Reactive oxygen species-responsive cascade drug release nanopreparation and method of preparation thereof

Abstract

The application discloses a reactive oxygen species (ROS) responsive cascade drug release nano-prodrug and a preparation method thereof, and specifically relates to the following steps: dispersing a polymer prodrug and triethylamine in an organic solvent to prepare a dispersion A; dispersing an iron salt in an organic solvent to prepare a dispersion B; mixing the dispersion A and the dispersion B to obtain a dispersion C; under ultrasonic waves or stirring, dropping the dispersion C into deionized water, mixing uniformly, removing the organic solvent through dialysis, and obtaining the ROS responsive cascade drug release nano-prodrug; and experiments prove that the ROS responsive cascade drug release nano-prodrug has good drug loading stability; can release drugs in response to ROS, and realizes the combination of chemotherapy and chemical kinetic therapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to an active oxygen response cascade drug release nano-prodrug and a preparation method thereof. BACKGROUND

[0002] Chlorambucil is an organic compound, which is mainly used for chronic lymphocytic leukemia, ovarian cancer and low-grade malignant non-Hodgkin's lymphoma. The mechanism of action is the same as that of other nitrogen mustard drugs, which mainly causes cross-linking of DNA strands to affect the function of DNA, and the cells undergo apoptosis to kill tumor cells and achieve the purpose of treating tumors.

[0003] Cinnamaldehyde is the main active component of Cinnamomum cassia, and researches have reported that cinnamaldehyde exhibits strong anti-tumor activity by stimulating the generation of intracellular reactive oxygen species and inducing reactive oxygen species-mediated oxidative damage of tumor cells. The therapeutic cinnamaldehyde is combined with an active oxygen response drug delivery system to compensate for exogenous hydrogen peroxide, increase intracellular reactive oxygen species of tumor cells, realize complete drug release, and improve the anti-tumor efficacy.

[0004] The two main methods of drug-loaded molecules are physical encapsulation and chemical coupling. However, the encapsulated nano-prodrug usually has unsatisfactory stability, leading to inevitable drug leakage. In order to solve this problem, prodrug is considered to be a good strategy. By using a reactive linker to bind the therapeutic drug to the polymer backbone, the prodrug can achieve high drug loading stability. Therefore, the development of a prodrug-based nano-prodrug capable of generating reactive oxygen species and triggering drug release by reactive oxygen species is an effective strategy for combining chemical kinetics therapy and activated chemotherapy. In order to rationally design the drug delivery system, it is necessary to have a very good understanding of the physiological conditions of the disease site, and to rationally design the drug carrier that stimulates the reaction. The drug carrier can undergo dramatic chemical or physical changes according to the stimulus, so that the drug can escape. The multi-stimulus response drug delivery system can better target certain pathological conditions that have multiple stimuli at the same time, thereby improving the drug efficacy. With the help of Fenton reaction, reactive oxygen species can be generated, and theoretically, controlled drug delivery can be achieved at any desired site, which further expands the application in the treatment of a wide range of diseases. However, there is no report on an active oxygen sensitive, active oxygen response cascade drug release nano-prodrug connected with chlorambucil and its preparation method. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide an active oxygen response cascade drug release nano-prodrug.

[0006] The second purpose of the present application is to provide a preparation method of the active oxygen response cascade drug release nano-prodrug.

[0007] A third object of the present application is to provide the use of the active oxygen response cascade drug release nano-prodrug in the preparation of an antitumor drug.

[0008] The technical solution of the present application is summarized as follows:

[0009] The preparation method of the active oxygen response cascade drug release nano-prodrug comprises the following steps

[0010] 1) dispersing the polymer prodrug and triethylamine in an organic solvent to prepare a dispersion A with a concentration of the polymer prodrug of 0.1 mg / mL to 10 mg / mL; the final concentration of the triethylamine is 2-5 μL / mL;

[0011] 2) dispersing an iron salt in an organic solvent to prepare a dispersion B with a concentration of 0.1 mg / mL to 10 mg / mL;

