WO2021226762A1 - Tumor microenvironment response-type nano-composite drug loading system, and preparation method therefor and use thereof - Google Patents

Abstract

Provided are a tumor microenvironment response-type nano-composite drug loading system, and a preparation method therefor and the use thereof. The drug loading system comprises a block polymer, an anti-tumor drug and curcumin. The block polymer is a tumor microenvironment response-type polymer, and the anti-tumor drug and curcumin are wrapped in the block polymer. The preparation method is simple in process, low in cost and high in universality. The prepared tumor microenvironment response-type nano-composite drug loading system has the advantages of good biocompatibility, high drug loading rate, sensitive response to tumor microenvironments, capability of reversing multi-drug resistance, etc., can effectively reduce the systemic toxicity of anti-tumor drugs, and also inhibits the expression of P-gP protein in drug-resistant tumor cells, thereby effectively achieving the reversal of multidrug-resistant tumors and having an important guiding and application value in the aspect of cancer treatment.

Description

Tumor microenvironment responsive nano composite drug-carrying system and preparation method and application thereof Technical field

The invention relates to the field of tumor medicines, in particular to a tumor microenvironment responsive nanocomposite drug-carrying system, a preparation method thereof, and application in reversing tumor multidrug resistance (MDR).

Background technique

With the increase in the incidence of cancer year by year, people are paying more and more attention to the research of cancer treatment. Chemotherapy, as an important means of cancer treatment, has developed rapidly in recent years, and new chemotherapy drugs have emerged one after another. However, tumor multidrug resistance (MDR) has always been a major bottleneck that chemotherapy is difficult to break through. MDR is usually an acquired phenotype of tumor cells, which makes the cells resistant to a variety of chemotherapeutics, leading to the failure of a variety of chemotherapeutics. Studies have shown that the mechanisms of tumor multidrug resistance against different drugs mainly include: (a) Transmembrane protein overexpression (P-glycoprotein (P-gp), multidrug resistance protein (MRP) and breast cancer Resistance protein (BCRP), etc.); (b) Enhanced DNA repair ability; (c) Changes in drug targets and (d) Abnormal signal transduction pathways. Among them, the overexpression of ABC (ATP-binding cassette) transporter on the cell membrane leads to the efflux of chemotherapeutic drugs is one of the most important mechanisms of MDR.

Nanoparticles are used more and more widely in the field of biomedicine due to their unique size effect, easy surface modification, diversified composition, and good biocompatibility. Nanoparticle-based drug delivery systems have shown obvious advantages in improving the efficiency of tumor treatment. However, in the face of complex biological systems and individual differences in tumor patients, nano-drug delivery systems are still facing huge challenges in their clinical applications: on the one hand, how to deliver and release drugs in a controllable and precise manner, and on the other hand, how to reduce non-specific drugs. Drug residues of the opposite sex reduce the systemic toxicity of nanoparticles.

Therefore, it is currently an important issue to achieve good tumor targeting and efficient drug delivery rate of the nanocomposite drug delivery system, and to significantly reverse the effect of MDR.

Summary of the invention

In view of some technical problems in the prior art, the present invention provides a tumor microenvironment-responsive nanocomposite drug delivery system based on in-depth research, which can efficiently load drugs and can significantly inhibit drug-resistant cells P-gp Expression, has a good therapeutic effect on tumor MDR. The tumor microenvironment-responsive nanocomposite drug-carrying system prepared by the invention is a nanoparticle with good biocompatibility, which lays a foundation for its wide application in the field of biomedicine. With the in-depth research on drug-carrying systems, it has been discovered that nano-drug carriers in the prior art still have many defects, such as low targeting, poor tumor permeability, unselective drug release locations, and uncontrolled release speed, which hinder The further development and application of nano-carriers in the field of tumor medicine. In order to solve the technical problems existing in the prior art, the present invention provides a tumor microenvironment-responsive nanocomposite drug-carrying system and a preparation method and application thereof. Specifically, the present invention includes the following.

The present invention provides a tumor microenvironment-responsive nanocomposite drug-carrying system, which comprises a block polymer, an anti-tumor chemotherapeutic drug and curcumin (Curcumin, CAS number: 458-37-7), wherein the block polymer The substance is a tumor microenvironment responsive polymer, and the anti-tumor drug and the curcumin are encapsulated in the block polymer.

In some specific embodiments, the nanocomposite drug delivery system is a nanoparticle, and it is prepared by using the block polymer as a carrier to simultaneously encapsulate the antitumor drug and the curcumin in a self-assembly manner.

In certain specific embodiments, the tumor microenvironment responsive polymer is a polymer that disassembles in the tumor microenvironment.

In certain specific embodiments, the tumor microenvironment responsive polymer is PLGA-ss-PEG.

