CN108619526B

CN108619526B - Preparation method of nano delivery system of targeted reduction sensitive co-carried chemotherapeutic drug and P-gp drug resistance reversal agent

Info

Publication number: CN108619526B
Application number: CN201810488982.1A
Authority: CN
Inventors: 张琳华; 朱敦皖; 秦玉; 樊帆; 张志明
Original assignee: Institute of Biomedical Engineering of CAMS and PUMC
Current assignee: Institute of Biomedical Engineering of CAMS and PUMC
Priority date: 2018-05-21
Filing date: 2018-05-21
Publication date: 2021-02-09
Anticipated expiration: 2038-05-21
Also published as: CN108619526A

Abstract

The invention relates to a preparation method of a nano delivery system of a targeted reduction-sensitive co-carried chemotherapeutic drug and a P-gp drug resistance reversal agent. By using amphiphilic copolymer PCL with reduction sensitivity₇₅₀₀‑ss‑PEG₇₅₀₀‑ss‑PCL₇₅₀₀The polymer vesicle with reduction sensitivity is constructed, hydrophobic drugs paclitaxel and Tariquard are entrapped through intermolecular hydrophobic force, doxorubicin is loaded in a hydrophilic inner cavity of the polymer vesicle by adopting a pH gradient method, and a folic acid targeting group is modified on the surface of the vesicle through a covalent bond, so that the polymer vesicle with the synergistic effects of targeting, reduction response and chemotherapy is constructed. The small molecule P glycoprotein (P-gp) inhibitor, Tariquidar, reduces drug resistance of drug-resistant cells by blocking the efflux of P-gp to a substrate, and increases the uptake of drugs by the drug-resistant cells, thereby reversing multidrug resistance. The nano delivery carrier can simultaneously entrap hydrophobic chemotherapeutic drugs and hydrophilic chemotherapeutic drugs, has tumor targeting and reduction responsiveness to a tumor microenvironment, and can realize multi-drug resistance tumor killing of tumor reversion.

Description

Preparation method of nano delivery system of targeted reduction sensitive co-carried chemotherapeutic drug and P-gp drug resistance reversal agent

Technical Field

The invention relates to a preparation method of a nano delivery system of a targeted reduction-sensitive co-carried chemotherapeutic drug and a P-gp drug-resistant reversal agent, in particular to a preparation method of a nano delivery system of a polymersome of a targeted reduction-sensitive co-carried chemotherapeutic drug and a P-gp drug-resistant reversal agent. The nano delivery carrier can simultaneously entrap hydrophobic chemotherapeutic drugs and hydrophilic chemotherapeutic drugs, has tumor targeting and reduction responsiveness to a tumor microenvironment, can realize multi-drug resistance reversal of tumors, and kills the tumors through the synergistic effect of different chemotherapeutic drugs.

Background

Chemotherapy is the most important means for treating tumors clinically at present, and most of antitumor drugs are poor in water solubility, large in adverse reaction and poor in selectivity, so that the clinical application of the antitumor drugs is severely limited. In addition, a major challenge in cancer therapy is the development of drug resistance in the patient's tumor tissue. Tumor cell Multidrug resistance (MDR) is one of the major causes of chemotherapy failure, limiting cure rates, and reducing survival rates in tumor patients. Therefore, a proper drug carrier is developed, drugs with different action mechanisms are delivered into the same tumor cell at the same time, and the MDR reversal agent is combined to overcome the problem that the drugs are pumped out by the tumor cell, so that the drug effect can be improved, the toxic and side effects can be reduced, and the multi-drug resistance of the tumor can be overcome.

Overcoming tumor MDR from a delivery system perspective is also one of the research hotspots of many formulation researchers. Compared with the traditional chemotherapy drugs, the nano drug-loaded system can effectively reduce the drug toxicity, improve the drug stability and improve the drug distribution. Polymersomes, as one of the nanoformulations, exhibit a number of advantages: hydrophobic and hydrophilic drugs can be encapsulated at the same time; the EPR effect of the tumor can be utilized to effectively transport the medicine to a pathological area; the ideal polymersome can consume ATP of cells through endocytosis, so that the efflux function of P-gp is weakened; meanwhile, by designing the polymer vesicles with tumor microenvironment response, the medicine can be quickly released at the target part of the tumor, so that the curative effect is improved. The researchers of the invention try to prepare the polymer vesicle with the reduction responsiveness by using the amphiphilic polymer, and simultaneously entrap the hydrophobic and hydrophilic chemotherapeutic drugs, and then combine the P-gp inhibitor and the folic acid targeting to promote the drug accumulation and increase the bioavailability of the drug. In addition, because the multi-drug resistant cells have multiple drug resistant mechanisms, the effective drug concentration in the cells can not be formed, and the drug-loaded nanoparticles can improve the drug concentration of tumor tissues due to the unique size effect and surface effect of the drug-loaded nanoparticles, thereby achieving the reversal effect of the multi-drug resistance. With the progress of nanotechnology research, it is reasonable to believe that it has a profound effect on the treatment of tumors.

The P glycoprotein (P-gp/ABCB 1), as a member of the earliest discovery of the ATP-binding cassette transporter (ABC) superfamily, plays an important role in mediating drug transport and directly influences the bioavailability of drugs. A great deal of facts show that the existence of P-gp makes drug-resistant tumor cells insensitive to anti-tumor drugs, and a few scholars research drugs aiming at P-gp targets to overcome tumor MDR. Mainly comprises 1) regulating the efflux function of P-gp by using P-gp regulator or inhibitor; 2) the recognition effect of P-gp is avoided by wrapping the P-gp substrate by the nano-carrier; 3) the nanometer preparation and P-gp regulator or inhibitor are combined for administration to exert better effect. The study adopts Tariquar as a P-gp inhibitor combined nano preparation to overcome tumor drug resistance. The Tariquard as a third-generation P-gp inhibitor has improved performances in various aspects compared with the first and second generation P-gp inhibitors. Moreover, the application of the P-gp inhibitor can obviously improve the bioavailability of chemotherapeutic drugs, effectively enhance the lethality to tumors, and can be used as a potential treatment method.

