1 was tested. The specific method includes the following steps:

[0068] (1) Adjust rat red blood cells into a 2% cell suspension with physiological saline;

[0069] (2) Prepare an SH181 solution with physiological saline as the experimental group, so that the final concentrations are 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 mg / mL respectively;

[0070] (3) The positive control group is deionized water, and the negative control group is physiological saline;

[0071] (4) Mix the above solutions with an equal volume of red blood cell suspension, gently shake, incubate at 37 °C for 2 h, and centrifuge at 3000 rpm for 10 min;

[0072] (5) Collect the supernatant, measure its absorbance at 540 nm using a microplate reader, and calculate the hemolysis rate.

[0073] As Figure 3 shown, plot the drug concentration on the abscissa and the hemolysis rate on the ordinate to obtain the blood compatibility characterization data of the carrier; the results show that the red blood cells in the deionized water group have completely swelled and broken, there is no obvious change in the saline group, and the hemolysis rates of the experimental groups at different concentrations are far less than 5%, proving that the blood compatibility of this product is good.

[0074] Example 3 :

[0075] This example tested the cell safety of the nano-drug carrier (SH181) with P-glycoprotein inhibitory function prepared in Example 1. The specific method includes the following steps:

[0076] (1) Prepare fresh culture medium solutions with the nano-drug carrier (SH181) prepared in Example 1 at concentrations of 1, 5, 25, 50, 100, and 200 μg / mL respectively.

[0077] (2) Using L929 and MDA-MB-231 / T as model cells, in a 96-well plate inoculated with 5×10 3 cells, add 100 μL of the culture medium prepared in step (1) respectively, and add an equal amount of empty culture medium to the control group, and incubate for 24 h.

[0078] (3) After incubation, remove the culture medium, wash with PBS, and then detect with CCK-8.

[0079] (4) Use a microplate reader to detect the absorbance of each well at 450 nm, and calculate the cell survival rate of each well according to the standard curve of fluorescence intensity-concentration.

[0080] As Figure 4 shown, plot the drug concentration on the abscissa and the cell survival rate on the ordinate to obtain the cell safety data of the drug carrier (SH181); the results show that whether it is normal cells L929 or drug-resistant triple-negative breast cancer cells MDA-MB-231 / T, under the action of the drug carrier (SH181) at different concentrations, their survival rates remain above 80%, proving that our product is basically non-toxic to cells and has good cell safety, and can be used as a drug carrier for subsequent development.

[0081] Example 4 :

[0082] This example tested the redox responsiveness of the nano-drug carrier (SH181) with P-glycoprotein inhibitory function prepared in Example 1. The specific method includes the following steps:

[0083] (1) Dissolve the carrier SH181 in PBS (pH 7.4), add exogenous glutathione (GSH, 5 mM), and incubate at 37 °C.

[0084] (2) At fixed time points (0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 h), collect the samples and monitor the absorption peaks and molecular weights using liquid chromatography - mass spectrometry (LC - MS).

[0085] (3) By plotting the change in the absorption peak area corresponding to ER against the time elapsed after adding GSH, the release rate of free ER from the carrier SH181 can be obtained.

[0086] As Figure 5 shown, plot the time on the abscissa and the release rate of ER on the ordinate to obtain the glutathione responsiveness characterization data of the carrier. The results show that in PBS (pH 7.4) with exogenous glutathione (GSH, 5 mM), SH181 is completely converted to free ER, and its conversion half - life is 1.947 ± 0.088 h. In PBS (pH 7.4) without glutathione, the chromatogram of SH181 does not change, demonstrating its physiological stability and GSH responsiveness.

[0087] Example 5 :

[0088] This example tested the P - gp inhibitory effect of the nano - drug carrier (SH181) with P - glycoprotein inhibitory function prepared in Example 1. The specific method includes the following steps:

[0089] (1) Prepare fresh medium solutions of the P - gp inhibitor ER and the carrier SH181 at different concentrations (0.1, 0.5 μM) respectively. The positive control group is verapamil (10 μM), and the negative control Control group is blank medium.

[0090] (2) Seed MDA - MB - 231 / T cells in a 6 - well plate and incubate overnight.

[0091] (3) Remove the old medium, add the solutions from step (1) into it, and continue to incubate for 8 h.

[0092] (4) After incubation, remove the old medium, add 2 mL of Rho123 (5 μg / mL) to each well and continue to incubate for 1 h.

[0093] (5) After incubation, wash twice with cold PBS, digest with trypsin, resuspend in 500 μL of PBS after centrifugation, and perform flow cytometry analysis. The obtained fluorescence data is processed using FlowJo 10 software.