In certain specific embodiments, the anti-tumor drugs include selected from camptothecin, irinotecan, cytarabine, paclitaxel, docetaxel, doxorubicin, gemcitabine, platinum-based chemotherapeutics (cisplatin, At least one of the group consisting of carboplatin, nedaplatin, cycloplatin, oxaliplatin, and ropolamine) and 5-fluorouracil.

In some specific embodiments, the molar ratio of the tumor drug to curcumin is 1:2-10, and the molar ratio of the antitumor drug to the block polymer is 1:3.

The second aspect of the present invention provides a method for preparing a tumor microenvironment-responsive nanocomposite drug-carrying system, which includes the following steps:

(1) Preparation steps of coating solution a;

(2) Adding anti-tumor drugs, curcumin and block polymers to an organic solvent to obtain an organic mixed phase solution b;

(3) The step of mixing solution a and solution b, stirring and evaporating to obtain transparent liquid c;

(4) The step of filtering the transparent liquid c with a filter membrane, and then ultrafiltration and centrifugation to obtain the nanocomposite drug-carrying system.

In certain embodiments, the coating agent is at least one selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, poloxamer, and oleic acid.

The third aspect of the present invention provides the application of the tumor microenvironment-responsive nanocomposite drug-carrying system according to the first aspect in reversing tumor multidrug resistance.

The nano-composite drug-carrying system of the present invention uses block polymers responsive to tumor microenvironment as a carrier, and simultaneously encapsulates anti-tumor drugs and curcumin in a self-assembly manner. Among them, the nano drug delivery system is a water-soluble spherical nanoparticle, which is beneficial to realize its passive targeting effect on tumors. The nano drug-carrying system has a specific response to the tumor microenvironment, and the nanoparticle structure can be disassembled in the tumor microenvironment to realize the targeted release of the drug at the tumor site. In addition, the curcumin released by the nano drug delivery system can effectively reduce the expression of drug-resistant protein in drug-resistant tumor cells. The tumor microenvironment responsive nanocomposite drug-carrying system prepared by the invention has the advantages of good biocompatibility, high drug loading rate, sensitive tumor microenvironment response, reversing multi-drug resistance and the like. Cell experiments have proved that the tumor microenvironment-responsive nanocomposite drug-carrying system prepared by the present invention can significantly increase the concentration of chemotherapeutic drugs in multi-drug resistant tumor cells, increase the killing rate of drug-resistant tumors, and realize effective reversal of tumor MDR.

Description of the drawings

^{Figure 1 is a 1} H NMR chart of a polymer responsive to the tumor microenvironment and the molecular structure of the polymer.

Figure 2 is a scanning electron microscope image of the nanocomposite drug-carrying system.

Figure 3 shows the UV-Vis spectrum of the nanocomposite drug-carrying system.

Figure 4 is a diagram of the hydrated particle size of the nanocomposite drug-carrying system.

Figure 5 shows the results of multidrug resistance detection of tumor cells in the nanocomposite drug-carrying system.

Figure 6 shows the cell fluorescence detection result of the nanocomposite drug-carrying system.

Figure 7 shows the results of detection of drug-resistant protein expression by the nanocomposite drug delivery system.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. The detailed description should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only used to describe specific embodiments and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper limit and the lower limit of the range and each intermediate value between them are specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the field of the present invention. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the content of this manual shall prevail.

[Nano Composite Drug Delivery System]

The nanocomposite drug delivery system of the present invention comprises a block polymer, an antitumor chemotherapeutic drug and curcumin (Curcumin, CAS number: 458-37-7), wherein the block polymer is a tumor microenvironment responsive polymer, The anti-tumor drug and the curcumin are encapsulated in the block polymer.

Preferably, the nano-composite drug-carrying system of the present invention refers to the formation of drug delivery nanoparticles with a median diameter of 1-1000 nm by the drug and the medicinal material. Also preferably, the nano-drug carrier system is a water-soluble spherical nano-particle with a diameter of 75-270 nm. It is more preferably 100 nm, which is more conducive to achieving its passive targeting effect on tumors under this condition.

Preferably, the nanocomposite drug-carrying system is prepared by using a block polymer as a carrier and simultaneously encapsulating the anti-tumor drug and the curcumin in a self-assembly manner. The block polymer of the present invention can also be referred to as a "block copolymer", which is a linear copolymer formed by alternate polymerization of different chemical structures. This polymer has the advantages of controllable molecular weight, narrow molecular weight distribution, and designable molecular structure and composition.

Preferably, the block polymer of the present invention is a tumor microenvironment responsive polymer, and the tumor microenvironment responsive polymer is a polymer that disassembles in a tumor microenvironment. It has a specific response to the tumor microenvironment, and the nanoparticle structure can be disassembled in the tumor microenvironment. Through active targeting or the high permeability and retention effect of solid tumors, the targeted release of drugs at the tumor site can be achieved, and the drug's impact on the tumor can be improved. The specific selectivity of cells increases the concentration of the drug in the target area and reduces its distribution in the non-target area. Preferably, the tumor microenvironment responsive polymer is PLGA-ss-PEG. The principle of unpacking is based on that the disulfide bond of PLGA-ss-PEG is sensitive to the reducing environment and is easily unwound under reducing conditions to form two -SH groups, which disintegrate the polymer. This is the polymer As the basis of the tumor microenvironment-sensitive drug carrier.

