A stimulus-responsive dual-drug co-delivery micelle and its preparation method and application

Abstract

The present invention relates to a stimulus-responsive dual-drug co-delivery micelle and a preparation method and application thereof, belonging to the field of polymer chemistry and pharmaceutical preparation. The present invention uses doxorubicin (DOX) directly as a hydrophobic block, and bonds with the hydrophilic block mPEG through a disulfide bond that can respond to a high concentration of glutathione in the tumor microenvironment to form an mPEG-SS-DOX micelle material, and uses molecular dynamics simulation screening to obtain the Flory-Huggins interaction parameter between curcumin (Cur) and the hydrophobic end of mPEG-SS-DOX to be the smallest. The self-assembly process of drug-loaded micelles and the effect of drug loading on the morphology and structure of micelles are studied by dissipative particle dynamics simulation. The mPEG-SS-DOX / Cur micelles with redox responsiveness are successfully prepared, and the micelle particle size is small, the surface is negatively charged, and the drug loading and encapsulation efficiency are good.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations and polymer chemistry, and specifically relates to a stimulus-responsive dual-drug co-delivery micelle and a preparation method thereof. Background Art

[0002] In order to solve the problem that a single chemotherapy drug cannot meet clinical expectations, combination therapy of different drugs is widely used in clinical practice to improve the treatment effect. Compared with single drug therapy, multi-drug combination therapy for malignant tumors has significant advantages: multiple drugs can treat malignant tumors through the same or different anti-tumor mechanisms, and the same or better effects can be achieved through lower doses of drugs; the drug combination in multi-drug combination therapy can alleviate or neutralize the adverse reactions caused by single drug therapy, and can overcome treatment-related multidrug resistance through various mechanisms.

[0003] As a widely studied nano drug delivery system, polymer micelles can be loaded with multiple drugs at the same time to enhance the chemotherapy effect, which is crucial in the multi-drug combination treatment of malignant tumors. Hydrophobic drugs can be dissolved and stabilized in the hydrophobic core of micelles, improving drug stability. In addition, the hydrophilic shell can prolong the circulation time and improve the steric stability by reducing the conditioning effect in the blood circulation. At the same time, due to the enhanced permeability and retention effect (EPR), nano-sized micelles can selectively accumulate at the tumor site and improve the anti-tumor effect.

[0004] Stimuli-responsive co-delivery micelles can release drugs in response to endogenous stimuli and promote drug enrichment and release in tumor tissues. A large number of research results show that tumors induce the formation of abnormal blood vessels, resulting in a severe hypoxic state in the tumor microenvironment (TME), and tumor cells use anaerobic glycolysis as the main energy supply mode. However, lactic acid accumulated through anaerobic glycolysis produces an acidic pH value in the TME. The concentration of glutathione (GSH) in the TME is 4 times that of the normal tissue microenvironment, and the concentration of GSH in tumor cells is 10 to 1000 times that of the normal tissue microenvironment. Therefore, the redox reaction strategy is applied to the field of co-delivery micelles to achieve responsive release of co-loaded drugs. The sensitive disulfide bond of GSH can be broken under a certain concentration of GSH. The hydrophilic and hydrophobic parts are connected into amphiphilic polymers using disulfide bonds, which eventually lead to the decomposition of the co-delivery micelles and the release of drugs at designated locations. The micelle structure containing redox-sensitive fragments is stable in the blood circulation. Some studies have shown that GSH-responsive co-delivery micelles were used to encapsulate DOX and gene drugs (pTRAIL), in which disulfide bonds were used as reaction units. When the co-delivery micelles were delivered to the TME, the reduction of disulfide bonds ultimately led to the release of DOX and pTRAIL. Anti-tumor experiments showed that its tumor inhibition rate (94.0%) was significantly higher than that of other control groups.

[0005] Molecular dynamics (MD) simulation methods are powerful means to determine the properties of components based on chemical structure, and they can be used, for example, to evaluate the compatibility of drugs with excipients in formulations. Based on the principle of "like dissolves like", various methods have been used to study the compatibility of compounds. The Flory-Huggins interaction parameter has been shown to be a good indicator of the compatibility of drugs with micellar materials and can be calculated by MD simulation. Some researchers used MD simulation methods to calculate the Flory-Huggins interaction parameter to characterize the interaction between the amphiphilic copolymer excipient PEG-hexPLA and four different lipophilic candidate drugs. The Flory-Huggins interaction parameters obtained by MD simulation were compared with experimental data. The theoretical and experimental results showed the same trend, verifying the feasibility of MD simulation.

