CN111557909B

CN111557909B - PH-responsive polymer micelle with reversible change of form, preparation method and application

Info

Publication number: CN111557909B
Application number: CN202010448798.1A
Authority: CN
Inventors: 孙春萌; 涂家生; 孙梦娟
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2022-05-20
Anticipated expiration: 2040-05-25
Also published as: CN111557909A

Abstract

The invention discloses a pH-responsive polymer micelle with reversibly changed morphology, a preparation method and application thereof. The polymer micelle core expands due to the protonation of an amino acid sequence of the micelle core and the electrostatic repulsion of another polypeptide in a tumor microenvironment, but the micelle cannot disintegrate due to the crosslinking of the disulfide bond, and shrinks if the micelle leaves the tumor microenvironment, the release of a medicament is reduced, so that the damage of the medicament to normal cells is reduced; in addition, the application of dabigatran etexilate in the tumor direction provides a direction for the subsequent combination therapy of dabigatran etexilate with chemotherapeutic drugs.

Description

PH-responsive polymer micelle with reversible change of form, preparation method and application

Technical Field

The invention relates to a tumor targeting drug, in particular to a pH-responsive polymer micelle with reversible change of form, a preparation method and application thereof.

Background

In recent years, nanoparticles have great potential, wherein liposomes, polymer micelles and albumin nanoparticles are all provided with marketed drugs, due to the complexity of the tumor microenvironment, however, nanoparticles still have a huge barrier to delivery, such as abnormal vasculature, reduces the chance of nanoparticles entering the tumor area, dense extracellular matrix hinders the nanoparticles from entering the tumor tissue, while higher interstitial pressures tend to pump nanoparticles entering the tumor tissue out of the blood, these are barriers to nanoparticle delivery, and studies have shown that the size of the nanoparticles can affect the penetration and retention of the nanoparticles at the tumor site, in order to increase the delivery effect of nanoparticles, many researchers have designed a nanoparticle that responds to endogenous or exogenous factors, the particle size can change from large to small or from small to large, but the particle size change lacks reversible design. The polymer micelle is formed by self-assembly of amphiphilic block copolymers, usually two-block polymers or three-block polymers, and different shapes such as star, worm, capsule, petal and the like can be generated according to different block ratios and block designs of the polymers, so the morphological design of the polymer micelle has greater potential compared with other nanoparticles. Meanwhile, the polymer micelle can increase the solubility of insoluble drugs and has high encapsulation efficiency, but the polymer micelle has the biggest defects of relatively poor stability, easy drug leakage in advance and great toxic and side effects.

The tumor cells have stronger proliferation and differentiation capacity, consume a large amount of oxygen at tumor parts, ensure that the oxygen supply at the tumor parts is insufficient, generate a large amount of lactic acid, and the pH value (6.5-6.8) outside the cells is generally lower than the pH value (7.2-7.4) in normal tissues and blood. Therefore, in recent years, many researchers have studied pH-sensitive drugs for the difference between the pH value in the tumor region and that in the normal physiological environment, so that the drugs exert the greatest effect in the tumor region. However, many pH-sensitive drugs only consider that the drug is released after reaching the tumor region, but do not consider that the drug is slowly released in the tumor region, the drug resistance of the tumor may be caused due to the fact that the therapeutic concentration of the drug is not reached, and the drug which is not released in time returns to the normal physiological environment along with blood circulation, so that the drug has an effect of damaging normal cells.