[0012] 3) mixing the dispersion A and the dispersion B in a volume ratio of 1:0.05-10 to obtain a dispersion C, and under ultrasonic wave or stirring, adding the dispersion C to deionized water in a volume ratio of 1:1-10, mixing uniformly, removing the organic solvent through dialysis, and obtaining the active oxygen response cascade drug release nano-prodrug;

[0013] The polymer prodrug is shown in formula (I):

[0014]

[0015] Among them:

[0016] 1≤m≤30;

[0017] 1≤n≤50;

[0018] R1 is a polyethylene glycol monomethyl ether with a weight average molecular weight of 1000-10000;

[0019] R2 is -H, -CH3 or -CH2CH3;

[0020] R3 is -H, -CH3 or -CH2CH3;

[0021] R4 has a structure shown in formula (II), (III) or (IV);

[0022]

[0023] The organic solvents in the step 1) and the step 2) are the same, and are preferably dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide.

[0024] The iron salt is ferric chloride, ferrous chloride or ferric nitrate.

[0025] The active oxygen response cascade drug release nano-prodrug prepared by the preparation method.

[0026] The active oxygen response cascade drug release nano-prodrug is used for preparing an antitumor drug.

[0027] Advantages of the present application:

[0028] Experiments prove that the active oxygen response cascade drug release nano-prodrug has good drug loading stability; can release drugs in response to active oxygen, and realizes the combination of chemotherapy and chemical kinetic therapy. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The active oxygen response cascade drug release nano-prodrug is the nuclear magnetic resonance hydrogen spectrum of product 1 described in Example 1.

[0030] Figure 2 The active oxygen response cascade drug release nano-prodrug is the nuclear magnetic resonance hydrogen spectrum of product 2 described in Example 1.

[0031] Figure 3 The active oxygen response cascade drug release nano-prodrug is the nuclear magnetic resonance hydrogen spectrum of product 3 described in Example 1.

[0032] Figure 4 The active oxygen response cascade drug release nano-prodrug is the nuclear magnetic resonance hydrogen spectrum of product 5 described in Example 1.

[0033] Figure 5 The active oxygen response cascade drug release nano-prodrug is the nuclear magnetic resonance hydrogen spectrum of polymer 1 described in Example 1.

[0034] Figure 6 The active oxygen response cascade drug release nano-prodrug is the nuclear magnetic resonance hydrogen spectrum of the polymer prodrug described in Example 1.

[0035] Figure 7 The active oxygen response cascade drug release nano-prodrug is the transmission electron microscope image of the active oxygen response cascade drug release nano-prodrug described in Example 1.

[0036] Figure 8 The active oxygen response cascade drug release nano-prodrug is the analysis of the effect of the concentration of the active oxygen response cascade drug release nano-prodrug on the cell survival rate detected by MTT in Example 6.

[0037] Figure 9 The active oxygen response cascade drug release nano-prodrug is the release rate of the active oxygen response drug of the active oxygen response cascade drug release nano-prodrug prepared in Example 1.

[0038] Figure 10 The active oxygen response cascade drug release nano-prodrug is the fluorescence quantitative analysis of the active oxygen produced in the cell by the active oxygen response cascade drug release nano-prodrug detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) in Example 8. DETAILED DESCRIPTION

[0039] The present application will be further described below in combination with specific examples.

[0040] The structure of the polymer prodrug is shown as formula I.

[0041]

[0042] Table 1

[0043]

[0044] 5000 in the table refers to polyethylene glycol monomethyl ether with a weight average molecular weight of 5000; the same explanation applies to others.

[0045] R4, the structure is shown as formula (II), (III) or (IV);

[0046]

[0047] II is chlorambucil, III or IV and chlorambucil derivatives.