In a specific embodiment, the anti-tumor drugs encapsulated in nanoparticles by self-assembly include those selected from camptothecin, irinotecan, cytarabine, paclitaxel, docetaxel, adriamycin, gemcitabine, and platinum. At least one of the group consisting of chemotherapeutic drugs (cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and ropolamine) and 5-fluorouracil.

Preferably, the molar ratio of the anti-tumor drug to curcumin is 1:2-10, and the molar ratio of the anti-tumor drug to the block polymer is 1:3. Also preferably, the molar ratio of the anti-tumor drug to curcumin is 1:2-6.

In a specific embodiment, the preparation steps of the tumor microenvironment responsive polymer of the present invention are as follows:

1) Take thiodipropionic acid and reflux at 55-75°C and concentrate;

2) Adding excess ethoxyethane to obtain a white solid and vacuum drying;

3) Add dithiodipropionic anhydride and the catalyst 4-dimethylaminopyridine and triethylamine solution to the anhydrous dimethylformamide containing carboxyl group PLGA and place it at 40-50℃, stir and concentrate, add excess ether to precipitate the product, To PLGA-ss-COOH;

4) The dehydration condensation reaction connects PEG to one end of PLGA-ss-COOH to obtain PLGA-ss-PEG.

It also includes proton nuclear magnetic resonance spectroscopy ( ¹ H NMR) to characterize the structure of the obtained product. The specific results are shown in Figure 1. The ¹ H NMR spectrum obtained is consistent with the molecular structure of the designed polymer (annotated in the figure), indicating the successful synthesis of PLGA-ss-PEG.

[Preparation method of nano composite drug-carrying system]

The preparation method of the nanocomposite drug-carrying system of the present invention includes the following steps:

(1) Preparation steps of coating solution a;

(2) Adding anti-tumor drugs, curcumin and block polymers to an organic solvent to obtain an organic mixed phase solution b;

(3) The step of mixing solution a and solution b, stirring and evaporating to obtain transparent liquid c;

(4) The step of filtering the transparent liquid c with a filter membrane, and then ultrafiltration and centrifugation to obtain the nanocomposite drug-carrying system.

In the preparation method of the nanocomposite drug-carrying system, the coating agent in step (1) is selected from at least one of the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, poloxamer and oleic acid. Preferably, the concentration of the coating agent is 0.4-3.5% by mass to volume, more preferably 0.5-3.0%.

In the preparation method of the nanocomposite drug-carrying system, the organic mixed phase solution b of step (2) is obtained by magnetic stirring. In a specific embodiment, the magnetic stirring conditions are 200-400 rpm, preferably 300 rpm.

In the preparation method of the nanocomposite drug-carrying system, step (3) includes placing the a solution obtained from step (1) at 800-1200 rpm for vigorous stirring. Preferably, the stirring condition is 950 rpm. The solution b obtained in the extraction step (2) is slowly added to the solution a, and the solution a gradually changes from clear and transparent to an emulsion, and stirring is continued, and the obtained mixed solution is transferred to a reduced-pressure rotary evaporator. Preferably, the reduced-pressure rotary evaporation conditions are under 850mbr negative pressure, 40°C, and the time is 0.5-1.2h. More preferably, the transparent liquid c is obtained after 1h of rotary evaporation.

In the preparation method of the nanocomposite drug-carrying system, the pore size of the filter membrane in step (4) is 0.22 μm, and the molecular weight cut-off of the ultrafiltration tube is 3000 kDa. The centrifugation conditions were 10000 rpm, 4°C, and the centrifugation time was 20 min. Repeat three times.

Preferably, the preparation method of the nano-composite drug-carrying system of the present invention further includes using high performance liquid chromatography (HPLC) to detect the concentration of the drug in the nanoparticles, and conducting cell experiments on sensitive and drug-resistant tumor cells to investigate the effects of nanoparticles. Reversal and killing effects of drug-resistant tumors, including cell fluorescence, cell viability testing, q-PCR testing and Western blot testing, etc.