[0006] Dissipative particle dynamics (DPD) simulation can be used to study the self-assembly of amphiphilic polymers into nanomicelles of macroscopic systems from a mesoscopic perspective. DPD simulation can study the co-micelle process of different polymers, the structural analysis of mixed polymer micelles at the microscopic and mesoscopic scales, the factors affecting the self-assembly structure, and the mechanism of controlled drug release, providing a powerful supplementary tool for experimental results. Some researchers used coarse-grained DPD simulation to study the morphology, drug encapsulation and release of polyethylene glycol-polylactic acid-polyethylene glycol (PEG-PLA-PEG) amphiphilic block copolymer systems, and observed that the degree of drug encapsulation and release was mainly affected by the concentration of the copolymer in the mixture, and the average aggregation number and average micelle volume increased with the encapsulation of the drug in the micelles. The shape of the micelles was mainly spherical.

[0007] Doxorubicin (DOX) is currently the most active monotherapy drug, which inhibits nucleic acid synthesis by intercalating into DNA to induce cell apoptosis. Due to its broad-spectrum anti-tumor activity, it is widely used to treat a variety of malignant tumors such as breast, ovarian, prostate, brain, lung, leukemia, etc. However, DOX has severe cardiotoxicity, a narrow therapeutic window, and the occurrence of multidrug resistance, which seriously limits its clinical application. Summary of the invention

[0008] The present invention adopts MD simulation and DPD simulation strategies to screen, design and synthesize stimulus-responsive dual-drug co-delivery micelles, and predict and verify the self-assembly process and drug loading performance of the designed stimulus-responsive dual-drug co-delivery micelles. The redox-responsive DOX and Cur dual-drug co-delivery micelles constructed by the present invention have good physicochemical properties and anti-tumor effects.

[0009] The technical solution adopted by the present invention is: a stimulus-responsive dual-drug co-delivery micelle, which uses mPEG as a hydrophilic block, a disulfide bond as a redox-sensitive fragment, and doxorubicin DOX as a hydrophobic block to construct a polymer-drug linker mPEG-SS-DOX as a micelle material, and encapsulates drug X to form a stimulus-responsive dual-drug co-delivery micelle mPEG-SS-DOX / X.

[0010] Furthermore, in the above-mentioned stimulus-responsive dual-drug co-delivery micelle, the drug X includes paclitaxel (PTX), curcumin (Cur) and a curcuminol derivative (CD-2e).

[0011] Furthermore, in the above-mentioned stimulus-responsive dual-drug co-delivery micelle, the mass ratio of mPEG-SS-DOX:drug X is (6:1) to (3:1).

[0012] Further, in the above-mentioned stimulus-responsive dual-drug co-delivery micelle, the mPEG is polyethylene glycol monomethyl ether mPEG 2000 .

[0013] Furthermore, in the above-mentioned stimulus-responsive dual-drug co-delivery micelle, the polymer-drug linker mPEG-SS-DOX has the structural formula shown in (I):

[0014]

[0015] A method for preparing a stimulus-responsive dual-drug co-delivery micelle comprises the following steps:

[0016] 1) 3,3'-dithiodipropionic acid (DTDP) and acetyl chloride were reacted under reflux at 65°C for 2 h with N2 protection to prepare 3,3'-dithiodipropionic anhydride (DTDPA);

[0017] 2) 3,3'-dithiodipropionic anhydride (DTDPA) and mPEG were dissolved in anhydrous dimethylformamide (DMF), 4-dimethylaminopyridine (DMAP) and triethanolamine (TEA) were added under stirring, and the resulting mixed solution was reacted at 35°C with N2 protection for 24 hours to prepare mPEG-SS-COOH;

[0018] 3) mPEG-SS-COOH dissolved in dimethyl sulfoxide (DMSO) was activated by N-hydroxysuccinimide (NHS) and |N|,|N|-dicyclohexylcarbodiimide (DCC) under stirring. After stirring for 12 hours, the mPEG-SS-COOH solution was slowly added to the deprotonated doxorubicin (DOX) solution, and 4-dimethylaminopyridine (DMAP) was added to catalyze the reaction. The solution was evacuated and stirred in the dark under N2 protection for 24 hours. The solution was placed in a dialysis bag, first dialyzed in DMSO to remove free DOX, then dialyzed in deionized water to remove the organic solvent DMSO, and freeze-dried to obtain mPEG-SS-DOX;

[0019] 4) mPEG-SS-DOX and drug X were dissolved in dimethyl sulfoxide (DMSO), and the resulting mixed solution was slowly added dropwise to deionized water. After stirring for 12 h in the dark, the mixed solution was transferred to a dialysis bag, and DMSO was removed by dialysis. The dialysate was replaced regularly. After the dialysis was completed, the solution was filtered through a 0.45 μm microporous filter membrane to obtain the stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / X.