Dabigatran etexilate is a competitive direct thrombin inhibitor and can be combined with a thrombin receptor on blood vessels, and thrombin is a central enzyme of a coagulation cascade, is a typical PAR-1 agonist and is a main effect protease of the coagulation cascade generated by the action of coagulation factors such as procoagulant factor TF and the like. Thrombin converts fibrinogen into fibrin which is deposited in the tumor vasculature, providing a scaffold for the formation of new blood vessels, promoting angiogenesis. Fibrin deposition provides a good microenvironment for the adhesion and growth of tumor cells, which is beneficial to the metastasis of tumors. In addition, thrombin is a potent activator of platelet adhesion, aggregation and secretion. Platelet activation not only contributes to the hypercoagulable state of cancer patients, but also promotes tumor growth, angiogenesis, and tumor metastasis. By reducing the chance of thrombin contacting thrombin receptors, angiogenesis is reduced. The dabigatran etexilate capsules currently used in the market are oral preparations, are only used as antithrombotic agents, belong to BCS II class medicines, and have low solubility and low bioavailability.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a polymer micelle with reversible change of form and a preparation method thereof, wherein the polymer micelle can improve the solubility of a medicament, simultaneously can cause burst release of the medicament at a tumor part, increases the local release concentration of the medicament and reduces the pH response of tumor medicament resistance; the invention also aims to provide the polymer micelle antitumor drug with high bioavailability.

The technical scheme is as follows: a pH-responsive polymer micelle with reversible change of form adopts disulfide bonds to form core crosslinking; the polymer micelle is formed by respectively connecting a hydrophobic structure of an amphiphilic segmented copolymer with a section of amino acid sequence, and the tail end of the amino acid sequence contains a disulfide bond.

Further, one segment of the amino acid sequence contains histidine and glutamic acid; the other section contains arginine and glycine;

further, the amino acid sequences are respectively (HE)_nCC and (RG)_nCC。

Further, the amino acid sequences are linked to the hydrophobic end of the block copolymer, respectively, wherein (HE)_nCC as a C sequence, (RG)_nCC is regarded as a D sequence, forming a structure of A-B-C and A-B-D and a mixed structure of A-B, A-B-C and A-B-D.

Further, the mass ratio of the mixed structure A-B to A-B-C to A-B-D is 0-10:0.8-1.2: 0.8-1.2.

A preparation method of a pH-responsive polymer micelle with reversible change of morphology comprises the following steps:

(1) synthesizing an amphiphilic block copolymer using a ring-opening polymer method;

(2) connecting an amino acid sequence at the hydrophobic end of the amphiphilic block copolymer;

(3) feeding the carriers of the amino acid sequences respectively connected in the step 2 according to the mass ratio of 1:1, adding a fat-soluble drug, dissolving the fat-soluble drug by using an organic reagent, then spin-drying the organic reagent, passing a 0.22 or 0.45 mu m water film after hydration, exposing the mixture in the air, and freeze-drying the mixture.

Further, in step 2, the amino acid sequence is covalently linked to the hydrophobic segment of the amphiphilic block copolymer.

Further, in step 3, the mass ratio of the total amount of the carriers of the respectively connected amino acid sequences to the fat-soluble drug is 1:1-20: 1.

Further, in step 3, the fat-soluble drug is dabigatran etexilate.

Use of a pH-responsive reversibly morphotropically changing polymeric micelle characterized by: the polymer micelle is applied to antitumor drugs.

Has the advantages that: compared with the prior art, the invention has the following remarkable effects:

1. the tail ends of the amphiphilic block copolymer are respectively connected with a section of amino acid sequence, two amino acids at the tail ends of the sequence are cysteine, and disulfide bonds are crosslinked when a core structure of the micelle is formed, so that the core structure of the micelle is more stable.

2. If the expanded polymer micelle is not taken up by tumor cells and the medicine is not completely released, the polymer micelle returns to a normal physiological environment along with blood, the hydrophobic part of the polymer micelle can generate electrostatic attraction, so that the whole polymer micelle is reduced, the particle size is reduced, the medicine is not easy to release, and the damage effect on normal cells is reduced.

3. The invention can not only increase the solubility of the medicine and improve the bioavailability of the medicine, but also apply the dabigatran etexilate to the direction of tumors, thereby providing a direction for the subsequent combined treatment of the dabigatran etexilate and the chemical medicine.