[0048] Example 1

[0049] The preparation method of the reactive oxygen response cascade drug release nano prodrug comprises the following steps:

[0050] 1) Disperse the polymer prodrug and triethylamine in dimethyl sulfoxide to prepare a dispersion A of the polymer prodrug with a concentration of 1 mg / mL; the final concentration of the triethylamine is 5 μL / mL; the polymer prodrug is shown in Table 1;

[0051] 2) Disperse the ferric chloride in dimethyl sulfoxide to prepare a dispersion B with a concentration of 1 mg / mL;

[0052] 3) Mix the dispersion A and the dispersion B in a volume ratio of 1:0.05 to obtain a dispersion C; under ultrasonic wave (or stirring), drop the dispersion C into deionized water in a volume ratio of 1:4, mix well, remove the organic solvent dimethyl sulfoxide by dialysis to obtain the reactive oxygen response cascade drug release nano prodrug;

[0053] Preparation of the polymer prodrug in this example:

[0054] 1) Dissolve 20 mmol of cinnamaldehyde and 56 mmol of 2-mercaptoethanol in 30 mL of ethyl acetate to prepare a mixture A. Add 4 mmol of trifluoroacetic acid dropwise to the mixture A under ice bath conditions to prepare a mixture B. Stir the mixture B in an ice bath for 30 minutes, then transfer to room temperature and stir overnight. After stirring overnight, purify the mixture B by silica gel column chromatography, and the eluent is petroleum ether / ethyl acetate (1:1, v / v) to obtain a white solid product 1, which is confirmed by nuclear magnetic resonance hydrogen spectrum, see Figure 1 .

[0055] 2) Dissolve 3.7 mmol of product 1 and 3 mmol of triethylamine in 30 mL of anhydrous dichloromethane to form mixture C. Add 3 mmol of methacryloyl chloride dropwise to mixture C under ice bath conditions to form mixture D. Allow mixture D to reach room temperature and stir overnight. Purify mixture D by silica gel column chromatography using petroleum ether / ethyl acetate (1:1, v / v) as the eluent to obtain product 2 as a yellow oil. Product 2 is confirmed by proton nuclear magnetic resonance spectroscopy as shown in Figure 2 .

[0056] 3) Dissolve 1.2 mmol of product 2 and 1 mmol of chlorambucil and 0.25 mmol of 4-diaminopyridine in 20 mL of anhydrous dichloromethane to form mixture E. Bubble argon gas through mixture E and stir for 30 minutes under ice bath conditions. Dissolve 2.4 mmol of 1,2-dichloroethane in 20 mL of anhydrous dichloromethane and add dropwise to mixture E to form mixture F. Stir mixture F under ice bath conditions for 1 hour, then allow mixture F to reach room temperature and stir overnight. Purify mixture F by silica gel column chromatography using petroleum ether / ethyl acetate (20:1, v / v) as the eluent to obtain product 3 as an orange-yellow oil. Product 3 is confirmed by proton nuclear magnetic resonance spectroscopy as shown in Figure 3 .

[0057] 4) Dissolve 20 mmol of hydroxyethyl methacrylate and 24 mmol of triethylamine in 30 mL of anhydrous dichloromethane to form mixture G. Stir mixture G under ice bath conditions for 5 minutes. Dissolve 24 mmol of p-nitrophenyl chloroformate in 20 mL of anhydrous dichloromethane and add dropwise to mixture G to form mixture H. Stir mixture H at room temperature for 24 hours. Wash mixture H with water twice, then wash with saturated brine until the solution is colorless. Concentrate mixture H by rotary evaporation to obtain product 4. Add product 4 dropwise to n-hexane to obtain mixture I. Place mixture I in a freezer at -80 °C for one night to form a white precipitate. Discard the supernatant and dry the precipitate to obtain product 5. Product 5 is confirmed by proton nuclear magnetic resonance spectroscopy as shown in Figure 4 .

[0058] 5) Dissolve 0.15 mmol of product 3, 0.35 mmol of product 5, 0.01 mmol of polyethylene glycol monomethyl ether with a molecular weight of 5000, and 0.0025 mmol of AIBN in 3 mL of dioxane to obtain mixture J. Bubble argon gas through mixture J for 30 minutes, then place mixture J in a 70 °C oil bath and stir for 24 hours. After 24 hours, add mixture J dropwise to n-hexane to obtain mixture K. Centrifuge mixture K for 5 minutes, discard the supernatant, and dry the precipitate to obtain polymer 1. Polymer 1 is confirmed by proton nuclear magnetic resonance spectroscopy as shown in Figure 5 .