[0020] Furthermore, in the above-mentioned preparation method, the deprotonated doxorubicin DOX is prepared by dissolving DOX·HCl in DMSO, adding triethanolamine dropwise, and obtaining the deprotonated DOX under continuous stirring.

[0021] Furthermore, in the above-mentioned preparation method, the dialysis bag has a molecular weight of 2000Da.

[0022] The present invention provides a stimulus-responsive dual-drug co-delivery micelle for use in preparing chemotherapeutic drugs.

[0023] The beneficial effects of the present invention are:

[0024] 1. The stimulus-responsive dual-drug co-delivery micelle mPEG-SS-DOX / Cur of the present invention realizes dual-drug co-delivery, has a good drug loading capacity, can greatly improve the drug loading efficiency, and utilizes the different anti-tumor mechanisms of the two drugs to achieve a more effective tumor killing effect.

[0025] 2. The stimulus-responsive dual-drug co-delivery micelle mPEG-SS-DOX / Cur of the present invention can achieve responsive release under the redox conditions of the tumor environment, which helps the accumulation of drugs in the tumor part. Under normal conditions, DOX will not be easily hydrolyzed and broken for release. The present invention increases the in vivo stability of DOX, achieves a sustained release effect, reduces the cardiotoxicity of the anti-tumor drug DOX, and has good safety.

[0026] 3. In the prior art, DOX is usually used in combination with other drugs. Cur inhibits cell proliferation by inhibiting the activity of human epidermal growth factor receptor-2 (HER 2) and the activation of nuclear factor κB (NF-kB), which is different from the anti-tumor mechanism of DOX. The present invention uses polymer micelles to co-deliver DOX and Cur, which reduces toxic side effects, increases accumulation in tumor sites, and ultimately improves anti-tumor effects.

[0027] 4. The mPEG-SS-DOX / Cur provided by the present invention can responsively release drugs under redox conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 3D molecular models of DOX, PTX, Cur and CD-2e.

[0029] Figure 2 This is the periodic system diagram of DOX, PTX, Cur and CD-2e.

[0030] Figure 3 3D molecular models of DOX, PTX, Cur and CD-2e after geometry optimization.

[0031] Figure 4 This is the periodic system diagram of DOX, PTX, Cur and CD-2e after DPD simulation.

[0032] Figure 5 Bead partitioning diagram for various molecules in DPD simulation.

[0033] Figure 6 Schematic diagram of the self-assembly of mPEG-SS-DOX / Cur micelles.

[0034] Figure 7 Schematic diagram of the simulated equilibrium state of mPEG-SS-DOX / Cur micelles at different drug to micelle material feed ratios.

[0035] Figure 8 This is the synthetic route of mPEG-SS-DOX.

[0036] Fig. 9 H NMR spectra of DTDPA (A), DTDP (B), mPEG (C), mPEG-SS-COOH (D) and mPEG--SS-DOX (E).

[0037] Fig.10 Mass spectra of mPEG (A) and mPEG-SS-DOX (B).

[0038] Fig.11 This is the particle size diagram of mPEG-SS-DOX / Cur.

[0039] Fig.12 This is the potential diagram of mPEG-SS-DOX / Cur.

[0040] Fig.13 This is the transmission electron microscopy image of mPEG-SS-DOX / Cur.

[0041] Fig.14 This is a diagram of the stability study of mPEG-SS-DOX / Cur.

[0042] Fig.15 This is the result of the hemolysis experiment of mPEG-SS-DOX / Cur.

[0043] Fig.16 This is the in vitro release curve of mPEG-SS-DOX / Cur under different conditions.

[0044] Fig.17 This is a comparison of the inhibitory effects of mPEG-SS-DOX, mPEG-SS-DOX / Cur, DOX and Cur on the proliferation of HepG2 and S180 cells (statistical differences are represented by *P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below by specific implementation examples. Those skilled in the art should understand that the implementation examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0046] Example 1 A stimulus-responsive dual-drug co-delivery micelle mPEG-SS-DOX / X

[0047] The polymer-drug linker mPEG-SS-DOX was constructed with mPEG as the hydrophilic block, disulfide bonds as the redox-sensitive fragments, and doxorubicin DOX as the hydrophobic block.

[0048] The polymer-drug linker mPEG-SS-DOX was used as the micelle material, and the MD simulation method was used to select drugs with good compatibility with the hydrophobic end. The self-assembly process of the drug-loaded micelles and the effect of drug loading on the morphology and structure of the micelles were studied through DPD simulation. The stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / X were prepared by nanoprecipitation and dialysis methods.