Disclosure of Invention

The invention aims to provide a preparation method of a nano delivery system of a targeted reduction-sensitive co-carried chemotherapeutic drug and a P-gp drug resistance reversal agent, in particular to a preparation method of a nano delivery system of a polymersome of a targeted reduction-sensitive co-carried chemotherapeutic drug and a P-gp drug resistance reversal agent. The nano delivery carrier can simultaneously entrap hydrophobic chemotherapeutic drugs and hydrophilic chemotherapeutic drugs, has tumor targeting and reduction responsiveness to a tumor microenvironment, and can realize reversing of multi-drug resistance of tumors and killing of the tumors through the synergistic effect of the chemotherapeutic drugs.

The preparation method of the nano delivery system of the targeted reduction-sensitive co-carried chemotherapeutic drug and the P-gp drug resistance reversal agent provided by the invention comprises the following steps:

1) fully dissolving an amphiphilic copolymer PCL-ss-PEG-ss-PCL (25 mg), 0.2-5% (wt) DSPE-PEG (2000) Folate (distearoyl phosphatidyl ethanolamine-polyethylene glycol-folic acid), 4-5% (wt) TQR and 2.5mg PTX in dichloromethane or a mixed organic solvent of methanol and dichloromethane (volume ratio of 1: 1), removing the organic solvent by using a rotary evaporator to form a uniform film on the inner wall of a reactor, and drying in vacuum for 12-20 hours;

2) preparing 150 mM ammonium sulfate aqueous solution, dispersing the sample membrane in 5ml ammonium sulfate solution, and hydrolyzing at 60-65 ℃ for 5 h. Mixing, standing to room temperature, and performing ultrasonic treatment for 30min (5 mm probe, Ampl 30%) in ice bath to obtain stable and uniform drug-loaded reduction sensitive polymer vesicle dispersion;

3) the polymersome suspension was placed in a treated dialysis bag (MWCO: 8000-;

4) loading DOX: adding 5.0mg DOX into the dialyzed sample, and incubating for 1-2 h at 60-65 deg.C under magnetic stirring (300 r/min).

5) Removing the unencapsulated drug: dialyzing with PBS (pH 7.4) for 20h, and storing at 4 deg.C to obtain reduction response polymer vesicle dispersion liquid of co-carried chemotherapeutic drug and P-gp inhibitor of targeted tumor;

the molecular weight of the PCL-ss-PEG-ss-PCL copolymer is 12000-24000, wherein the mass percentage of the PEG hydrophilic chain segment is 20-40%.

The ratio of the volume (mL) of the ammonium sulfate aqueous solution for hydration to the mass (mg) of the carrier material is 1:4-1:20, and the ammonium sulfate aqueous solution is between 150 mM and 300 mM.

The pH7.4PBS used for dialysis may be physiological saline.

The hydrophilic chemotherapeutic is adriamycin, or daunorubicin, doxorubicin, epirubicin, idarubicin, or mitoxantrone.

The hydrophobic chemotherapeutic drug is paclitaxel, or docetaxel, vindesine, and vinorelbine.

The P-gp inhibitor is Tariquidar, and also is a third-generation P-gp inhibitor of Elacridar, Zosuquidar and Laniquidar.

The nano delivery system of the target reduction-sensitive co-carried chemotherapeutic drug and the P-gp drug resistance reversal agent prepared by the method is applied to preparing the drugs for treating tumors.

The tumor treatment drug is a polymer vesicle nanoparticle which is targeted, reduced and sensitive and carries a chemotherapeutic drug and a P-gp reversal agent together.

The drug delivery system is constructed by PCL-ss-PEG-ss-PCL polymer with reduction sensitivity. Wherein, the hydrophobic drugs Paclitaxel (PTX) and Tariquar (TQR) are loaded on the hydrophobic membrane layer of the polymersome, the folic acid targeting group is modified on the surface of the polymersome through covalent bonds, and the pH gradient method is applied to load the adriamycin (DOX) in the hydrophilic inner cavity of the polymersome. The prepared vesicle shell is hydrophilic PEG, has the characteristics of long circulation and stable space, can quickly release medicine after targeting a tumor through reduction response, and effectively overcomes the multidrug resistance of tumor cells through the loading of Tariquodar.

The reduction response type nano delivery system of the tumor-targeted co-loaded chemotherapeutic drug and the P-gp inhibitor can be particularly applied to high-expression P-gp tumor drug-resistant cells such as breast cancer resistant MCF-7/ADR drug-resistant cells, human liver cancer cell HepG2/ADR and human ovarian cancer paclitaxel drug-resistant strain A2780/Taxol.

In summary, the present invention has substantial features:

1) the carrier material used in the invention has good biocompatibility and biodegradability. Can self-assemble to form polymer vesicles with small particle size and good and stable dispersity.

2) The polymer vesicle designed by the invention can entrap hydrophilic and hydrophobic drugs at the same time, and overcomes the defects of low bioavailability caused by the in vivo instability and easy in vivo clearance of free adriamycin and the insolubility of the hydrophobic drugs.

3) The polymer vesicle is made of reduction sensitive materials with disulfide bonds, and can quickly release drugs by responding to a tumor microenvironment after reaching a local tumor, so that the treatment effect is improved, and the toxic and side effects are reduced.

4) The hydrophilic layer PEG on the surface of the polymer vesicle has flexibility, so that the polymer micelle has long circulation characteristic and space stability, and the circulation time of the polymer micelle in blood is prolonged.

5) The polymer vesicle with proper size can be effectively enriched to a tumor part by utilizing an EPR effect; enter cells through endocytosis, and effectively avoid the substrate recognition effect of P-gp.

6) The active targeting of the nanoparticles to tumor cells with high expression of folate receptors can be improved by adopting folic acid modification.

7) The invention adopts the combined administration of two chemotherapeutics, enhances the chemotherapeutical effect and reduces the drug resistance risk brought by single administration.