4. The invention is applied to a nano-targeting drug delivery system, the polymer micelle carrier has reversible form, the micelle particle size is reduced in normal physiological environment, the drug release is not carried out or is slow, the micelle expands and increases the particle size in a tumor microenvironment, the drug can be burst released in a tumor area, the release concentration of the drug in local part is improved, but the polymer micelle can be finally taken by cells, the glutathione concentration in the cells is higher, the micelle contains disulfide bonds and can carry out disulfide bond response, two drugs with synergistic action can be entrapped, one drug can respond outside the cells, and the other drug can respond in the cells, so that the drug effect is enhanced, and the side effect is reduced. In addition, the polymer micelle has smaller particle size when entering a tumor microenvironment, can penetrate into the tumor, then has larger particle size, and increases the detention of the polymer micelle at the tumor part.

Drawings

FIG. 1 is a nuclear magnetic map of mPEG-PLA in the present invention;

FIG. 2 is a nuclear magnetic map of mPEG-PLA-COOH in the present invention;

FIG. 3 is an infrared identification chart of mPEG-PLA-COOH in the invention;

FIG. 4 shows mPEG-PLA- (HE) according to the invention₆Nuclear magnetic map of CC;

FIG. 5 shows mPEG-PLA- (HE) according to the invention₆An infrared identification chart of CC;

FIG. 6 shows mPEG-PLA- (RG) in the present invention₆Nuclear magnetic map of CC;

FIG. 7 shows mPEG-PLA- (RG) in the present invention₆An infrared identification chart of CC;

FIG. 8 is a graph showing the comparison of the particle sizes of the polymer micelles of the present invention at different pH values;

FIG. 9 is a graph showing the comparison of the particle sizes of the polymer micelles of the present invention under different medium conditions;

FIG. 10 is a graph of in vitro release according to the present invention;

FIG. 11 is a drawing of a tube forming experiment in the present invention;

FIG. 12 is a semi-quantitative graph of the present invention;

FIG. 13 is a graph showing the cytotoxicity of MCF-7 in vectors and formulations of the invention;

FIG. 14 is a semi-quantitative graph of immunofluorescence in accordance with the present invention;

FIG. 15 is a schematic view of the structure of the present invention.

Detailed Description

Example 1

Synthesis of mPEG-PLA: adding mPEG2000(25g) and D, L-lactide (27g) into a 100ml dry two-necked bottle, vacuumizing, charging nitrogen, repeating for 3 times to ensure that the reaction system is anhydrous and anaerobic, placing the two-necked bottle in an oil bath, slowly heating to 135 ℃, adding a catalyst stannous octoate 0.2% (W/V) when the materials are completely dissolved and the temperature is reduced to 110 ℃, diluting the stannous octoate and toluene according to a certain proportion for quickly adding the catalyst, quickly adding a certain amount of catalyst under the condition of nitrogen flow, sealing the system, vacuumizing, charging nitrogen, repeating for three times, heating the reaction system to 145 ℃, and reacting for 6 hours. After the reaction is finished, cooling the two-necked bottle to room temperature, opening the sealing device, adding a proper amount of dichloromethane for dissolving, precipitating a product by using glacial ethyl ether, filtering, and repeating for 3 times to obtain the product. Putting the product into a dryer, adding phosphorus pentoxide into the dryer, vacuumizing, and drying for 24h to obtain the product. As shown in fig. 1, the obtained mPEG-PLA hydrogen nuclear magnetic spectrum (1H-NMR) showed that δ 3.3(a) and δ 3.6(b) correspond to the methyl and methylene proton peaks of mPEG, δ 5.1(c) and δ 1.5(d) correspond to the methine and methyl proton peaks of PLA, respectively, and δ 7.26 is deuterated chloroform (CDCl)₃) Indicating that the polymer mPEG-PLA was successfully synthesized.