[0049] (I) Using molecular dynamics simulation to screen for drugs with the best compatibility with the hydrophobic end

[0050] Here are the steps:

[0051] 1. First, the 3D molecular model of the hydrophobic end DOX, PTX, Cur and CD-2e of the micelle material (mPEG-SS-DOX) was drawn using the Materials Visualizer module in the Materials Studio software. The results are as follows: Figure 1 .

[0052] 2. Use the Forcitce module in Materials Studio software to perform geometry optimization on the 3D molecular models of the hydrophobic end DOX, PTX, Cur and CD-2e of the micelle material (mPEG-SS-DOX). Select Calculation, select GeometryOptimization for the task, and select Fine for precision. Select Smart for the algorithm, and set the energy convergence tolerance and force convergence tolerance to 1.0×10 -4 kcal / mol and 0.005 The maximum number of iterations was set to 10000, COMPASSII was selected for the force field, and Atom based was selected for the summation method of van der Waals forces and electrostatic energy. After the parameters were set, click Run. After running for a while, the geometry optimization of the four 3D molecular models was completed. The energy changes before and after the geometry optimization are shown in Table 1. The optimization results are shown in Figure 3 .

[0053] Table 1 Total energy of DOX, PTX, Cur, and CD-2e before and after geometry optimization

[0054]

[0055] 3. Use the Amorphous Cell module in Materials Studio to build a periodic system of DOX, PTX, Cur, and CD-2e. Select Calculation in the Amorphous Cell module, add 55 3D molecular models to each periodic system, select Construction as the task, and select Fine as the precision. The results are as follows: Figure 2 .

[0056] 4. Anneal the four periodic systems that have completed geometry optimization. The starting temperature is 298K, the maximum temperature is set to 598K, the number of cycles is set to 5, the temperature gradient in each cycle is 6, and the kinetic steps of each temperature gradient are 10,000. Select the NPT ensemble and the pressure is 1.0×e -4GPa, the simulation time is set to 1fs, the temperature is controlled by the Andersen method, and the pressure is controlled by the Berendsen method. COMPASS II force field is used to calculate the potential energy of the system, while the van der Waals energy calculation method uses the atom-based method, and the electrostatic energy calculation method is the Ewald method. After annealing, dynamics is performed, and the entire system selects the NPT ensemble, the temperature is set to 298K, and the pressure is 1.0e -4 GPa, the total energy before and after the simulation and the system density after the dynamic simulation are shown in Table 2, and the simulation results are shown in Figure 4 .

[0057] Table 2 Total energy before and after simulation and system density after simulation for DOX, PTX, Cur, and CD-2e

[0058]

[0059] 5. The periodic system of DOX, PTX, Cur, and CD-2e obtained a reasonable structural configuration after three processes of geometry optimization, annealing, and kinetics. The cohesive energy density was calculated using the Forcite module in the Materials Studio software. The Cohesive Energy Density task was selected in the task bar. After the calculation, the solubility parameter (δ) was obtained. The Flory-Huggins interaction parameter was calculated according to the following formula:

[0060]

[0061] Among them, χ 药物-疏水端 is the Flory-Huggin interaction parameter between the drug and the hydrophobic end; δ 药物 and δ 疏水端 is the solubility parameter of the encapsulated drug and the hydrophobic end of the micelle material; V 药物 is the average molar volume of the drug, R is the ideal gas constant, and T is the temperature.

[0062] The results of the Flory-Huggins interaction parameters between DOX and PTX, Cur, and CD-2e are shown in Table 3. The Flory-Huggins interaction parameter between DOX and Cur is the smallest, indicating that Cur has the best compatibility with the micelle material mPEG-SS-DOX, so mPEG-SS-DOX is used to encapsulate Cur.

[0063] Table 3 Flory-Huggins interaction parameters between DOX and PTX, Cur, and CD-2e

[0064]

[0065] (II) DPD simulation study on the self-assembly process of micellar material mPEG-SS-DOX and encapsulated drugs to form micelles and the influence of encapsulated drug content on the morphology and structure of self-assembled micelles

[0066] Here are the steps:

[0067] 1. DPD simulation is used for coarse-grained simulation. The molecular structure is divided. Several atoms or a functional group are represented as DPD beads, which are highlighted with colors. The polyethylene glycol monomer is divided into P beads, which are blue in color; DOX is divided into three D1 beads, one D2 and one D3 beads, which are all represented by red in color; Cur is divided into two C beads, which are yellow in color; and four water molecules are divided into W beads, which are gray in color. The results are shown in Figure 1. Figure 5 .