8) The third generation P-gp inhibitor is adopted, so that the cytotoxicity and instability generated by P-gp are effectively reduced, the uptake of chemotherapeutic drugs is enhanced, and the MDR of tumors is effectively overcome.

Drawings

FIG. 1 is an atomic force microscope (a) and a particle size distribution diagram (b) of a reduction-sensitive double drug-loaded polymer vesicle (Co-PS);

FIG. 2 is the in vitro stability of Blank polymersome (Blank PS) and Co-PS;

FIG. 3 is a graph of the in vitro release of Tariquard-, paclitaxel-and doxorubicin-loaded reduction-sensitive polymersomes (TQR-Co-PS) under different conditions;

FIG. 4 shows Western blot detection of P-gp expression in MCF-7 and MCF-7/ADR of breast cancer cells;

FIG. 5 is a graph of the growth inhibition curves of various concentrations of Free doxorubicin and paclitaxel (Free DOX + PTX), Free Tariquar, paclitaxel and doxorubicin (Free DOX + PTX + TQR), Co-PS, TQR-Co-PS, folate-targeted Tariquar, paclitaxel and doxorubicin-loaded reduction-sensitive polymersomes (FA-TQR-Co-PS) and Blank PS for MCF-7/ADR and MCF-7 breast cancer cells for 48 h (n =6,

±s）；

FIG. 6 is a graph showing the dead/viable cell distribution of Free DOX + PTX (b), Free DOX + PTX + TQR (c), Co-PS (d), TQR-Co-PS (e), FA-TQR-Co-PS (f) after they have been allowed to act on MCF-7/ADR cells for 48 hours at the same drug concentration (DOX concentration of 0.5. mu.g/mL, PTX concentration of 1.25. mu.g/mL, TQR concentration of 0.375. mu.g/mL); wherein a is a double staining pattern of cells of a control group without a drug; green fluorescence is a fluorescence signal excited by living cells, and red fluorescence is a fluorescence signal excited by dead cells;

FIG. 7 IC50 values for Free DOX + PTX, Free DOX + PTX + TQR, Co-PS, TQR-Co-PS, and FA-TQR-Co-PS for MCF-7 and MCF-7/ADR cells;

FIG. 8 is a confocal laser microscope used to observe the uptake of Free DOX + PTX, Free DOX + PTX + TQR, Co-PS, TQR-Co-PS, and FA-TQR-Co-PS by MCF-7/ADR cells;

FIG. 9 shows the flow cytometry semi-quantitative uptake of doxorubicin by MCF-7/ADR cells for different dosing groups;

FIG. 10 shows the HPLC detection of paclitaxel uptake by MCF-7/ADR cells for different administration groups;

FIG. 11 is a graph of Free DOX + PTX, Free DOX + PTX + TQR, Co-PS, TQR-Co-PS, and FA-TQR-Co-PS induced levels of apoptosis in MCF-7 and MCF-7/ADR cells;

FIG. 12 is a graph of the effect of Free DOX + PTX, Free DOX + PTX + TQR, Co-PS, TQR-Co-PS (a) and FA-TQR-Co-PS (b) on MCF-7/ADR cell cycle;

table 1 shows the particle size, potential, dispersion and encapsulation efficiency of different polymersome formulations;

table 2 shows the Resistance Index (RI) and the Reversal Factor (RF) values of the resistance to drug-Resistant cells for the different formulations.

Detailed Description

The present invention will be further described with reference to the following examples. The experimental methods in the examples, in which specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer; general equipment, materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

A method for preparing Blank reduction-sensitive polymersome (Blank PS) comprises the following steps:

(1) 25mg of PCL₇₅₀₀-ss-PEG₇₅₀₀-ss-PCL₇₅₀₀（PCL₇₅₀₀-ss-PEG₇₅₀₀-ss-PCL₇₅₀₀The preparation method is shown in the application on the same date of the applicant, and the name is as follows: folic acid target reduction sensitive drug-loaded polymer nano micelle and preparation method and application thereof) is fully dissolved in dichloromethane (or mixed organic solvent of methanol and dichloromethane), a rotary evaporator is used for removing the organic solvent, so that a layer of uniform film is formed on the inner wall of an eggplant-shaped bottle, the residual dichloromethane is dried by nitrogen, and the mixture is put into a vacuum drying oven and dried for 12 hours in vacuum at room temperature;

(2) adding 5mL Phosphate Buffer Solution (PBS) with pH7.4 into an eggplant-shaped bottle, shaking to disperse the film in the solution, hydrating for 5h at 65 ℃, mixing uniformly, placing to room temperature, carrying out ultrasonic treatment (5 mm probe, Ampl 30%) for 30min under ice bath to obtain stable and uniform blank polymer micelle dispersion, and storing at 4 ℃. The particle size, potential and polydispersity of the polymer vesicle PS are shown in Table 1, the average particle size of the Blank polymer vesicle PS is 159.17 nm, and the dispersity coefficient is 0.068, which indicates that the nanoparticles prepared by the method have uniform particle size and are suitable for being used as drug carriers. The stability of Blank PS is observed in figure 2, and the particle size of Blank PS still keeps the original particle size when the Blank PS is stored for 21 days at 4 ℃, which shows that the nanoparticle has good stability and is beneficial to long-time storage.

Example 2

A preparation method of reduction-sensitive polymer vesicles (Co-PS) carrying adriamycin and paclitaxel comprises the following steps:

(1) fully dissolving an amphiphilic copolymer PCL-ss-PEG-ss-PCL (25 mg) and 2.5mg of paclitaxel in dichloromethane (or a mixed organic solvent of methanol and dichloromethane), removing the organic solvent by using a rotary evaporator to form a uniform film on the inner wall of the eggplant-shaped bottle, and drying for 12 hours in vacuum;

(2) a300 mM ammonium sulfate aqueous solution was prepared, and the sample film was dispersed in 5ml of ammonium sulfate solution and hydrolyzed at 65 ℃ for 5 hours. Mixing, standing to room temperature, and performing ultrasonic treatment for 30min (5 mm probe, Ampl 30%) in ice bath to obtain stable and uniform drug-loaded reduction sensitive polymer vesicle dispersion;

(3) the polymersome suspension was placed in a treated dialysis bag (MWCO 8000-14000 Da) and dialyzed against a sucrose solution (102.69 g sucrose +1.152g histidine in 1L water) for 20 h.