Preparation Example 1

This preparation example is the preparation step of the polymer PLGA-ss-PEG-COOH that responds to the tumor microenvironment. The specific steps are as follows:

(1) Take 1g of 3,3-dithiodipropionic acid (DTDP) in a round-bottom flask, add 3mL of acetyl chloride, then transfer the round-bottom flask to a water bath, reflux for 2h in a water bath at 65℃, and put it in the bottle Obtain a light yellow solution, continue heating to concentrate the solution to about 0.5mL;

(2) After the above concentrated solution is cooled to room temperature, add excess ether (about 10 mL) to it, stir at room temperature for 3 hours at a constant speed, the solution turns from clear and transparent to turbid, and then filter the turbid liquid with a Buchner funnel and organic filter membrane to remove the ether A white solid is obtained, and the obtained white solid is repeatedly washed with ether for 3 times to remove impurities. The washed white solid was vacuum dried at 40°C for 24 hours to obtain dithiodipropionic anhydride (DTDPA);

(3) Dissolve 800.0mg carboxyl PLGA (0.8mM) in 10.0mL anhydrous dimethylformamide (DMF), add 154.0mg DTDPA (0.8mM), 97.6mg 4-dimethylaminopyridine (0.8mM) ) And 112μL of TEA (0.8mM), stir in a fume hood under a water bath at 45℃ to mix the components uniformly, then continue to stir for 36h to volatilize the solvent, and the volume of the solution is concentrated to about 1.0mL, adding excess ether, solution From clarification to turbidity, after stirring at room temperature for 4 hours at a constant speed, filter the turbid liquid with a Buchner funnel and organic filter membrane to remove the ether to obtain a white solid. The obtained white solid is washed with ether for 3 times to remove impurities. The washed white solid was vacuum dried at 40°C for 24h to obtain PLGA-ss-COOH;

(4) Weigh 800.0mg PLGA-ss-COOH (0.8mM) powder, 15.3mg EDC (0.8mM) powder and 26.0mg sulfo-NHS (1.2mM) powder and dissolve in 10mL anhydrous dimethylformamide (DMF) _{PLGA-ss-COOH activated by adding NH 2} -PEG-COOH powder to the PLGA-ss-COOH activation solution under vigorous stirring after cooling the solution to room temperature for 12 hours in the dark. React for 12 hours in the dark at room temperature to obtain PEG-modified PLGA-ss-COOH, that is, PLGA-ss-PEG-COOH; continue stirring for 36 hours in a fume hood to evaporate the solvent and concentrate the solution volume to about 1.0 mL. Add excess ether The solution changed from clear to turbid. After stirring at room temperature for 4 hours, filter the turbid liquid with a Buchner funnel and organic filter membrane to remove the ether to obtain a white solid. The obtained white solid was washed 3 times with ether to remove impurities. The washed white solid was vacuum dried at 40°C for 24 hours to obtain purified PLGA-ss-PEG-COOH;

(5) Characterize the structure of the product obtained by hydrogen nuclear magnetic resonance spectroscopy ( ^{1 H NMR).}

The results are shown in Figure 1. The ¹ H NMR spectrum obtained is consistent with the molecular structure of the designed polymer (annotated in the figure), indicating that PLGA-ss-PEG was successfully synthesized.

Preparation Example 2

This preparation example is the preparation step of tumor microenvironment-responsive nano-drug-loaded particles DOX-NPs, wherein only a single component doxorubicin (DOX) is wrapped in the nanoparticles. The specific steps are as follows:

(1) Weigh 50 mg of poloxamer 188, add 5 mL of deionized water, and stir at 25°C for 10-30 minutes to fully dissolve the water phase 1% coating agent solution;

(2) Weigh 5 mg of doxorubicin and 20 mg of the carrier PLGA-ss-PEG-COOH prepared in Preparation Example 1, add 1 mL of dichloromethane, magnetically stir at 300 rpm, and stir overnight at room temperature to obtain an organic phase mixed solution;

(3) Take the solution a obtained in step (1) of this preparation example and place it under vigorous stirring at 950 rpm, use a syringe to extract the solution b obtained in step (2) of this preparation example, and slowly inject it into the a solution under stirring In, a solution gradually changed from clear and transparent to emulsion, and continued to stir for 1-2h. Transfer the obtained mixture to a reduced-pressure rotary evaporator, and rotatably evaporate at 40°C for 1 hour under 850mbr negative pressure to obtain a transparent liquid c;

(4) Take the liquid c obtained in step (3) of this preparation example through a 0.22μm filter membrane, and centrifuge it with an ultrafiltration centrifuge tube with a molecular weight cut-off of 3000kDa at 10000rpm and 4°C for 20min, repeat 3 times to remove The drug is wrapped, and the tumor microenvironment-responsive nano-drug-loaded particles DOX-NPs are obtained.