Synthesis of mPEG-PLA-COOH: succinic anhydride (200mg,2mmol) was added to anhydrous CH₂Cl₂Dissolving mPEG-PLA (4000mg,1mmol) and DMAP (24.5mg,2mmol), stirring for 12h, pouring the solution into excess glacial ethyl ether, filtering, dissolving the filtered product in anhydrous CH₂Cl₂In the next step, the product is precipitated with glacial ethyl ether and repeatedAnd standing in a dryer overnight for three times to obtain the final product. As shown in figure 2-3, a small peak is included in the range of 12-13 ppm, the peak is a characteristic peak of carboxyl, which proves that mPEG-PLA-COOH has been synthesized, and figure 3 further proves that mPEG-PLA-COOH is at 3500cm^-1The peak at the hydroxyl group at the upper end of mPEG-PLA disappeared, confirming successful attachment of carboxyl group at the end of mPEG-PLA.

Synthesis of mPEG-PLA- (HE)₆CC and mPEG-PLA- (RG)₆CC: precisely weighed mPEG-PLA-COOH (50mg), EDC (2.18mg) and NHS (1.58mg), and added 2ml of DMF to completely dissolve the material in N₂Stirring to react for 4h under protection for activation, and then precisely weighing (HE)₆CC(23mg)/(RG)₆Dissolving CC (18.75 mg) in 1ml PBS (pH 7.4), slowly dripping the organic phase into the aqueous phase under the ice-bath condition, continuing the reaction for 24 hours in the ice-bath under the protection of N2, after the reaction is finished, putting the solution into acetate buffer solution with pH 5 for dialysis (the molecular weight of a dialysis bag is 3500), dialyzing for two days, and freeze-drying to obtain the compound. Preparation of mPEG-PLA- (HE) without disulfide bond in the same manner₆And mPEG-PLA- (RG)₆. Following lyophilization and nuclear magnetic characterization, mPEG-PLA- (HE) as shown in FIGS. 4-6₆CC and mPEG-PLA- (RG)₆CC Homological (HE)₆CC and (RG)₆The characteristic peaks of the CC reference sample are identical. As shown in FIGS. 5-7, infrared ray is used for mPEG-PLA- (HE)₆CC and mPEG-PLA- (RG)₆CC was further characterized and matched to the characteristic peaks of the control.

Preparation of polymer micelle: mixing mPEG-PLA- (HE)₆CC and mPEG-PLA- (RG)₆And (3) feeding the CC carrier according to the mass ratio of 1:1, wherein the medicine is dabigatran etexilate, the ratio of the total mass of the carrier to the medicine is 20:1, adding an organic reagent to dissolve the carrier and the medicine, carrying out spin drying and hydration on the organic reagent, passing through a 0.22 or 0.45 mu m water film, exposing in the air, and freeze-drying to obtain the pH-responsive reversibly-morphology-changeable polymer micelle, as shown in FIG. 15, which is a structural schematic diagram of the invention. The polymer micelle is a disulfide bond-containing polymer micelle.

Example 2

The mPEG-PLA, mPEG-PLA- (HE) prepared in example 1 was added₆CC and mPEG-PLA-, (RG)₆And (3) feeding a CC carrier according to the mass ratio of 10:0.8:1.2, wherein the medicine is dabigatran etexilate, the ratio of the total mass of the carrier to the medicine is 20:1, adding an organic reagent to dissolve the carrier and the medicine, spin-drying and hydrating the organic reagent, passing through a 0.22 or 0.45 mu m water film, exposing in the air, and freeze-drying to obtain the pH-responsive reversibly-morphology-changeable polymer micelle.

Comparative example 1

The same as in example 1, except that the vector used was mPEG-PLA- (HE)₆And mPEG-PLA- (RG)₆The prepared polymer micelle does not contain disulfide bonds.