[0068] 2. Before using DPD to simulate the self-assembly process of micelle material mPEG-SS-DOX and drug-loaded Cur, it is necessary to calculate the interaction parameters (a ij )

[0069] The interaction parameter a between different types of beads ij The following relationship applies:

[0070] a ij ≈a ii +3.27χ ij

[0071] In this relationship, χ ij is the Flory-Huggins parameter, a ii is the interaction of beads of the same type, which follows the relationship:

[0072]

[0073] Where K B T = 1, at room temperature, dimensionless compressibility k -1 =15.9835. N m is the number of water molecules that make up the coarse water droplets, α = 0.101-0.001; ρ DPD is the number density of the system, so the exclusion parameter a ii =25.

[0074] Here, the Flory-Huggins parameter χ ij It can be calculated according to the following equation:

[0075]

[0076] where δ i and δ jis the solubility parameter of beads i and j, V bead is the average molar volume of all beads, and the gas constant R ≈ 8.314.

[0077] The Flory-Huggins interaction parameters between the beads in the system were calculated by molecular dynamics simulation, and the results are shown in Table 4.

[0078] Table 4 DPD interaction parameters α between beads ij

[0079]

[0080] 3. Use the Mesocite module to perform dissipative particle dynamics simulation. First, establish a periodic condition: 15% mPEG-SS-DOX, 5% Cur and 80% water were added to the periodic box. The temperature was set to 298K, so that the thermal energy K B T = 1, bead density ρ is set to 3, dissipation force parameter γ is set to 4.5, elastic constant C is set to 4, cutoff radius r is set to 4. c Set to 1. These conditions ensure that the simulation can be carried out reasonably and efficiently. The number of simulation steps is set to 300,000 steps, and the integration step size Δt = 0.05 to achieve thermodynamic equilibrium. The results are shown in Figure 6 .

[0081] 4. The DPD simulation method was used to study the effect of the content of the drug-loaded Cur component on the micelle self-assembly structure and equilibrium morphology. The results are shown in Figure 7 , the amount of micelle material mPEG-SS-DOX in the periodic box is set to 15%; the feeding ratio of micelle material mPEG--SS-DOX to encapsulated drug Cur is set to 6:1, 3:1, 2:1, 1:1 respectively; the amount of water beads changes with the amount of encapsulated drug Cur, and the simulation experimental parameters and conditions are consistent with the above. When the feeding ratio is 6:1 and 3:1, the micelle material mPEG-SS-DOX and encapsulated drug Cur in the system self-assemble to form a stable spherical structure. The cross-sectional view shows that the encapsulated drug Cur is stably encapsulated in the core of the spherical micelle, DOX is the middle layer, and mPEG is the outer shell. When the feed ratio is 2:1, the amount of drug Cur encapsulated in the core of the micelle increases, and the mPEG layer of the outer layer of the micelle becomes thinner, so that part of DOX is exposed to the surface of the micelle. When the encapsulated drug Cur is further added to the simulation system, when the feed ratio is 1:1, the core of the micelle is larger, and the micelle material cannot completely cover the surface of the sphere, so that part of the drug appears on the surface of the spherical micelle. It can be seen that the best feed ratio of the micelle material mPEG-SS-DOX and the encapsulated drug Cur when the micelle system is loaded with drugs is 6:1 to 3:1, and the micelles formed can be stable.

[0082] (III) Synthesis of polymer-drug conjugate (mPEG-SS-DOX)

[0083] Synthesis route such as Figure 8 , the steps are as follows:

[0084] 1. Synthesis of DTDPA

[0085] 1.0 g DTDP and 5 mL acetyl chloride were added to a 50 mL round-bottom flask, the mixed solution was maintained at 65 ° C and refluxed under N2 protection. After 2 h of reaction, acetyl chloride was removed by rotary evaporation, and the product was precipitated by adding glacial ether to the mixed solution. After filtering, vacuum drying for 24 h, DTDPA was obtained.

[0086] 2. Synthesis of mPEG-SS-COOH

[0087] mPEG-SS-COOH was synthesized using mPEG and DTDPA as raw materials. DTDPA (192 mg, 1.0 mmol) and mPEG (1000 mg, 0.5 mmol) were dissolved in 3.0 mL of anhydrous DMF in a round-bottom flask. Then, DMAP (122 mg, 1.0 mmol) and TEA (140 μL, 1.0 mmol) were added to the solution under stirring. The mixed solution was maintained at 35 °C and the reaction was protected with N2. After 24 h of reaction, ice ether was added to the mixed solution to precipitate mPEG-SS-COOH. The ether was removed by centrifugation to obtain a white precipitate, which was freeze-dried and stored at -20 °C.

[0088] 3. Synthesis of mPEG-SS-DOX

[0089] DOX·HCl (116 mg, 0.2 mmol) was dissolved in 2 mL DMSO, and 84 μL TEA was added dropwise to obtain deprotonated DOX under continuous stirring.