Preparation Example 3

This preparation example is the preparation step of tumor microenvironment-responsive nano-drug-loaded particles CUR-NPs, wherein only a single component curcumin (CUR) is encapsulated in the nanoparticles. The specific steps are as follows:

(1) Weigh 50 mg of poloxamer 188, add 5 mL of deionized water, and stir at 25°C for 10-30 minutes to fully dissolve the water phase 1% coating agent solution;

(2) Weigh 10 mg of curcumin and 20 mg of the carrier PLGA-ss-PEG-COOH prepared in Preparation Example 1, add 1 mL of dichloromethane, magnetically stir at 300 rpm, and stir overnight at room temperature to obtain an organic phase mixed solution;

(3) Take the solution a obtained in step (1) of this preparation example and place it under vigorous stirring at 950 rpm, use a syringe to extract the solution b obtained in step (2) of this preparation example, and slowly inject it into the a solution under stirring In, a solution gradually changed from clear and transparent to emulsion, and continued to stir for 1-2h. Transfer the obtained mixture to a reduced-pressure rotary evaporator, and rotatably evaporate at 40°C for 1 hour under 850mbr negative pressure to obtain a transparent liquid c;

(4) Take the liquid c obtained in step (3) of this preparation example through a 0.22μm filter membrane, and centrifuge it with an ultrafiltration centrifuge tube with a molecular weight cut-off of 3000kDa at 10000rpm and 4°C for 20min, repeat 3 times to remove The drug is wrapped, and the tumor microenvironment-responsive nano drug-loaded particles CUR-NPs are obtained.

Preparation Example 4

This preparation example is the preparation step of the tumor microenvironment-responsive nanocomposite drug-loaded particles DC-NPs, wherein the nanoparticles are simultaneously encapsulated with two components, doxorubicin (DOX) and curcumin (CUR), and the specific steps are as follows:

(1) Weigh 50 mg of poloxamer 188, add 5 mL of deionized water, and stir at 25°C for 10-30 minutes to fully dissolve the water phase 1% coating agent solution;

(2) Weigh 5 mg of doxorubicin, 10 mg of curcumin and 20 mg of the carrier PLGA-ss-PEG-COOH prepared in Preparation Example 1, add 1 mL of dichloromethane, stir with magnetic force at 300 rpm, and stir overnight at room temperature to obtain an organic phase mixed solution ；

(3) Take the solution a obtained in step (1) of this preparation example and place it under vigorous stirring at 950 rpm, use a syringe to extract the solution b obtained in step (2) of this preparation example, and slowly inject it into the a solution under stirring In, a solution gradually changed from clear and transparent to emulsion, and continued to stir for 1-2h. Transfer the obtained mixture to a reduced-pressure rotary evaporator, and rotatably evaporate at 40°C for 1 hour under 850mbr negative pressure to obtain a transparent liquid c;

(4) Take the liquid c obtained in step (3) of this preparation example through a 0.22μm filter membrane, and centrifuge it with an ultrafiltration centrifuge tube with a molecular weight cut-off of 3000kDa at 10000rpm and 4°C for 20min, repeat 3 times to remove The drug is wrapped to obtain the tumor microenvironment-responsive nanocomposite drug-loaded particles DC-NPs.

The scanning electron micrograph of the nanoparticles is shown in Figure 2. The obtained composite drug-loaded nanoparticles can be observed under the scanning electron microscope as relatively uniform spherical particles with a diameter of about 100 nm.

Example 1

This embodiment is the drug loading detection step of the tumor microenvironment responsive nanocomposite drug loading system obtained in Preparation Example 4. details as follows.

The nano drug-loaded particles prepared in Preparation Example 2 were dissolved in methanol, ultrasonicated for 3-5 min, and the solution was passed through a 0.22 μm filter membrane to obtain a drug solution after disassembly. At the same time, doxorubicin (DOX) and curcumin (CUR) were dissolved in methanol to obtain a single drug solution with a concentration of 25 μg/mL and 2.5 μg/mL, respectively. The above-mentioned three kinds of solutions draw 100μL into 96-well plates respectively, and detect each sample with a microplate reader, and the detection wavelength range is 300-700nm. The result is shown in Fig. 3. The UV-Vis absorption spectrum of CUR is a single peak, and its maximum absorption wavelength is 430nm. There are multiple peaks on DOX's UV-Vis spectrum, and its maximum absorption wavelength is 500nm. The ultraviolet-visible spectrum of the nanoparticle solution has a maximum absorption wavelength of 450nm, which is located between the maximum absorption peaks of CUR and DOX. At the same time, in the 490-550nm band, the spectrum of nanoparticles almost completely overlaps the spectrum of DOX. The above results indicate that two drugs are successfully encapsulated in the nanoparticles.

Example 2

This example is the step of detecting the hydrated particle size of the tumor microenvironment-responsive nanocomposite drug-carrying system obtained in Preparation Example 4. details as follows.

The nano drug-carrying particles prepared in Preparation Example 2 were made into a 1 mg/mL solution with pure water, pure water was used as a control, and the hydrated particle size of the nanoparticles was measured with a dynamic light scattering instrument. The measurement was repeated 3 times. The result is shown in Figure 4. Shown. The prepared nanoparticles have a hydrated particle size of 140±14nm, which is larger than the results of scanning electron microscopy. This is caused by normal particle swelling and the hydrogen bond interaction between the particle surface and water molecules.

Example 3

This embodiment is the procedure for detecting multidrug resistance of tumor cells in the tumor microenvironment-responsive nanocomposite drug-carrying system obtained in Preparation Example 4. details as follows.