(4) Loading DOX: the dialyzed sample was added with 5.0mg DOX under magnetic stirring at a constant temperature of 65 ℃ (300 r/min) and incubated for 2 h.

(5) Removing the unencapsulated drug: dialyzed against PBS (pH7.4) for 20h and stored at 4 ℃. The atomic force microscope picture of the polymersome is shown in figure 1; samples were taken for particle size distribution and potential measurements and the results are shown in Table 1 and FIG. 1.

(6) Encapsulation efficiency of Co-PS

The prepared polymersome suspension is centrifuged 3 times at 23000 rpm for 30min each time, and washed with deionized water to remove the drug which is not loaded, and finally the precipitate is resuspended in 5mL of water and lyophilized. Weighing a certain mass of freeze-dried powder, dissolving the freeze-dried powder in DMSO, detecting the absorbance of the freeze-dried powder by an ultraviolet spectrophotometer (UV) at 480 nm, and quantifying the drug content of the adriamycin by combining an ultraviolet absorption standard curve of the adriamycin-DMSO solution at 480 nm. Additionally weighing a certain mass of lyophilized powder, dissolving in acetonitrile, and detecting the amount of paclitaxel by HPLC (mobile phase is acetonitrile: 50 mM ammonium acetate buffer solution with pH4.0, flow rate ratio is 60% by mass: 40% by mass, and total flow rate is 1.0 mL/min). The Encapsulation Efficiency (EE) is calculated according to the following formula:

encapsulation efficiency = (actual drug loading amount/actual drug loading amount) × 100%

The drug loading rate and the encapsulation efficiency of the polymersome are the evaluation of the drug loading performance of the polymersome and one of the important characteristics for evaluating whether the polymersome can be used as a drug carrier. As shown in table 1, the encapsulation efficiency of doxorubicin by Co-PS was 18.94%, indicating that doxorubicin could be loaded into the lumen of the polymersome by the pH gradient method. And for the hydrophobic drug paclitaxel, the encapsulation rate of the polymer vesicle reaches 97.70%, which shows that the hydrophobic drug paclitaxel can be effectively encapsulated in the hydrophobic membrane layer of the polymer vesicle by adopting a thin-film hydration ultrasonic method, and the good drug-loading performance of the polymer vesicle is shown.

Example 3

A method for preparing reduction-sensitive polymer vesicle (TQR-Co-PS) carrying Tariquard, paclitaxel and adriamycin is provided. The method comprises the following steps:

(1) in the preparation of TQR-Co-PS, 5% (wt) of TQR was added during the rotary evaporation coating in the step (1) of example 2. TQR-Co-PS was prepared by following the same procedures as (1), (2), (3), (4) and (5) of example 2.

(2) Encapsulation efficiency of TQR-Co-PS

The prepared polymersome suspension is centrifuged for 3 times at 23000 rpm for 30min each time, and washed by deionized water to remove the unencapsulated drug, and finally the precipitate is resuspended in 5mL of water and lyophilized. Weighing a certain mass of freeze-dried powder, dissolving the freeze-dried powder in DMSO, detecting the absorbance of the freeze-dried powder by an ultraviolet spectrophotometer (UV) at 480 nm, and quantifying the drug content of the adriamycin by combining an ultraviolet absorption standard curve of the adriamycin-DMSO solution at 480 nm. Additionally weighing a certain mass of freeze-dried powder, dissolving in acetonitrile, and detecting the amount of paclitaxel and Tariquidar by HPLC (mobile phase is acetonitrile: 50 mM ammonium acetate buffer solution with pH4.0, flow rate ratio is 60% by mass: 40% by mass, and total flow rate is 1.0 mL/min). The Encapsulation Efficiency (EE) is calculated according to the following formula:

The experimental result shows that the entrapment rate of the doxorubicin of the TQR-Co-PS is 18.16%, which is similar to the Co-PS, and shows that the loading of the TQR does not influence the drug loading performance of the whole polymer vesicle on the doxorubicin. And for the hydrophobic drug paclitaxel, the encapsulation efficiency of the polymer vesicle also reaches 99.46, and the encapsulation efficiency of the Tariquard also reaches 83.44%, so that the polymer vesicle has good drug-loading performance. The results are shown in Table 1.

(3) In vitro drug release study of TQR-Co-PS

The sample prepared in example 3 was divided into 6 portions, and 1mL of each sample (material 10 mg/mL) was placed in a dialysis (MWCO 8000- + 14000 Da) bag, the dialysis bag was placed in 25 mL of 0.01M PBS solution of 1M sodium salicylate (pH7.4) or 0.01M PBS solution of 1M sodium salicylate (10 mM Glutathione (GSH)) at pH7.4, placed on a shaker, shaken at 37 ℃ and 120 rpm, and after a certain time (4 h, 8 h, 1 d, 3 d), 5mL of the sample was sampled to be assayed, and then 5mL of fresh release solution was supplemented. After the absorbance of doxorubicin was measured by an ultraviolet spectrophotometer for the released solution, paclitaxel and Tariquidar in the released solution were extracted with dichloromethane (7 to 9 mL), the dichloromethane was evaporated, dissolved in a small amount (0.5 mL) of acetonitrile (HPLC purity), and the amounts of paclitaxel and Tariquidar were measured by HPLC using the same method as in example 2 (6) for the encapsulation efficiency.