[0090] mPEG-SS-COOH (438 mg, 0.2 mmol) dissolved in 3 mL DMSO was activated by NHS (184 mg, 1.6 mmol) and DCC (82 mg, 0.4 mmol) under stirring. After stirring for 12 h, the mPEG-SS-COOH solution was filtered and the filtrate was slowly added to the deprotonated DOX solution, and DMAP (49 mg, 0.4 mmol) was added to the solution to catalyze the reaction, evacuate, and stir in the dark under N2 protection. After stirring for 24 h, the solution was placed in a dialysis bag (MWCO: 2000 Da) and first dialyzed in DMSO to remove free DOX. The solution was then dialyzed in deionized water to remove the organic solvent DMSO. mPEG-SS-DOX was obtained by freeze drying.

[0091] H NMR spectrum of mPEG-SS-DOX ( 1 H-NMR) results are as follows Fig. 9 Shown: From 1 H-NMR spectrum shows that Fig. 9 As shown in Figure B, the peak (c) of hydrogen in the carboxyl group of DTDP is at about δ = 12 ppm. Fig. 9 As shown in Figure A, the peak of hydrogen in the carboxyl group at about δ = 12 ppm disappears, proving that DTDP is successfully dehydrated to form anhydride DTDPA. The methylene peak (d) and methyl peak (e) of mPEG appear at about δ = 3.60 ppm and δ = 3.25 ppm, respectively. Fig. 9 Middle C and Fig. 9 It can be seen in D. Fig. 9 In D, the methylene peaks of DTDP (a, b) appear at δ = 2.95 ppm and δ = 2.55 ppm. The peak at δ = 4.25 ppm (f) is the ester bond peak of the reaction product of DTDPA and mPEG, which proves that mPEG and DTDPA successfully react to form mPEG-SS-COOH. Fig. 9 E is mPEG-SS-DOX 1 H-NMR spectrum, Fig. 9 In Figure E, characteristic peaks of doxorubicin (j, k) and mPEG-SS-COOH (d, f) with a δ value of 4.0-8.0 ppm appeared, indicating that DOX was successfully bonded to mPEG via disulfide bonds.

[0092] The mass spectrometry results of mPEG-PLGA-SS-DOX are as follows Fig.10 As shown: the molecular weight of mPEG is about 2000, and the molecular weight of mPEG-SS-DOX is about 2700, indicating that DOX is successfully bonded to mPEG.

[0093] (IV) Preparation of stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / Cur

[0094] Here are the steps:

[0095] Weigh 15 mg mPEG-SS-DOX and 3 mg Cur and fully dissolve them in 2.0 mL DMSO. Slowly drop the resulting mixed solution into 5.0 mL deionized water, stir for 12 h in the dark, transfer to a dialysis bag (MWCO: 2000 Da), and dialyze in deionized water to remove DMSO. Replace the dialysate every 2, 4, 6, 8, 10, and 12 h. After dialysis, filter through a 0.45 μm microporous membrane and adjust the volume to 10 mL to obtain mPEG-SS-DOX / Cur micelles.

[0096] (V) Determination of particle size and potential of stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / Cur

[0097] An appropriate amount of mPEG-SS-DOX / Cur micelles were absorbed into a particle size dish and a potential dish, and the particle size, potential and PDI of the mPEG-SS-DOX / Cur micelles were determined by dynamic light scattering (DLS) method. The test temperature was set at 25°C. The particle size and potential of mPEG-SS-DOX / Cur were as follows: Fig.11 , Fig.12 As shown, the particle size of the polymer prodrug micelles is 142.7±2.8nm, and the particle size is less than 200nm, so it is suitable for maintaining a low level of reticuloendothelial uptake and minimal renal excretion, indicating that it can promote drug enrichment in tumor tissue through the EPR effect as an intelligent carrier and enhance the tumor treatment effect; the potential is -10.9±0.85mV, and the surface is negatively charged, which can prevent protein absorption in the blood circulation, minimize nonspecific interactions with the reticuloendothelial system, and prolong the in vivo circulation time of the micelles.

[0098] (VI) Morphological study of stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / Cur

[0099] The mPEG-SS-DOX / Cur micelles were dropped onto a clean copper grid and placed in a ventilated place to dry. After drying, the size and morphology of the micelles were observed using a transmission electron microscope (TEM). Fig.13 As shown, the mPEG-SS-DOX / Cur micelles are regular spheres without aggregation, and the particle size is about 130 nm, which is basically consistent with the particle size measurement results.