Cultivate MCF7/ADR cells (adriamycin-resistant human breast cancer cells) to grow to a cell fusion rate of about 80%. After digestion with trypsin, make ^{a cell suspension with a concentration of 5×10 4} /mL, and then inoculate the cell suspension. Transfer 100 μL of the solution to a 96-well plate, place it at 37°C and 5% CO _{2 and} incubate overnight to allow the cells to grow adherently. Prepare DOX solution and NPs solution separately with culture medium (DOX concentration: 0.0013, 0.0064, 0.032, 0.16, 0.8, 4, 20μg/mL), change the cell culture medium in the well plate to different concentrations of drug culture medium, and simultaneously The wells containing only culture medium were set as blank control, and the wells containing cells but no drug were set as normal control. Set 5 replicate wells for each concentration, and incubate the culture plate in an incubator for 72 hours. After incubation, add 10 μL of CCK-8 solution to each well, and measure the absorbance at 450 nm with a microplate reader after 1 hour. The experiment was repeated three times.

The results are shown in Figure 5. After the cells were co-cultured with DOX for 72 hours, the IC50 value of the cells was 6.1 μg/mL, while the IC50 value of the cells and NPs was 0.4 μg/mL after 72 hours of co-culture, which was significantly lower than that of the DOX culture group. It shows that the nano drug-carrying system prepared by the present invention can effectively overcome the multi-drug resistance of tumor cells and improve the drug effect.

Example 4

This embodiment is the cell fluorescence detection step of the tumor microenvironment-responsive nanocomposite drug-carrying system obtained in Preparation Example 4. details as follows.

Cultivate MCF7/ADR cells (adriamycin-resistant human breast cancer cells) to grow until the cell fusion rate reaches about 80%. After digestion with trypsin, ^{a cell suspension with a concentration of 1×10 5} /mL is prepared. A matched sterile glass slide is pre-placed in the 24-well plate, rinsed with PBS, and blotted dry. Inoculate 1 mL of cell suspension into a 24-well plate, and _{incubate overnight at 37° C. and 5% CO 2} to allow the cells to grow adherently. Prepare 1μg/mL DOX solution, 1μg/mL DOX+1μM CUR mixed solution and NPs solution of equal drug concentration with culture medium respectively, replace the cell culture medium in the 24-well plate with the above three solutions, and add no drug The wells of the culture medium served as the control group. Set at least 3 holes in each group. After incubating for 24 hours, proceed as follows:

(1) Aspirate the culture medium and wash twice with PBS;

(2) Fix the cells with 75% alcohol for 30 minutes (300μL/well);

(3) Absorb the alcohol, wash twice with PBS, 5 minutes each time;

(4) DAPI was diluted with PBS at a ratio of 1:2000, added to each well, and stained for 5 minutes;

(5) Aspirate DAPI, wash 3 times with PBS, 5 min each time;

(6) Add 5μL of the mounting tablet dropwise to the glass slide, take out the climbing tablet upside down on the mounting tablet, and store in the dark until dry;

(7) Take pictures with an inverted fluorescence microscope.

The results are shown in Figure 6A. The red fluorescence intensity is the strongest in the cells co-cultured with NPs. The red fluorescence in the DOX+CUR group was the second, and was mainly distributed in the cytoplasm. However, the amount of red fluorescence entering the nucleus of the NPs group was significantly increased, as shown in Figure 6B, indicating that NPs can increase the cellular uptake of DOX and at the same time increase its nuclear penetration ability.

Example 5

This embodiment is the step of detecting the expression of the drug-resistant protein MDR1 by the tumor microenvironment-responsive nanocomposite drug-carrying system obtained in Preparation Example 2. details as follows.

Cultivate MCF7 cells (human breast cancer cells) and MCF7/ADR cells (adriamycin-resistant human breast cancer cells) to grow to a cell fusion rate of about 80%. After digestion with trypsin, they are inoculated into 6-well plates, of which 1 MCF7 cells were seeded in wells No. 2 and MCF7/ADR cells were seeded in wells 2-5. After culturing for 24 hours, the culture medium in wells 3-6 was replaced with 1μg/mL DOX, 1μM CUR, 1μg/mL DOX+1μM CUR, and the same DOX concentration NPs, and cultured for another 24h. MCF7 cells without any treatment were used as a negative control, and MCF7/ADR cells without any treatment were used as a positive control. After 24 hours of incubation, perform the following steps to extract cell protein.

(1) Aspirate the culture solution of each well and wash twice with ice PBS;

(2) Add RIPA containing EDTA and protease inhibitor, 100μL/well, lyse on ice for 20min;

(3) The lysate is sucked into the pre-cooled EP tube;

(4) Centrifuge at 15000g for 15min at 4℃;

(5) Aspirate the supernatant to obtain cell protein, and measure the protein concentration by the BCA method.