[0059] 6) Dissolve 0.01 mmol of polymer 1 in 2 mL of N,N-dimethylformamide to obtain mixture L. Dissolve 0.07 mmol of dopamine hydrochloride and 5 μL of triethylamine in 5 mL of N,N-dimethylformamide to obtain mixture M. Add mixture M to mixture L to obtain mixture N. Stir mixture N at room temperature for 6 hours, then add it to 30 mL of 0.1 M hydrochloric acid solution to obtain mixture O. Dialyze mixture O in pure water for 48 hours, then lyophilize to obtain the polymer prodrug. The structures of products 1, 2, 3, 5, and polymer 1 are as shown in formulas (V), (VI), and (VII).

[0060] As shown in (VIII) and (IX);

[0061]

[0062] The prodrug's 1H NMR spectrum is shown below. Figure 6 .

[0063] Transmission electron microscopy (TEM) image of the reactive oxygen species-responsive cascade drug release nanoprodrug is shown below. Figure 7 .

[0064] The preparation of the polymer prodrugs in the following embodiments is the same as that in this embodiment.

[0065] Example 2

[0066] A method for preparing reactive oxygen species-responsive cascade drug release nanoprodrugs includes the following steps:

[0067] 1) The polymer prodrug and triethylamine were dispersed in N,N-dimethylformamide to prepare a polymer prodrug with a concentration of 0.1%.

[0068] Dispersion A at mg / mL; final concentration of triethylamine 2 μL / mL; polymer prodrugs are shown in Table 1;

[0069] 2) Ferrous chloride was dispersed in N,N-dimethylformamide to prepare dispersion B with a concentration of 0.1 mg / mL;

[0070] 3) Mix dispersion A and dispersion B in a 1:1 volume ratio to obtain dispersion C. Under ultrasonic (or stirring) conditions, add dispersion C dropwise to deionized water in a 1:1 volume ratio, mix well, and remove impurities by dialysis.

[0071] The organic solvent N,N-dimethylformamide was used to obtain reactive oxygen species-responsive cascade drug release nanoprodrugs;

[0072] Example 3

[0073] A method for preparing reactive oxygen species-responsive cascade drug release nanoprodrugs includes the following steps:

[0074] 1) The polymer prodrug and triethylamine were dispersed in N,N-dimethylacetamide to prepare a dispersion A with a polymer prodrug concentration of 10 mg / mL; the final concentration of the triethylamine was 4 μL / mL; the polymer prodrug is shown in Table 1;

[0075] 2) Disperse ferric nitrate in N,N-dimethylacetamide to prepare dispersion B with a concentration of 10 mg / mL;

[0076] 3) Mix dispersion A and dispersion B at a volume ratio of 1:10 to obtain dispersion C. Under ultrasonic (or stirring) conditions, add dispersion C dropwise to deionized water at a volume ratio of 1:10, mix well, and remove the organic solvent N,N-dimethylacetamide by dialysis to obtain reactive oxygen species responsive cascade drug release nanoprodrug.

[0077] Example 4

[0078] The preparation method of reactive oxygen species-responsive cascade drug release nanoprodrugs is the same as in Example 1, and the polymer prodrugs are shown in Table 1.

[0079] Example 5

[0080] The preparation method of reactive oxygen species-responsive cascade drug release nanoprodrugs is the same as in Example 1, and the polymer prodrugs are shown in Table 1.

[0081] Example 6

[0082] MTT assay. To demonstrate the cytotoxic effect of the reactive oxygen species (ROS)-responsive cascade drug-release nanoprodrug of this invention on tumor cells, the CT26 cell line was used as a cell model, and the MTT assay was employed to verify the cytotoxic effects of different concentrations of the ROS-responsive cascade drug-release nanoprodrug.