[0100] (VII) Stability study of stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / Cur

[0101] The stability of the micelle solution was evaluated using particle size and PDI as evaluation indicators. The micelle solution was stored at 4°C, and samples were taken on days 0, 2, 4, 6, 8, and 10 to measure the particle size and PDI of the micelles using the dynamic light scattering (DLS) method. Fig.14 As shown, the average particle size and PDI have little change over time, and the average particle size is stable within 200 nm and always remains in the effective particle size range, indicating that the micelles have good stability when stored at 4°C.

[0102] (VIII) Study on the hemolytic activity of the stimulus-responsive dual-drug co-delivery micelle mPEG-SS-DOX / Cur

[0103] An appropriate amount of blood was collected from the rat's eye sockets, placed in an EDTA anticoagulant tube, centrifuged (1000r / min, 10min), the upper serum was discarded, and then an appropriate amount of PBS buffer was added, centrifuged again (1000r / min, 10min), the upper solution was discarded, and the operation was repeated several times until the upper solution was clear and transparent. After washing, 200μL of the lower precipitated red blood cells were taken and added to 10mL PBS buffer to obtain a 2% red blood cell suspension. In the experimental group, 500μL of 2% red blood cell suspension, 1.5mL of different concentrations of free drug (DOX+Cur) solution and mPEG-SS-DOX / Cur micelle solution were added to the centrifuge tube. In the negative control group, 500μL of 2% red blood cell suspension and 1.5mL PBS buffer were added to the centrifuge tube. In the positive control group, 500μL of 2% red blood cell suspension and 1.5mL deionized water were added to the centrifuge tube. All centrifuge tubes were placed in a 37°C water bath for 2 hours, then centrifuged (1000 r / min, 10 minutes), hemolysis was observed, and photos were taken to record the experimental phenomenon. The absorbance value (A) of the upper layer solution after centrifugation at 545 nm was measured by UV-visible spectrometer, and the hemolysis rate was calculated according to the following formula:

[0104]

[0105] Hemolytic study results Fig.15 As shown in the data, mPEG-SS-DOX / Cur micelles can significantly improve the hemolysis of free drugs. The hemolysis rates of mPEG-SS-DOX / Cur micelles with different drug concentrations are all less than 4%, and hemolysis will not occur, which proves that mPEG-SS-DOX / Cur micelles have good biocompatibility.

[0106] (IX) In vitro release experiment of stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / Cur

[0107] The redox-responsive release of micelles was studied by dialysis. First, different release media were prepared: PBS buffer solution without GSH and PBS buffer solution with a GSH concentration of 10mM, and 1.0% Tween-80 was added respectively. Then, an equal amount of mPEG-SS-DOX / Cur micelle solution was added to a dialysis bag of the same length (MWCO: 2000Da), and then placed in a centrifuge tube with 20mL of release medium added, and placed in a constant temperature oscillator for shaking, with the temperature set to 37°C and the speed set to 100rpm. After incubation for 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72h, 1mL of release medium was drawn, and an equal amount of release medium under the same conditions was immediately added.

[0108] The results are as follows Fig.16 As shown in the figure, from the release curves of DOX in micelles with or without GSH, it can be seen that GSH concentration has a significant effect on the cumulative release amount and release speed of the drug. In the absence of GSH, the release of DOX is slow, and the cumulative release within 72h is less than 20%. Because the DOX molecules are chemically connected to the mPEG chain, and mPEG-SS-DOX is stable under the condition of no GSH. Under the condition of 10mM GSH, the release rate is rapidly accelerated. The redox-sensitive bond between the DOX molecule and the polymer is broken, resulting in a cumulative release of about 23% and more than 72% at 2h and 72h, respectively. The key reason may be that the redox-sensitive bond between DOX and mPEG is broken, the micelles are decomposed, and the free DOX molecules are easily diffused into the solution. From the release curves of Cur in micelles with or without GSH, it can be seen that although the release curve of Cur depends on GSH, the release rate of Cur is faster than that of DOX under the condition of no GSH, and the cumulative release of Cur at 2h and 72h is greater than 10% and 30%, respectively. The reason may be that Cur is physically loaded into the micelle core through hydrophobic interaction during the self-assembly process. Due to the swelling of the micelles, some Cur molecules may be released and diffuse into the solution during incubation without GSH. Under 10mM GSH conditions, the release is faster, and the cumulative release of Cur within 2h and 72h under 10mM GSH conditions is 28% and 87%, respectively. The above situation is due to the cleavage of the redox-sensitive bond between DOX and mPEG, which leads to the swelling and disintegration of the micelles and the release of the encapsulated drugs. In general, the release rate of DOX and Cur increases with the increase of GSH. Therefore, the release of drugs (DOX and Cur) from micelles is triggered by GSH.