The results show that the cumulative release amounts of the adriamycin in the reductive release solution (containing GSH) and the release solution without GSH of the drug-loaded polymer vesicles in the first three days are 74.97% and 64.97% respectively, and the total release amount of the adriamycin is obviously higher than that of the release solution without GSH. This is because the reducing environment can destroy disulfide bonds in the polymersome, so that the polymersome is broken, and water-soluble adriamycin in the inner cavity is rapidly released. As for the hydrophobic drug paclitaxel, the release speed is slow due to the low solubility in water, and the cumulative release curve of paclitaxel can be seen, even by day 20, in the release solution in normal environment, the release amount of paclitaxel is only released by 50.66%, while the release amount of paclitaxel in the reductive release solution is increased to 81.67%, which shows that the reduction sensitivity of the polymer vesicle not only accelerates the release of the hydrophilic drug, but also accelerates the release of the hydrophobic drug. The same rule is shown for the release of the hydrophobic P-gp inhibitor tariquidar, which indicates that the release of the three drugs can be accelerated under the reducing condition, the drug concentration is increased, and the combined treatment effect of the drugs is favorably realized. The release results are shown in FIG. 3.

Example 4

A preparation method of Tariquard-loaded, reduction-sensitive polymer vesicles (FA-TQR-Co-PS) carrying Tariquard, paclitaxel and adriamycin is provided. The method comprises the following steps:

(1) in the preparation of FA-TQR-Co-PS, 5% (wt) TQR and 3% (wt) DSPE-PEG (2000) template were added during the rotary evaporation and film coating in the step of (1) in example 2. FA-TQR-Co-PS was prepared according to the same procedures as (1), (2), (3), (4) and (5) of example 2.

(2) Encapsulation efficiency of FA-TQR-Co-PS

The method for detecting the entrapment rate of FA-TQR-Co-PS is the same as that in example 3 (2), and the entrapment rate of the doxorubicin of the FA-TQR-Co-PS is 18.52 percent, which indicates that the surface modification of folic acid does not influence the loading of the doxorubicin into the inner cavity of the polymersome by a pH gradient method. The encapsulation efficiency of the hydrophobic drug paclitaxel reaches 96.48, and the encapsulation efficiency of the Tariquard is 84.53%, thus showing the high-efficiency drug-loading performance of the polymer vesicle. The results are shown in Table 1.

TABLE 1

Example 5

Western blot detection of P-gp expression of breast cancer cells MCF-7 and MCF-7/ADR

MCF-7 and MCF-7/ADR cells were cultured in 6-well plates until they were confluent, and then the cells were collected, lysed with RIPA Lysis Buffer at 4 ℃ for 30min, and centrifuged at 12000 rpm for 15 min to collect the supernatant. The total protein content of the supernatant was assayed using the BCA protein assay kit, and a volume of protein extract (each 50. mu.g loaded) was used to separate each protein by SDS-PAGE using an 8% separation gel, and then the proteins on the gel were transferred to a polyvinyl idene ﬂ uoride membrane, blocked with TBST containing 5% skimmed milk powder at room temperature for 3 hours, and then washed three times with TBST for 10 min each. Primary anti-MDR 1/ABCB1 Rabbit mAb (1:1000, v/v) was incubated overnight at 4 ℃ and then washed three times with TBST for 10 min each. After incubating the secondary antibody Horseradish peroxidase (HRP) -conjugated coat anti-platelet secondary antibody (1:10000, v/v) for 90 min at 37 ℃, the mixture was washed three times with TBST, and the band was detected with a high-sensitivity luminescence kit. The internal reference is Na, K-ATPase Antibody.

FIG. 4 shows that MCF-7/ADR cells highly express P-gp, while MCF-7 cells do not detect P-gp expression under the same conditions, indicating that MCF-7/ADR cells can be used as drug-resistant model cell lines, and MCF-7 cells are good control sensitive cell lines.

Example 6

Cytotoxicity and reversal drug resistance performance of drug-loaded reduction sensitive polymer vesicle

(1) In order to examine the reversal effect of the drug-loaded polymer vesicle on multidrug resistance of drug-resistant cells, the study examined non-drug-loaded polymer vesicles with different concentrationsThe same DOX + PTX preparation has killing effect on MCF-7 sensitive strains and drug-resistant strains. Single cell suspensions (MCF-7 and MCF-7/ADR cells) were prepared in 10% fetal bovine serum-free RPMI1640 medium and inoculated into 96-well plates at a rate of about 3500 MCF-7 cells per well and 5000 MCF-7/ADR per well, at a volume of 100. mu.L per well. After 24 h of cell adherence, the culture solution is replaced, culture medium containing nanoparticles with different concentrations is added, and the nanoparticles comprise Free DOX + PTX, Free TQR + DOX + PTX, Co-PS, TQR-Co-PS, FA-TQR-Co-PS, Blank PS and TQR-PS (adriamycin concentration: 0.016, 0.16, 0.8, 4, 20, 100, 1000 ng/mL; paclitaxel concentration: 0.04, 0.4, 2, 10, 50, 250, 2500 ng/mL; and carrier material (PS) concentration in Blank PS and TQR-PS is 0.4, 4, 20, 100, 500, 2500, 25000 ng/mL). Additionally arranging a negative control group and a blank control group, arranging 6 multiple wells in each group, transferring the culture plate into CO₂In an incubator at 37 ℃ with 5% CO₂And culturing for 48 hours under the saturated humidity condition, then absorbing and discarding supernatant in the pore plate, adding 120 mu L of mixed MTS culture medium into each pore, and culturing at 37 ℃ and 5% CO₂After 1 h of ambient incubation. The absorbance was measured at 490 nm with a microplate reader. Cell viability was calculated as follows and cell growth inhibition curves were plotted.

Fig. 5 shows that the blank vesicles have no obvious cytotoxicity at a given concentration, indicating that the blank vesicles have good biocompatibility as drug carriers. The Free DOX + PTX group can see that the killing effect of the Free drug on sensitive strain cells is obviously stronger than that of drug-resistant cell strains; the result of the obvious reduction of the cell survival rate of the drug-resistant cell strain after the addition of TQR in the free DOX + PTX is similar to that of the sensitive strain shows that the TQR can overcome the drug resistance of the cells, thereby enhancing the effect of the chemotherapeutic drug on killing the tumor. The toxicity of Co-PS and TQR-Co-PS to cells shows the same trend as that of free drugs, and the cytotoxicity of folate targeting nanoparticles (FA-TQR-Co-PS) is superior to that of nanoparticles without folate targeting, which shows that the chemotherapeutic drugs combined with P-gp inhibitors TQR and folate targeting can realize good tumor killing effect.