Example 3

And (5) investigating the influence of different pH values on the particle size of the polymer micelle. Mixing mPEG-PLA- (HE)₆CC and mPEG-PLA- (RG)₆Weighing a proper amount of CC carrier according to the mass ratio of 1:1, respectively dissolving by using Hepes buffer solution with pH of 7.4, measuring the particle size, then adjusting the pH to 6.5 by using acetic acid, adjusting the pH to 7.4 by using ammonia water, finally adjusting the pH to 6.0 by using acetic acid, and measuring the particle size of the polymeric micelle under different pH conditions by using a particle size analyzer, wherein the particle size of the polymeric micelle is changed along with the change of the pH value as shown in figure 8.

Example 4

The stability of the different polymeric micelles was investigated. The disulfide bond-containing polymer micelle prepared in example 1 and the disulfide bond-free polymer micelle prepared in comparative example 1 were dissolved in a buffer solution of pH 7.4, and the particle sizes of the two polymer micelles were measured by dynamic light scattering, as shown in fig. 9, it was found that the particle sizes of the disulfide bond-containing polymer micelles were relatively small, then the pH values of the two polymer micelle solutions were adjusted to 6.5, and the particle sizes of the two polymer micelles were measured again by dynamic light scattering, respectively, and as a result, it was found that the particle sizes of the two polymer micelles were changed at different pH, but the disulfide bond-containing polymer micelles were changed slightly to about 170nm, while the disulfide bond-free polymer particles were large to about 500nm, and were almost dissociated to give poor stability, and the disulfide bonds were found to improve the stability of the polymer micelles.

Example 5

The release rate of the polymer micelle in different release media is examined. The polymer micelle prepared in example 4 was precisely weighed, dissolved with ultrapure water so that the concentration of the drug was 0.5mg/ml, 0.5ml was taken out into a dialysis bag, 0.5ml of PBS solution having pH values of 6.5 and 7.4 was added thereto, and the solution was repeated 3 times each, the dialysis bag was tightened, placed in a 15ml centrifuge tube, and 10ml of dissolution media, which were PBS/35% ethanol solution having pH values of 6.5 and 7.4, and 10mM reduced glutathione solution, were added thereto, respectively. Shaking at 37 deg.C and 100r/min, respectively taking 0.2ml dissolution medium at 1, 2, 4, 8, 12 and 24h, and timely supplementing with 0.2ml fresh medium. As shown in FIG. 10, it can be seen that in the media of pH 7.4 and 6.5, the drug release is faster at pH 6.5, and slower at pH 7.4, the micelle is shown to have a certain response to pH, while in the media of pH 7.4 and 6.5, when 10mM reduced glutathione solution is added, the release rates are closer, indicating that the disulfide bond plays a great role in maintaining the stable state of the polymer micelle.

Example 6

The effect of dabigatran etexilate in inhibiting angiogenesis was examined. Designing a group of experiments, freezing the gun head and the 96-well plate in a refrigerator at-20 ℃, taking matrigel from-20 ℃ to a refrigerator at-4 ℃ in advance, standing overnight to melt the matrigel, performing other operations on the ice, adding 50 mu l of matrigel into the 96-well plate, incubating for 1h in an incubator at 37 ℃, and adding HUVEC cells 2 x 10 into the wells containing the matrigel⁴And (3) setting a blank group, a thrombin group and a thrombin + thrombin inhibitor group in each cell/hole, repeating the steps for 3 multiple holes, incubating for 4 hours, and then photographing under an inverted fluorescence microscope. As shown in FIG. 11, C represents a blank group, T represents thrombin, D represents dabigatran etexilate, D1, D2 and D3 represent dabigatran etexilate at different concentrations. Therefore, the addition of dabigatran etexilate can reduce the formation of cell network structures and branches. Semi-quantitative analysis of the vessel branches formed in FIG. 11 and the total branch lengths was performed, as shown in FIG. 12, and it can be seen that the network structure formed by the cells and the branches decreased after the thrombin inhibitor was added, compared to the thrombin-only cell group, and the dose of dabigatran etexilate increasedAdditionally, cells formed even fewer networks and branches than the blank. Therefore, the dabigatran etexilate can inhibit the formation of blood vessels.