[0109] (X) Cytotoxicity study of stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / Cur

[0110] The cytotoxicity of mPEG-SS-DOX / Cur was determined by MTT assay. HepG2 and S180 cells grown to logarithmic phase were cultured at a density of 1×10 4 cells / mL cell suspension, and then inoculated into a 96-well plate (100 μL / well) and cultured for 24 h to allow the cells to adhere to the wall and grow. Then 100 μL of DOX, Cur, mPEG-SS-DOX micelles and mPEG-SS-DOX / Cur micelle solutions were added to each well. The concentrations of DOX were 0.1, 1, 5, 10, and 20 μg mL -1 The concentrations of Cur were 0.07, 0.7, 3.5, 7, and 14 μg mL -1 , set up 3 parallels. After adding drugs, culture for 48h. Then add 20μL MTT solution to each well and culture for 4h. Then take out the 96-well plate and discard the upper culture medium and MTT solution, add 150μL DMSO to each well, measure the absorbance of each well at 570nm, and calculate the inhibition rate.

[0111] The results are as follows Fig.17 As shown in the figure, the inhibitory effects of DOX, Cur, mPEG-SS-DOX micelles and mPEG-SS-DOX / Cur micelles on the proliferation of tumor cells are dose-dependent. The toxicity of DOX to tumor cells is higher than that of mPEG-SS-DOX micelles. This result may be because DOX and mPEG-SS-DOX micelles enter tumor cells in different ways. DOX enters tumor cells by free diffusion, while mPEG-SS-DOX micelles enter tumor cells by endocytosis, which makes mPEG-SS-DOX micelles exert their drug effects more slowly. In addition, both mPEG-SS-DOX and mPEG-SS-DOX / Cur micelles can significantly inhibit the proliferation of tumor cells, and the cytotoxicity of mPEG-SS-DOX / Cur micelles is significantly increased compared with mPEG-SS-DOX micelles, indicating that the co-administration of DOX and Cur in micelles can enhance the inhibitory effect on the proliferation of tumor cells.

Claims

1. A stimulus-responsive dual-drug co-delivery micelle, characterized in that: The polymer-drug linker mPEG-SS-DOX, which is constructed with mPEG as a hydrophilic block, disulfide bonds as redox-sensitive fragments, and doxorubicin DOX as a hydrophobic block, is used as a micelle material to encapsulate curcumin Cur to form a stimulus-responsive dual-drug co-delivery micelle mPEG-SS-DOX / curcumin Cur; According to the mass ratio, mPEG-SS-DOX: curcumin Cur = (6:1) ~ (3:1); The mPEG is polyethylene glycol monomethyl ether mPEG 2000 ; The polymer-drug linker mPEG-SS-DOX has the structural formula shown in (I): （Ⅰ）。 2. The method for preparing a stimulus-responsive dual-drug co-delivery micelle according to claim 1, characterized in that: The preparation method comprises the following steps: 1) Take 3,3'-dithiodipropionic acid and acetyl chloride, and reflux for 2 h at 65 °C with N2 protection to prepare 3,3'-dithiodipropionic anhydride; 2) Dissolve 3,3'-dithiodipropionic anhydride and mPEG in DMF, add 4-dimethylaminopyridine and triethanolamine under stirring, and react the resulting mixed solution at 35°C with N2 protection for 24 hours to prepare mPEG-SS-COOH; 3) Activate mPEG-SS-COOH dissolved in DMSO with N-hydroxysuccinimide and |N|,|N|-dicyclohexylcarbodiimide under stirring. After stirring for 12 h, slowly add the mPEG-SS-COOH solution to the deprotonated doxorubicin DOX solution, and add 4-dimethylaminopyridine to catalyze the reaction. Vacuum and stir in the dark under N2 protection for 24 h. Then, place the solution in a dialysis bag, first dialyze in DMSO to remove free DOX, then dialyze in deionized water to remove the organic solvent DMSO, and freeze-dry to obtain mPEG-SS-DOX. 4) mPEG-SS-DOX and curcumin were dissolved in DMSO, and the resulting mixed solution was slowly added dropwise to deionized water. After stirring for 12 h in the dark, the mixed solution was transferred to a dialysis bag with a molecular weight of 2000 Da. DMSO was removed by dialysis, and the dialysate was replaced regularly. After dialysis, the solution was filtered through a 0.45 μm microporous filter membrane to obtain the stimulus-responsive dual-drug co-delivery micelles mPEG-SS-DOX / curcumin Cur.

3. The preparation method according to claim 2, characterized in that: The deprotonated doxorubicin DOX is prepared by dissolving DOX·HCl in DMSO, adding triethanolamine dropwise, and obtaining the deprotonated DOX under continuous stirring.

4. Use of the stimulus-responsive dual-drug co-delivery micelle described in claim 1 in the preparation of chemotherapeutic drugs.

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