Next, perform a Western blot experiment, the steps are as follows:

(1) With 10% Western blot electrophoresis gel;

(2) Add 5×SDS to each histone, boil at 95℃ for 5min, and cool;

(3) The protein loading amount is 10μg, add 1-3μL protein marker at both ends, after electrophoresis at a constant voltage of 80V for 30 minutes, pressurize to 120V for gel electrophoresis detection;

(4) Remove the electrophoresis gel, transfer it to the membrane at a constant current of 200mA for 2.5 hours, so that the bands on the gel are completely transferred to the membrane, and cut the bands corresponding to the positions of β-actin and MDR1 protein;

(5) Block with TBST containing 5% skimmed milk powder at room temperature for 1 hour, then add β-actin and MDR1 primary antibodies (diluted), and incubate at 37°C for 1 hour;

(6) Wash the strip 3 times with TBST, 10 min/time;

(7) Add the corresponding secondary antibody (diluted) with HRP and incubate for 1 hour at room temperature;

(8) Wash the strip 3 times with TBST, 10 min/time;

(9) The ECL luminescent liquid emits light on the strips and takes pictures with imaging equipment;

(10) The blackness results obtained by shooting after color development are counted with Image J software.

The results are shown in Figure 7. After co-cultivation with NPs, the expression of MDR1 protein in MCF7/ADR cells decreased significantly, about 60% of the positive control cells, indicating that NPs can significantly inhibit the resistance of MDR1 protein in MCF7/ADR cells. Expression, thereby increasing the drug uptake of cells, is a key factor in overcoming the multidrug resistance of tumor cells.

Example 6

This example is a biocompatibility test of the tumor microenvironment-responsive nanocomposite drug-carrying system obtained in Preparation Example 4, and the test object is Balb/c mice. Specific steps are as follows:

(1) The experimental subjects were three-week-old female Balb/c mice, purchased from the Guangdong Provincial Animal Experiment Center, and all experimental operations were in compliance with the regulations of the Animal Research Ethics Committee. All animals are kept in a sterile (SPF) breeding room, the temperature is maintained at 24 ± 2 ℃, 12h light/12h dark environment, free water and food;

(2) The experimental mice were divided into 6 groups, namely Control group (injection of physiological saline), Vehicle group (injection of empty nanoparticles), DOX group, CUR group, DOX+CUR group and DC-NPs group. The injection methods were all Tail vein injection, 6-8 mice per group. The dosage of CUR is 4mg/kg mouse, the dosage of DOX is 10mg/kg mouse, and the injection volume does not exceed 200μL;

(3) Three days after the injection, the mice were anesthetized with 3% sodium pentobarbital, and their chest cavity was quickly opened with a scalpel, and the heart, liver, spleen, lung, kidney and other major organs were removed. Cut the collected organ samples with a blade of 0.5 mm ³ tissue block, place it in 10% paraformaldehyde fixative, fix at 4℃ for 24h, rinse with pure water for 2h, dehydrated with ethanol gradient, and finally use paraffin wax It is embedded and used for hematoxylin and eosin (H&E) staining;

(4) Hematoxylin dye solution: Weigh 0.5g hematoxylin powder and 24g ammonium alum dissolved in 70ml distilled water, then take 31g NaIO, 5ml water, add 30ml glycerin and 2ml glacial acetic acid, mix well, filter with filter paper, spare. Eosin dye solution: Weigh 0.5g of water-soluble eosin dye solution and dissolve it in 100ml of distilled water. Dilute hydrochloric acid ethanol solution: Prepare 1% hydrochloric acid with 75% ethanol. A series of concentrations of ethanol, xylene, and neutral gum.

(5) Paraffin section dewaxing: put the sections in xylene Ⅰ 10min-xylene Ⅱ 10min-anhydrous ethanol Ⅰ 5min-anhydrous ethanol Ⅱ 5min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min- 70% alcohol for 5min-wash with distilled water. Hematoxylin stained cell nucleus: slice into Harris hematoxylin stain for 3-8min, wash with tap water, 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with 0.6% ammonia water, rinse with running water. Eosin stained cytoplasm: slices into eosin staining solution and stained for 1-3 min. Dehydration and mounting: Put the slices in 95% alcohol I 5min-95% alcohol II 5min-anhydrous ethanol I 5min-anhydrous ethanol II 5min-xylene I 5min-xylene II 5min to dehydrate and transparent, and remove the slices from two Take the toluene out and let it dry, then cover the slides with neutral gum. Microscope inspection, image acquisition and analysis;

It can be seen from the experimental results that the main organs of each group of mice have pathological changes, indicating that the tumor microenvironment-responsive nanocomposite drug delivery system obtained in Preparation Example 4 has no obvious toxic and side effects and has good biocompatibility. .