[0094] As Figure 6 shown, with the group as the abscissa and the percentage of intracellular Rho123 fluorescence intensity as the ordinate, a graph was plotted to obtain the characterization data graph of the P-gp inhibitory effect of the nano-drug carrier (SH181). In the figure, Control is the negative control group; * P < 0.05, ** P < 0.01 represents a significant statistical difference. The results show that compared with the Control group and the verapamil group, the Rho123 fluorescence intensity of the SH181 group of the carrier is more obvious and is consistent with the ER group, confirming that ER conjugated through a disulfide bond to the mPEG-PDLLA copolymer can still exert its P-gp inhibitory activity and can be applied to the preparation of nano-drugs for the treatment of drug-resistant tumors.

[0095] Example 6 :

[0096] In this example, the cellular uptake ability of the nano-drug carrier with P-glycoprotein inhibitory function prepared in Example 1 was detected. The specific method includes the following steps:

[0097] (1) Using Cou-6 as a marker, nano-micelles SH181@Cou-6 NPs encapsulating Cou-6 were prepared, and this was used as the experimental group. The positive control group was free Cou-6, and the negative control was PBS;

[0098] (2) MDA-MB-231 / T cells were seeded in 6-well plates. After incubation for 12 h, fresh media containing SH181@Cou-6 NPs and Cou-6 were added to the experimental group and the positive control group respectively, with a final concentration of 2 μg / mL. The negative control group was added with fresh media and continued to be incubated;

[0099] (3) After the incubation ended, the cells were rinsed 3 times with PBS, digested with trypsin, collected by centrifugation, and resuspended in 500 μL of PBS for flow cytometry analysis. The obtained fluorescence data was processed using FlowJo 10 software.

[0100] As Figure 7 shown, with the group as the abscissa and the intracellular fluorescence intensity as the ordinate, a graph was plotted to obtain the characterization of the cellular uptake ability of the carrier; in the figure, Control is the negative control group; * P < 0.05, ** P < 0.01 represents a significant statistical difference. The results show that compared with the intracellular fluorescence intensity of the Control group and the Cou-6 group, the SH181@Cou-6 NPs group is significantly enhanced, indicating that the SH181 carrier enhances the cellular uptake of Cou-6, meaning that this product has the ability to enhance drug uptake.

[0101] Example 7 :

[0102] Taking PTX as an example, the application of the nano-drug carrier with P-glycoprotein inhibitory function prepared in Example 1 in the preparation of PTX nano-micelles, the specific method includes the following steps:

[0103] Weigh SH181 (100 mg) and dissolve it in 600 μL of dimethyl sulfoxide. Weigh 10 mg of PTX and dissolve it in 200 μL of dimethyl sulfoxide. Mix the two solutions evenly. Under 1000 rpm, add the above mixed solution drop by drop to 12 mL of deionized water, stir for 20 min, transfer it to a 3.5 KD dialysis bag, dialyze for 8 h, filter (0.22 μm), take an appropriate amount for particle size and transmission electron microscopy analysis, and divide the remaining into aliquots, freeze-dry, and reserve for use.

[0104] As Figures 8 - 11 shown, they are respectively the structural simulation diagram, particle size distribution diagram, Zeta potential and transmission electron microscopy diagram of the PTX nano-micelles (SH181@PTX NPs). The data shows that the nano-particles self-assembled by encapsulating PTX with this product have a uniform morphology, the average particle size is 26.1 nm and shows a normal distribution, the polydispersity index PDI is 0.147, the distribution is relatively narrow, and the surface potential distribution is -2.1. Using MDA-MB-231 / T paclitaxel-resistant triple-negative breast cancer cells as a cell model, the cytotoxicity test of SH181@PTX NPs was carried out, and the experimental results are as Figure 12 shown. In the figure, * P < 0.05, ** P < 0.01 represents a significant statistical difference. The results show that compared with free paclitaxel, paclitaxel encapsulated by the SH181 carrier shows a lower cell survival rate, indicating that after encapsulation by SH181, the killing effect of paclitaxel on drug-resistant triple-negative breast cancer cells can be significantly enhanced.

[0105] Example 8 :

[0106] This example tested the effect of the paclitaxel nano-micelles (SH181@PTX NPs) prepared in Example 7 on the apoptosis of MDA-MB-231 / T cells. The specific method includes the following steps:

[0107] (1) Prepare fresh culture medium solutions of the paclitaxel nano-micelles (SH181@PTX NPs) and free paclitaxel prepared in Example 8 with final concentrations of 50, 100, and 200 nM;

[0108] (2) Seed MDA-MB-231 / T cells in a 6-well plate, add 1 mL of the culture medium prepared in step (1), and add an equal volume of empty culture medium to the control group, and incubate for 24 h;

[0109] (3) After incubation, the reaction of the apoptosis kit was carried out for 10 - 15 min, and flow cytometry analysis was performed.

