A Reductive Responsive Chemotherapy-Immunotherapy Prodrug, Nanomicelles and Preparation Method

Abstract

The present invention belongs to the field of pharmaceutical technology and relates to a reduction-responsive chemo-immunotherapy prodrug, a nano-micelle and a preparation method thereof. The prodrug is obtained by performing multiple steps of reactions such as oxidation, alkylation, amidation and esterification on the basis of oxaliplatin to obtain the reduction-responsive chemo-immunotherapy prodrug. The present invention uses the emulsion-solvent evaporation method to prepare the nano-prodrug into nano-micelles. The nano-micelles have appropriate particle size, high encapsulation efficiency, good stability and biological safety, and have good particle size potential, dispersibility and in vitro release behavior; after the OID nano-micelles enter the body, they can also release an IDO inhibitor with immunomodulatory effects, synergize chemotherapy and immune regulation, and play a combined treatment effect, which can effectively inhibit the growth of HT29 tumors, providing new strategies and ideas for the treatment of cancer and immune escape, and having broad application prospects in the field of medicine.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a reduction-responsive chemo-immunotherapy prodrug, a nanomicelle and a preparation method thereof. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, and its incidence and mortality rank third and second respectively among all cancers, seriously threatening human life and health. Therefore, how to effectively treat colorectal cancer has become an urgent problem to be solved.

[0003] At present, the conventional treatment methods for colon cancer include surgical treatment, chemotherapy, radiotherapy and molecular targeted therapy. Chemotherapy is the most commonly used strategy in clinical practice. Platinum drugs are the basic chemotherapy drugs for the treatment of colorectal cancer and are applied in the vast majority of clinical treatment regimens. Among them, oxaliplatin is the third-generation derivative of platinum anticancer drugs and is the first-line drug for the treatment of metastatic colorectal cancer. This drug binds to two nucleotide bases on DNA to inhibit DNA replication and transcription, thereby playing a role in inhibiting tumor proliferation.

[0004] However, as a divalent platinum drug, oxaliplatin also has obvious deficiencies. When the clinical dosage of oxaliplatin is relatively large, there will be more adverse reactions, mainly involving hematopoietic system, digestive system and nervous system toxicity. This is because oxaliplatin is not selectively distributed in the body, so it will damage normal cells while killing tumor cells, thus producing inevitable systemic toxicity. Secondly, although oxaliplatin has certain immunotherapeutic potential, due to the influence of the tumor-suppressive immune microenvironment, the anti-tumor immune response induced by oxaliplatin in the body is limited when administered alone.

[0005] Indoleamine 2,3-dioxygenase inhibitor (IDO inhibitor) plays an important role in tumor immunity. It can inhibit the production of regulatory T cells (Tregs) in tumor tissues, thereby reducing the immune tolerance of tumor-associated antigens, interfering with the immune escape of tumor cells, and thus enhancing the killing effect of the body's immune system on tumors. Therefore, the combination of oxaliplatin and IDO inhibitor is used to improve the immunosuppression of the tumor environment, enhance the efficacy of tumor chemo-immunotherapy, and thus improve the efficacy of oxaliplatin.

[0006] In addition, using different formulation strategies such as liposomes, polymer nanocarriers, inorganic nanocarriers and self-assembled nanocarriers to load drugs can effectively improve the bioavailability of drugs, reduce side effects and enhance the stability of drugs during the delivery process. It can also increase the targeted accumulation of drugs at the tumor site through the enhanced permeability and retention effect (EPR effect).

[0007] Therefore, there is an urgent need to provide a reductive-responsive chemo-immunotherapy prodrug and apply it to anti-cancer drugs to overcome the above-mentioned deficiencies, providing a new idea for the treatment of colorectal cancer. Summary of the Invention

[0008] The present invention aims at the deficiencies existing in the above-mentioned prior art and provides a reductive-responsive chemo-immunotherapy prodrug, a nanomicelle and a preparation method thereof.