Example 7

This embodiment is an anti-tumor effect test of the tumor microenvironment-responsive nanocomposite drug-carrying system obtained in Preparation Example 4, and the test object is a nude mouse with tumor burden. Specific steps are as follows:

(1) The experimental subjects were three-week-old female nude mice, which were purchased from the Guangdong Provincial Animal Experiment Center. All experimental operations were in compliance with the regulations of the Animal Research Ethics Committee. All animals are kept in a sterile (SPF) feeding room, the temperature is maintained at 24±2℃, 12h light/12h dark environment, free water and food;

(2) Use a 1 mL syringe with a 25G needle to withdraw 200 μL ^{of MCF7/ADR cell culture medium containing 5×10 6} cells/mL, and inject the cancer cells into the back of the right front leg of the model mouse by subcutaneous injection. Observe the tumor growth the next day until the tumor grows to a size visible to the naked eye. Use a vernier caliper to measure the tumor size. When the tumor volume reaches about 100mm ³ , the next tumor suppression experiment can be carried out;

(3) The experimental mice were divided into 6 groups, namely Control group (injection of physiological saline), Vehicle group (injection of empty nanoparticles), DOX group, CUR group, DOX+CUR group and DC-NPs group. The injection methods were all Tail vein injection, 6-8 mice per group. The dosage of CUR is 4mg/kg mouse, the dosage of DOX is 10mg/kg mouse, and the injection volume does not exceed 200μL/mouse. Inject the drug every 3 days, to 15 days, a total of 5 injections;

(4) Record the body weight and tumor volume of the mice every 2 days. After 3 weeks, the mice were anesthetized with 3% sodium pentobarbital, and their necks were severed to death. The subcutaneous tumors of the mice were taken out with surgical scissors and weighed. Group mice tumor weight, tumor volume was measured, and tumor inhibition curve was drawn.

The results of the examples show that the body weight of the DOX group mice has decreased to a certain extent during the experiment, indicating that long-term use of free DOX will produce more obvious toxic side effects, while the body weight of the DC-NPs group mice not only did not decrease, but increased. , Which shows that nano-encapsulation has a significant effect on reducing the toxic and side effects of drugs. At the same time, the tumor volume of the DC-NPs group was significantly smaller than that of the Control group. Compared with the DOX group and the DOX+CUR group, it also showed a more significant tumor suppression effect, indicating that the tumor microenvironment responsive type obtained in Preparation Example 4 The nano-composite drug-carrying system has excellent tumor inhibitory effects.

Although the present invention has been described with reference to the exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Without departing from the scope or spirit of the present invention, various adjustments or changes can be made to the exemplary embodiments of the present specification. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims (9)

1. A tumor microenvironment-responsive nanocomposite drug-carrying system, which is characterized in that it comprises a block polymer, an anti-tumor drug and curcumin, wherein the block polymer is a tumor microenvironment-responsive polymer, and the anti-tumor Tumor drugs and the curcumin are encapsulated in the block polymer.
2. The tumor microenvironment-responsive nanocomposite drug delivery system according to claim 1, wherein the nanocomposite drug delivery system is a nanoparticle, and it uses the block polymer as a carrier, and is simultaneously assembled by a self-assembly method. It is prepared by wrapping the anti-tumor drug and the curcumin.
3. The tumor microenvironment responsive nanocomposite drug delivery system according to claim 1, wherein the tumor microenvironment responsive polymer is a polymer that disassembles in a tumor microenvironment.
4. The tumor microenvironment responsive nanocomposite drug delivery system according to claim 1, wherein the tumor microenvironment responsive polymer is PLGA-ss-PEG.
5. The tumor microenvironment-responsive nanocomposite drug-carrying system according to claim 1, wherein the anti-tumor drug comprises selected from camptothecin, irinotecan, cytarabine, paclitaxel, docetaxel, At least one of the group consisting of adriamycin, platinum-based anti-tumor chemotherapeutics (cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and lopo), gemcitabine, and 5-fluorouracil.
6. The tumor microenvironment-responsive nanocomposite drug delivery system according to claim 1, wherein the molar ratio of the antitumor drug to curcumin is 1:2-10, and the antitumor drug and the block polymer The molar ratio is 1:3.
7. A method for preparing a tumor microenvironment-responsive nanocomposite drug-carrying system is characterized in that it comprises the following steps:

   (1) Preparation steps of coating solution a;

   (2) Adding anti-tumor drugs, curcumin and block polymers to an organic solvent to obtain an organic mixed phase solution b;

   (3) The step of mixing solution a and solution b, stirring and evaporating to obtain transparent liquid c;

   (4) The step of filtering the transparent liquid c with a filter membrane, and then ultrafiltration and centrifugation to obtain the nanocomposite drug-carrying system.
8. The method for preparing a tumor microenvironment-responsive nanocomposite drug delivery system according to claim 7, wherein the coating agent is selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, poloxamer and oleic acid At least one of.
9. The application of the tumor microenvironment-responsive nanocomposite drug-carrying system according to any one of claims 1 to 6 in reversing tumor multi-drug resistance.

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