(2) Depending on the viability of the cells at different concentrations,IC analysis of each formulation Using SPSS software₅₀。

The resistance index RI (RI) is used for evaluating the drug resistance level of MCF-7/ADR cells, and higher RI value indicates higher drug resistance degree of the cells. The effect of the nano-formulation on reversing drug resistance was evaluated by the drug resistance reversing factor RF (RF). The RI and RF calculation equations are as follows:

RI=IC_{50(MCF-7/ADR)}/IC_50(MCF-7)

RF=IC_{50 (control group)}/IC_{50 (Experimental group)}(free DOX + PTX group is control group; other administration group is experimental group)

IC50 is an important index for evaluating cytotoxicity of a medicament, and according to the killing effect of nano preparations with different concentrations on cells, SPSS software is used for analyzing IC50, and the result is shown in figure 7 and combined with the medicament resistance index and the medicament resistance reversal factor of table 2, the IC50 of the medicament-resistant cells is obviously higher than that of MCF-7 sensitive cells, and the medicament resistance index reaches 15.18 for a Free DOX + PTX administration group, which indicates that the MCF-7/ADR cells have strong medicament resistance. However, for the Co-PS group, the difference of the IC50 values of the two cells is smaller than that of the Free DOX + PTX group, and the drug resistance index is reduced to 4.24, which indicates that the polymersome can obviously reduce the drug resistance of the cells; the method is mainly characterized in that (1) the nanometer material enters cells in an endocytosis mode due to the advantages of the particle size of the nanometer material and the like, and the P-gp recognition is escaped. (2) The nanoparticle needs to consume a certain amount of ATP when entering cells, and the reduction of ATP can influence the expression of P-gp, thereby reducing the excretion effect of the P-gp. (3) The environment-responsive polymer vesicle can quickly release the medicine in tumor cells, so that the local high concentration is realized, and the killing effect of the tumor is improved. From IC50 and a drug resistance index, it can be seen that the drug resistance of the tumor is not completely reversed by the polymersome and the chemotherapeutic drug, so that the invention introduces Tariquidar, and the result shows that the IC50 value of MCF-7/ADR cells is obviously reduced, the RI value is obviously reduced, and the RF value is obviously increased no matter Free TQR + DOX + PTX or TQR-Co-PS, which indicates that the Tariquidar and the chemotherapeutic drug can obviously increase the sensitivity of the MCF-7/ADR cells to the chemotherapeutic drug, and effectively overcome the drug resistance of the tumor. While the IC50 of FA-TQR-Co-PS for two cells is lower than that of other two groups of nanoparticle administration groups, which shows that the folic acid targeted nanoparticles enhance the tumor killing effect and have good targeted combined treatment effect.

TABLE 2

(3) To more clearly observe the cytotoxicity of the different preparations on MCF-7/ADR, the study employed calcein-AM and PI kit to stain the cells to observe the proportion of cell death or necrosis after administration. Single MCF-7/ADR cell suspensions were prepared with 10% fetal bovine serum in folate-free RPMI1640 medium at about 2X 10 per well⁴Cells were seeded in 12-well plates, 2 mL per well volume. After the cells adhered to the wall, drugs (Free DOX + PTX, Free TQR + DOX + PTX, Co-PS, TQR-Co-PS and FA-TQR-Co-PS, respectively; DOX concentration 0.5. mu.g/mL, PTX concentration 1.25. mu.g/mL, TQR concentration 0.375. mu.g/mL) diluted in culture medium were added, and after 48 hours, the cells were harvested, washed with PBS, stained with calcein-AM and PI according to the method described in the specification, and then observed under laser confocal conditions.

As can be seen in FIG. 6, only a few cells died in the cell visual field of the Free DOX + PTX and Co-PS treated groups without TQR, and compared with the Co-PS treated group, the cell death rate was higher than that of the Free DOX + PTX, indicating that the nanoparticles can alleviate the drug efflux effect of the cells. A large number of dead cells appear in both the nanoparticle group containing the TQR and the Free TQR + DOX + PTX group, which shows that the TQR can effectively play the role of reversing drug resistance, reduce the efflux of the drug by tumor cells to improve the concentration of the drug in the tumor cells, and improve the sensitivity of the tumor cells to chemotherapeutic drugs, thereby improving the killing capacity of the drug in the tumor cells.

Example 7

Study on phagocytosis of drug-loaded reduction-sensitive polymer vesicles and adriamycin cell localization

(1) Doxorubicin cell localization study: a single MCF-7/ADR cell suspension is prepared by 10% fetal bovine serum-free RPMI1640 culture solution, 6500 cells per hole are inoculated on a laser confocal dish, and then the cell suspension is cultured overnight. The culture medium in the confocal laser culture dish was aspirated and 2 mL of polymersome (Free DOX + PTX, Free TQR + DOX + PTX, Co-PS, TQR-Co-PS, and FA-TQR-Co-PS, respectively) diluted with the culture medium was added to each dish, the doxorubicin concentration in the medium was 0.5. mu.g/mL, the paclitaxel concentration was 1.25. mu.g/mL, and the Tariquidar concentration was 0.375. mu.g/mL). After incubation for 4 h, the nanoparticles were aspirated away, and the cells were washed 2 times with 1mL PBS; fixing the cells with 1mL (P0098) of immunostaining fixative of Biyuntian for 20 minutes at room temperature; washing with 1mL of immunostaining solution (P0106) for about 5 min for 3 times; adding 1mL DAPI, culturing at room temperature for 20 min, and washing with PBS 3 times (3-5 min each time, shaking). Finally, 600. mu.L of PBS was added and observed by confocal laser microscopy.