[0083] CT26 mouse colon cancer cells (commercial product) were administered at a dose of 4×10⁻⁶. 3 The cells were seeded at a density of 1:1 in each well of a 64-well plate and incubated at 37°C for 24 hours until complete cell adhesion. Then, the plate was replaced with fresh complete culture medium (the complete culture medium formulation was a 90:10:1 volume ratio of DMEM medium, fetal bovine serum, and penicillin-streptomycin mixture) containing the reactive oxygen species-responsive cascade drug-releasing nanoparticles from Example 1 (chlorambucil concentrations of 0, 4, 8, 16, 32, 64, 128, 256, and 512 μg / mL). After incubation at 37°C for 48 hours, MTT solution (5 mg / mL, 10 μL) was added to each well, and the plate was incubated at 37°C for another 3-4 hours. The waste culture medium was aspirated, and 100-150 μL of DMSO was added. The 96-well plate was placed on a shaker at 120 rpm for 10 minutes until the insoluble matter was completely dissolved. The absorbance at 570 nm was measured using a microplate reader to evaluate cell viability.

[0084] As shown in Figure 8 Figure 1, the active oxygen response cascade drug release nano-prodrug prepared in Example 1 has a cell survival rate of less than 50% at a phenylbutazone concentration of 256 micrograms per milliliter.

[0085] Example 7

[0086] Drug release experiment. To prove that the active oxygen response cascade drug release nano-prodrug of the present application has good drug loading stability and can realize drug release in response to active oxygen, endogenous active oxygen is used to stimulate the generation of active oxygen from cinnamaldehyde, and active oxygen is used to stimulate the cleavage of thioformyl bond.

[0087] The specific operation is as follows:

[0088] The experiment is divided into two groups.

[0089] Group 1: Take 2 mL of the active oxygen response cascade drug release nano-prodrug prepared in Example 1 and transfer it to a dialysis bag with a molecular weight cut-off of 3500. Place the dialysis bag in 10 mL of buffer solution with a hydrogen peroxide concentration of 100 μM, and place the system in a shaking bed (temperature 37°C, shaking speed 120 rpm) for dialysis. At the 48th hour, take 1 mL of buffer solution outside the dialysis bag for quantitative analysis to determine the concentration of phenylbutazone. Then calculate the amount of drug released and draw the drug release curve;

[0090] Group 2: Take 2 mL of the active oxygen response cascade drug release nano-prodrug prepared in Example 1 and transfer it to a dialysis bag with a molecular weight cut-off of 3500. Place the dialysis bag in 10 mL of buffer solution with a hydrogen peroxide concentration of 10 μM, and place the system in a shaking bed (temperature 37°C, shaking speed 120 rpm) for dialysis. At the 48th hour, take 1 mL of buffer solution outside the dialysis bag for quantitative analysis to determine the concentration of phenylbutazone. Then calculate the amount of drug released and draw the drug release curve;

[0091] As shown in Figure 9 Figure 1, the active oxygen response cascade drug release nano-prodrug prepared in Example 1 has a drug release rate of less than 10% in a buffer solution with a hydrogen peroxide concentration of 10 μM within 48 hours, while the drug release rate exceeds 50% in a buffer solution with a hydrogen peroxide concentration of 100 μM.

[0092] Example 8

[0093] Detection of intracellular active oxygen production:

[0094] The experiment is divided into two groups.

[0095] Group 1: Take 1 mL of CT26 mouse colon cancer cells (commercial product) and transfer it to a 6-well plate. Add 2 mL of the active oxygen response cascade drug release nano-prodrug prepared in Example 1 to the 6-well plate, and incubate at 37°C for 48 hours. Then take 1 mL of the supernatant for quantitative analysis to determine the concentration of phenylbutazone. 5Each well was inoculated into 12-well plates at a density, and incubated at 37°C for 24 hours until the cells were completely adherent. The fresh complete medium without the active oxygen response cascade drug release nanopreparation was replaced, and the cells were incubated at 37°C for 24 hours. The medium was discarded, and the cells were washed twice with phosphate buffer (pH 7.2-7.4, 0.01M). Then, 10 micromolar DCFH-DA probe was added, and the cells were incubated at 37°C for 30 minutes. The cells were washed twice with phosphate buffer (pH 7.2-7.4, 0.01M), and finally imaged by confocal fluorescence microscopy (excitation wavelength 488 nm / emission wavelength 525 nm) and analyzed for fluorescence intensity.