[0110] As Figure 13 and 14 shown, plotting with Annexin V and propidium iodide (PI) on the vertical axis, a flow cytometry graph of the effect of SH181@PTX NPs on the apoptosis of MDA-MB-231 / T cells was obtained; Control in the graph is the control group; Q1 represents necrotic cells (Dead), Q2 represents late apoptotic cells (Late), Q3 represents early apoptotic cells (Early), and Q4 represents live cells; in the graph, * P < 0.05, ** P < 0.01, *** P < 0.001 represents significant statistical differences. The results showed that the apoptosis rate of the SH181@PTX NPs group was significantly higher than that of the paclitaxel group, especially the number of late apoptotic cells was four times that of paclitaxel, indicating that the paclitaxel nanomicelles (SH181@PTX NPs) could effectively promote the apoptosis of drug-resistant triple-negative breast cancer cells MDA-MB-231 / T, had the potential to reverse breast cancer drug resistance, and could be further developed as a candidate drug for anti-breast cancer drug resistance.

[0111] The applicant declares that the present invention illustrates the preparation method and application of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned method steps, that is, it does not mean that the present invention must rely on the above-mentioned preparation method steps to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of the raw materials selected for the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A nano-drug carrier with P-glycoprotein inhibitory function, characterized in that: The nano-drug carrier is composed of methoxypolyethylene glycol-polylactic acid copolymer mPEG-PDLLA covalently coupled with P-gp inhibitor Encequidar through a disulfide bond linker, and the structural formula is as follows:

2. A preparation method of a nano-drug carrier with P-glycoprotein inhibitory function as described in claim 1, characterized in that, The preparation method comprises the following steps: (1) Using tetrahydrofuran as a solvent, coupling the P-gp inhibitor Encequidar and the disulfide bond linker; (2) Using anhydrous dichloromethane as a solvent, coupling the product obtained in step (1) with the mPEG-PDLLA copolymer under the action of a catalyst; (3) Dissolving the product obtained in step (2) in deionized water, vortexing and ultrasonically emulsifying to obtain an emulsion; (4) Subjecting the emulsion to low-temperature ultra-high-speed centrifugation, transferring the supernatant to an ultrafiltration centrifugal tube, centrifuging, washing, and freeze-drying to obtain the product.

3. The preparation method of the nano-drug carrier with P-glycoprotein inhibitory function according to claim 2, characterized in that: In step (1), the molar ratio of the P-gp inhibitor Encequidar to the disulfide bond linker is 1:

2.

4. The preparation method of the nano-drug carrier with P-glycoprotein inhibitory function according to claim 2, characterized in that: In step (2), the number-average molecular weight of the mPEG-PDLLA is 4 kD.

5. The preparation method of the nano-drug carrier with P-glycoprotein inhibitory function according to claim 2, characterized in that: In step (2), the catalyst is 4-dimethylaminopyridine and dicyclohexylcarbodiimide.

6. The preparation method of the nano-drug carrier with P-glycoprotein inhibitory function according to claim 2, characterized in that: The low-temperature ultra-high-speed centrifugation in step (4) refers to centrifuging at 4 °C and 12,000 rpm for 15 min, and the minimum molecular weight cut-off of the ultrafiltration centrifugal tube is 3.5 kD.

7. Application of the nano-drug carrier with P-glycoprotein inhibitory function according to claim 1 in the preparation of anti-drug-resistant tumor nano-drugs.

8. Use of the nano-drug carrier with P-glycoprotein inhibitory function as claimed in claim 7 in the preparation of anti-drug-resistant tumor nano-drugs, characterized in that: The nano-drug carrier with P-glycoprotein inhibitory function is used for encapsulating poorly soluble anti-tumor drugs.

9. Use of the nano-drug carrier with P-glycoprotein inhibitory function as claimed in claim 8 in the preparation of anti-drug-resistant tumor nano-drugs, characterized in that: The poorly soluble anti-tumor drugs include paclitaxel (Paclitaxel, PTX), docetaxel (Docetaxel, DTX), and cabazitaxel (Cabazitaxel, CTX).

10. Use of a nano-drug carrier with P-glycoprotein inhibitory function as described in claim 9 in the preparation of anti-drug-resistant tumor nano-drugs, characterized in that: The method for encapsulating the paclitaxel to prepare the corresponding nano-micelles comprises: (1) Dissolving an appropriate amount of the nano-drug carrier and the paclitaxel in an appropriate volume of dimethyl sulfoxide, vortexing to mix them evenly and dissolving them fully; the mass ratio of the nano-drug carrier to the paclitaxel is 10:1; (2) Gradually adding the mixed solution obtained in step (1) dropwise to an appropriate amount of deionized water, and continuously stirring at 1000 rpm at room temperature for 20 min; (3) Transferring the solution obtained in step (2) into a dialysis bag with a molecular weight cut-off of 3.5 kD and dialyzing at room temperature for 8 h; (4) Passing the liquid obtained in step (3) through a membrane, aliquoting, and freeze-drying to obtain paclitaxel nano-micelles, and the pore size of the membrane is 0.22 μm.

Images