Example 7

The safety of the polymeric micelle carrier was investigated. One set of experiments was designed, MCF-7 cancer cells were seeded into 96-well plates at 5000 cells/well, and after 24h incubation, blank polymer micelles were diluted to different concentrations and added to 96-well plates for 24h incubation. Adding 20 mu L of tetramethyl azodicarbonamide blue (MTT, 5mg/mL) into each hole, continuously incubating for 4h, discarding liquid in the hole, adding DMSO according to the volume of 200 mu L/hole, shaking to fully dissolve crystals, measuring the light absorption value of each sample by using a microplate reader at 490nm wavelength, and measuring the OD value of a blank group. As shown in FIG. 13, it can be seen that the polymeric micelle carrier is not significantly toxic under the conditions of pH 6.0, 6.5 and 7.4, and the increase of the carrier concentration to 1mg/ml is also not significantly toxic. Therefore, the polymer micelle carrier has high safety.

Example 8

The pharmacodynamic effect of the polymer micelle is examined. A set of experiments was designed in which 4T1 cells were inoculated into Balb/c mice at the site of the fourth mammary gland pair, 6 x 10 cells each⁵One cell, observing the tumor formation of the mouse after ten days, and waiting until the tumor grows to 100mm³The experiment was started right and left, and mice were divided into 4 groups, which were injected with PBS and free drug, respectively. The mPEG-PLA-encapsulated drug and the mixed polymer micelle-encapsulated drug, the concentration of which is 50mg/kg, were injected twice a week for three consecutive weeks, after the administration, the drug was dissected, paraffin-embedded and sectioned, the antibody was stained with CD31, the antibody was observed under an inverted fluorescence microscope, and the result of semi-quantitative analysis using image J was shown in FIG. 14, from which it was seen that the therapeutic effect of the mixed polymer micelle was superior to that of the other groups.

Claims

1. A pH-responsive reversibly morphotropically changing polymeric micelle comprising: disulfide bonds are adopted to form core cross-linking, the polymer micelle is formed by respectively connecting a hydrophobic structure of an amphiphilic block copolymer with a segment of amino acid sequence, the tail end of the amino acid sequence contains disulfide bonds,the amino acid sequence (HE)_nCC and (RG)_nCC is attached to the hydrophobic end of the block copolymer, respectively, wherein (HE)_nCC as a C sequence, (RG)_nAnd the CC is regarded as a D sequence to form a structure of A-B-C and A-B-D and a mixed structure of A-B, A-B-C and A-B-D, wherein the mass ratio of the mixed structure A-B to the A-B-C to the A-B-D is 0-10:0.8-1.2: 0.8-1.2.

2. The pH-responsive, reversibly morphologically changing polymeric micelle of claim 1, wherein: the amino acid sequence has pH response amino acid.

3. A method for preparing the pH-responsive reversibly changeable morphology polymeric micelle of claim 1, characterized in that: the method comprises the following steps:

(2) respectively connecting the amino acid sequences to the hydrophobic ends of the amphiphilic block copolymer;

(3) feeding the carriers of the amino acid sequences respectively connected in the step 2 according to the mass ratio of 1:1, adding the fat-soluble drugs according to the proportion, dissolving the fat-soluble drugs by using an organic reagent, then spin-drying the organic reagent, passing through a 0.22 or 0.45 mu m water film after hydration, exposing the organic reagent in the air, and freeze-drying the organic reagent.

4. The method for preparing a pH-responsive reversibly changeable polymer micelle according to claim 3, wherein: in the step 3, the mass ratio of the total amount of the carriers of the respectively connected amino acid sequences to the fat-soluble medicine is 1:1-20: 1.

5. The method for preparing a pH-responsive reversibly changeable polymer micelle according to claim 3 or 4, wherein: in step 3, the fat-soluble drug is dabigatran etexilate.

6. Use of the pH-responsive reversibly morphable polymeric micelle of claim 1 in the preparation of a medicament, wherein: the polymer micelle is applied to the preparation of antitumor drugs.