(2) Doxorubicin flow-through semi-quantitation: single MCF-7/ADR cell suspensions were prepared in 10% fetal bovine serum in folate-free RPMI1640 medium at about 2X 10 per well⁵Cells were seeded in 12-well plates, 2 mL per well volume. Adding a medicament (the administration method is the same as laser confocal) diluted by a culture medium for 6 hours after the cells adhere to the wall, and then collecting the cells; a negative control was also set. Washing with PBS 2 times, digesting with pancreatin without EDTA to collect cells; cells were gently resuspended in 500. mu.L PBS, sieved and subjected to flow-through assay.

(3) Paclitaxel cellular uptake: inoculating cells at the early stage and performing drug-adding treatment for cocurrent detection, adding drugs, incubating for 4 h, washing for 2 times by using ice PBS, digesting and collecting by using pancreatin without EDTA, centrifuging at 4-degree 1000 rpm for 5 minutes, and collecting cells; 100 μ L RAPI was lysed with shaking at 4 ℃ for 20 min and then centrifuged at 12000 rpm for 15 min to take the supernatant. Adding equal volume of acetonitrile into the supernatant, performing ultrasonic extraction for 20 min to obtain hydrophobic paclitaxel, filtering the supernatant, detecting by HPLC, centrifuging the precipitate, resuspending by PBS with proper volume, and detecting the total protein content by BCA kit.

The inhibition effect of P-gp on the cell membrane can be reflected visually by observing the drug intake of MCF-7/ADR drug-resistant cells. FIG. 8 is a confocal laser microscopy showing clearly that only a very small amount of doxorubicin entering the nucleus was seen for the Free DOX + PTX group; as can be seen in conjunction with the flow-through semiquantitative FIG. 9, the amount of doxorubicin taken into the cells was very small in the Free DOX + PTX group, almost close to that of the normal cultured control group, indicating that P-gp in the drug-resistant cells was able to recognize doxorubicin and pump it out of the cells. Not only was doxorubicin pumped out of the cells, but also in FIG. 10, it can be seen that the amount of paclitaxel was very small in the cells of the Free DOX + PTX administration group, indicating that P-gp was also able to recognize paclitaxel, reducing the amount of drug phagocytosis by the cells. For Free TQR + DOX + PTX groups, the laser confocal images show that the adriamycin is enriched in cell nucleus, and the flow type semi-quantitative histogram also shows that the amount of the adriamycin entering the cells is obviously increased by the TQR; compared with the Free DOX + PTX group without TQR, the taxol uptake is increased by more than 2 times, so that the TQR can effectively inhibit the effect of P-gp and improve the drug uptake of drug-resistant cells. The ingestion amount of the adriamycin in the Co-PS administration group is higher than that of Free DOX + PTX, which shows that the polymersome can mask the efflux function of P-gp to a certain extent; after the polymersome is combined with the TQR, the ingestion of the adriamycin and the taxol is obviously increased, and the rule is consistent with the trend of a free drug administration group. In order to enhance the tumor killing effect of TQR-Co-PS, the targeting effect of nanoparticles can be increased by applying folic acid targeting, so that the drug uptake of cells is more effectively improved through the folic acid receptor-mediated phagocytosis of the drug-loaded polymer vesicles, the fluorescence signal of adriamycin in laser confocal of the drug-loaded polymer vesicles is higher than that of other nanoparticle administration groups, and the corresponding uptake of paclitaxel is also greatly improved. To sum up: the drug-loaded polymer vesicle synthesized by the research can obviously inhibit the effect of P-gp and improve the cellular uptake of a P-gp substrate so as to reverse the multidrug resistance of tumors.

Example 8

Effect of drug-loaded reduction-sensitive polymersome on apoptosis

Single cell suspensions (MCF-7 and MCF-7/ADR cells) were prepared in 10% fetal bovine serum in folate-free RPMI1640 medium at about 2X 10 cells per well⁵Cells were seeded in 12-well plates, 2 mL per well volume. Adding a medicament (the administration method is the same as laser confocal) diluted by a culture medium for 4 hours after the cells adhere to the wall, and collecting the cells; negative control, Annexin V single staining group and PI single staining group are additionally arranged. Washing with precooled PBS for 2 times, digesting with pancreatin without EDTA and collecting cells; resuspend cells in 100. mu.L of 1 XBindingAdd 5. mu.L Annexin V-FITC and 5. mu.L PI staining solution into Buffer, avoid light, incubate 10 minutes at room temperature, then add 400. mu.L 1 XBinding Buffer, then sieve and transfer to flow tube, carry on the flow cytometry detection within 1 h.

FIG. 11 shows that there was no significant difference in the proportion of apoptotic cells (early and late cells, respectively) after treatment of sensitive cells with Free DOX + PTX and Free TQR + DOX + PTX; the apoptosis ratio of the sensitive strain treated by the Co-PS and the TQR-Co-PS is not obviously poor (49.7 percent and 54.6 percent respectively); the TQR does not influence the proliferation of the sensitive strain cells, the apoptosis percentage (58.6%) of the sensitive strain cells treated by FA-TQR-Co-PS is slightly higher than that of the sensitive strain cells treated by Co-PS, and the folic acid mediated nano vesicles can increase the cytotoxicity of chemotherapeutic drugs on the sensitive strain cells, so that the apoptosis is accelerated. For MCF-7/ADR cells, no matter in a free drug group or a polymer vesicle administration group, TQR in the preparation can obviously increase the apoptosis ratio, which indicates that the TQR can effectively reverse the MDR effect of drug-resistant cells and strengthen the killing effect of chemotherapeutic drugs.

Example 9

Effect of drug-loaded reduction-sensitive polymersome on drug-resistant cell cycle

The periodic experiment is similar to the treatment of the apoptosis experiment, the cells are collected after the cells are incubated for 24 h after the nanoparticle-containing culture medium is added, and the cells are fixed overnight by 1-2 mL of precooled 70% ethanol. Centrifuging at 1000 rpm for 5 min, discarding the fixative, collecting the fixed cells, washing with 1ml of precooled PBS once, staining according to the method of the instruction, and then detecting the cell cycle by flow.