[0096] Group 2: CT26 mouse colon cancer cells were inoculated at a density of 1×10 5 Each well was inoculated into 12-well plates at a density, and incubated at 37°C for 24 hours until the cells were completely adherent. The fresh complete medium without the active oxygen response cascade drug release nanopreparation was replaced, and the cells were incubated at 37°C for 24 hours. The medium was discarded, and the cells were washed twice with phosphate buffer (pH 7.2-7.4, 0.01M). Then, 10 micromolar DCFH-DA probe was added, and the cells were incubated at 37°C for 30 minutes. The cells were washed twice with phosphate buffer (pH 7.2-7.4, 0.01M), and finally imaged by confocal fluorescence microscopy (excitation wavelength 488 nm / emission wavelength 525 nm) and analyzed for fluorescence intensity.

[0097] As shown in FIG. 1, the green fluorescence intensity of the active oxygen response cascade drug release nanopreparation prepared in Example 1 was much higher than that of the blank control group, demonstrating its strong ability to induce active oxygen generation. Figure 10

[0098] The experiments demonstrated that the active oxygen response cascade drug release nanopreparation prepared in one of Examples 2-5 had a similar active oxygen response drug release rate and effect of generating active oxygen in cells as the active oxygen response cascade drug release nanopreparation prepared in Example 1.

[0099] ​The active oxygen response cascade drug release nano prodrug of the application copolymerizes dopamine and chlormethine monomer based on active oxygen response thioacetal linkage on polyethylene glycol to form an amphiphilic polymer prodrug, utilizes dopamine to chelate iron ions, and self-assembles to form a nano prodrug. After the nano prodrug enters tumor cells, under the activation of endogenous hydrogen peroxide, the thioacetal linkage is broken to release chlormethine and cinnamaldehyde, the former induces tumor cells to undergo apoptosis, and the latter induces intracellular hydrogen peroxide to regenerate, on one hand, accelerates the breaking of the thioacetal linkage, and on the other hand, provides raw materials for the Fenton reaction catalyzed by iron ions, promotes the further generation of active oxygen, and realizes the combination of chemotherapy and chemical kinetics therapy.

Claims

1. A method for preparing reactive oxygen species-responsive cascade drug release nanoprodrugs, characterized by: Includes the following steps 1) Disperse the polymer prodrug and triethylamine in an organic solvent to prepare a dispersion A with a polymer prodrug concentration of 0.1 mg / mL to 10 mg / mL; the final concentration of the triethylamine is 2-5 μL / mL; 2) Disperse the iron salt in an organic solvent to prepare dispersion B with a concentration of 0.1 mg / mL to 10 mg / mL; 3) Mix dispersion A and dispersion B at a volume ratio of 1:0.05 to 10 to obtain dispersion C. Under ultrasonic or stirring conditions, add dispersion C dropwise to deionized water at a volume ratio of 1:1 to 10, mix well, and remove the organic solvent by dialysis to obtain reactive oxygen species responsive cascade drug release nanoprodrug. The polymer prodrug is shown in formula (I): in: 1≤m≤30； 1≤n≤50； R1 is polyethylene glycol monomethyl ether with a weight average molecular weight of 1000 to 10000; R2 is -H, -CH3, or -CH2CH3; R3 is -H, -CH3, or -CH2CH3; The structure of R4 is shown in equation (II), (III) or (IV); 2. The preparation method according to claim 1, characterized in that: The organic solvent used in steps 1) and 2) is the same, namely dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide.

3. The preparation method according to claim 1, characterized in that: The iron salt is ferric chloride or ferric nitrate.

4. A reactive oxygen species-responsive cascade drug release nanoprodrug prepared by the preparation method of any one of claims 1-3.

5. The application of the reactive oxygen species-responsive cascade drug release nanoprodrug of claim 4 in the preparation of antitumor drugs.

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