Doxorubicin can intercalate into DNA to inhibit nucleic acid synthesis, thereby affecting the cell cycle; paclitaxel inhibits cell mitosis and blocks cells in the G2/M phase of the cell cycle. As shown in FIG. 12, in the present study, it is evident that the MCF-7/ADR cell cycles treated by Free TQR + DOX + PTX, TQR-Co-PS and FA-TQR-Co-PS are blocked in the G2/M phase, and the cells in the G0/G1 phase are greatly reduced; the cell block degree of the FA-TQR-Co-PS group in the G2/M phase is higher than that of the TQR-Co-PS group (P is less than 0.01), which indicates that the folic acid receptor mediated endocytosis can strengthen the influence of the chemotherapeutic drug on the cell cycle; free DOX + PTX has no obvious effect (P is more than 0.05) on the blocking effect of the G2/M phase of the cells, which indicates that the drug rejection effect of the drug-resistant cells enables the drug-resistant cells not to be influenced by the chemotherapeutic drugs adriamycin and paclitaxel, compared with a control group, the Co-PS group without TQR reduces the proportion of the G0/G1 phase in the cell cycle to a certain extent, improves the proportion of the G2/M phase (P is less than 0.01), and indicates that the polymer vesicles can weaken the MDR of the cells to a certain extent, thereby enhancing the killing power of the chemotherapeutic drugs on the cells. From the general trend, the drug-loaded polymer vesicle can block cells in the G2/M phase, thereby influencing the proliferation of the cells.

The invention can overcome the defects of the prior art, fully utilizes the advantages of the nano preparation, and prepares the multifunctional drug delivery system which has an active targeting function and tumor microenvironment reduction response and simultaneously entraps chemotherapeutic drugs and P-gp inhibitors. The delivery system can be self-assembled to form three-layer spherical nanoparticles of hydrophilic-hydrophobic-hydrophilic, paclitaxel and a P-gp inhibitor Tariquidar are efficiently loaded into a hydrophobic membrane layer of a polymersome through a hydrophobic acting force, and then doxorubicin and the like are loaded into a hydrophilic inner cavity of the polymersome through a pH gradient method. The PEG layer of the polymer vesicle outer shell endows the polymer micelle with long circulation and space stability, and the targeting group DSPE-PEG-Folate is connected to the outer shell of the polymer vesicle to form the drug-loaded polymer vesicle with uniform particle size (200 nm), good biocompatibility and stability.

The P-gp inhibitor Tariquard is carried together with the chemotherapeutic drugs paclitaxel and adriamycin, so that the tumor targeted delivery and the tumor part drug controlled release are realized, the bioavailability of the chemotherapeutic drugs is particularly effectively improved, and the tumor multidrug resistance is overcome. On one hand, the adriamycin and the paclitaxel (1: 2.5, mass ratio) are jointly administrated, so that the tumor drug resistance risk caused by single administration is reduced, and the chemotherapeutic treatment effect is enhanced. On the other hand, the use of the Tariquard can block the substrate recognition effect of P-gp, thereby reducing the resistance of tumor cells to adriamycin and paclitaxel, increasing the drug uptake and improving the bioavailability of chemotherapeutic drugs. The invention can realize targeted delivery and environment-responsive drug release, greatly reduces the side effect caused by the systemic distribution of free chemotherapeutic drugs, and reverses MDR of drug-resistant cells to the chemotherapeutic drugs.

Claims

1. A preparation method of a nano delivery system of a targeted reduction-sensitive co-carried chemotherapeutic drug and a P-gp drug resistance reversal agent is characterized by comprising the following steps:

1) fully dissolving 25mg of amphiphilic copolymer PCL-ss-PEG-ss-PCL, 5wt.% of DSPE-PEG2000-Folate, 5wt.% of Tariquar and 2.5mg of paclitaxel in dichloromethane, removing the organic solvent by using a rotary evaporator to form a uniform film on the inner wall of the reactor, and drying for 12 hours in vacuum;

2) preparing 300mM ammonium sulfate aqueous solution, dispersing the sample membrane in 5mL ammonium sulfate solution, and hydrolyzing for 5h at 65 ℃;

mixing, standing to room temperature, performing ultrasonic treatment for 30min in ice bath, and performing 5mm probe and Ampl30% to obtain stable and uniform drug-loaded reduction sensitive polymer vesicle dispersion;

3) putting the polymersome suspension into a treated dialysis bag, and dialyzing in a sucrose solution for 20 hours, wherein the sucrose solution is 102.69g of sucrose and 1.152g of histidine dissolved in 1L of water;

4) loading adriamycin: adding 5.0mgDOX into the dialyzed sample, and incubating for 2h at the constant temperature of 65 ℃ and under magnetic stirring at 300 r/min;

5) removing the unencapsulated drug: dialyzing with pH7.4PBS for 20h, and storing at 4 ℃ to obtain the reduction response polymer vesicle dispersion liquid of the co-carried chemotherapeutic drug and the P-gp inhibitor of the targeted tumor.

2. The nano delivery system of the targeted reduction-sensitive co-carried chemotherapeutic drug and the P-gp drug resistance reversal agent prepared by the method of claim 1.

3. The application of the nano delivery system of the targeted reduction-sensitive co-carried chemotherapeutic drug and the P-gp drug resistance reversal agent prepared by the method of claim 1 in preparing a drug for treating tumors.

4. The use according to claim 3, characterized in that it comprises the application to anti-breast cancer MCF-7/ADR resistant cells, human hepatoma cells HepG2/ADR or human ovarian cancer paclitaxel resistant strain A2780/Taxol high expression P-gp cells.

5. The use according to claim 3, characterized in that the tumor treatment drug is a polymersome nanoparticle of a targeted reduction-sensitive co-carried chemotherapeutic drug and a P-gp reversal agent.