[0009] The specific technical solution of the present invention is as follows:

[0010] The first object of the present invention is to provide a reductive-responsive chemo-immunotherapy prodrug, the structural formula of which is shown in Formula 1 below:

[0011]

[0012] The drug molecular unit of the structural formula 1 is an oxaliplatin derivative, wherein m is an integer, the range is 6-30, and m is preferably 12, 14, 16.

[0013] The second object of the present invention is to provide a preparation method of a reductive-responsive chemo-immunotherapy prodrug, comprising the following steps:

[0014] 1) The mono-carboxylated and alkylated oxaliplatin (Cm-OXA-COOH) undergoes an amide reaction with ethanolamine to obtain the amidated and alkylated oxaliplatin;

[0015] 2) Using the free hydroxyl group as the free arm, and then undergoing a condensation reaction with N-tert-butoxycarbonyl-D-tryptophan (N-Boc-1-MT) to obtain the tert-butoxycarbonyl-protected 1-methyl-tryptophan and alkylated oxaliplatin (Cm-OXA-Boc-1-MT);

[0016] 3) After the tert-butoxycarbonyl-protected 1-methyl-tryptophan and alkylated oxaliplatin (Cm-OXA-Boc-1-MT) is deprotected by an acid to remove the tert-butoxycarbonyl (Boc), the alkylated oxaliplatin conjugated with 1-methyl-tryptophan (1-MT) is obtained, which is the reductive-responsive chemo-immunotherapy prodrug (OID).

[0017] The reductive-responsive chemo-immunotherapy prodrug of the present invention is based on oxaliplatin and undergoes multiple steps of reactions such as oxidation, alkylation, amidation and esterification, and long alkyl chains and the immunomodulatory IDO inhibitor 1-MT are respectively bonded on both sides of the tetravalent platinum to obtain a reductive-responsive chemo-immunotherapy dual prodrug.

[0018] Furthermore, the specific process of the step 1) is as shown in the reaction route 1 below:

[0019]

[0020] Dissolve the monocarboxylated and alkylated oxaliplatin in an organic solvent, activate it with a catalyst for 1 hour, add 1 - 10 molar equivalents of ethanolamine, react at any constant temperature between 20 - 60 °C for 6 - 48 h, then rotary evaporate to remove the solvent, dissolve the obtained substance in methanol, and then use preparative chromatography for separation and purification, and freeze-dry to obtain amidated and alkylated oxaliplatin.

[0021] Further, the specific process of step 2) is as shown in reaction route 2 below:

[0022]

[0023] Dissolve the amidated and alkylated oxaliplatin in an organic solvent, activate it with a catalyst for 1 hour, add 1 - 10 molar equivalents of N - tert - butoxycarbonyl - D - tryptophan, react at any constant temperature between 20 - 60 °C for 6 - 48 h, then rotary evaporate to remove the solvent, dissolve the obtained substance in methanol, and then use preparative chromatography for separation and purification, and freeze-dry to obtain tert - butyloxycarbonyl - protected 1 - methyl - tryptophan and alkylated oxaliplatin.

[0024] Further, the specific process of step 3) is as shown in reaction route 3 below:

[0025]

[0026] Dissolve the tert - butyloxycarbonyl - protected 1 - methyl - tryptophan and alkylated oxaliplatin in an organic solvent, then add an acid with a mass of 1 - 10 wt% of the solvent mass, react in the dark at any constant temperature between 0 - 40 °C for 1 - 12 h, then rotary evaporate to remove the solvent, dissolve the obtained substance in methanol, and then use preparative chromatography for separation and purification, and freeze-dry to obtain the reduction-responsive chemo-immunotherapy prodrug (OID).

[0027] Among them, in steps 1), 2) and 3), the organic solvent used is at least one of dichloromethane, acetonitrile, tetrahydrofuran, acetone, N,N - dimethylformamide, N,N - dimethylacetamide and dimethyl sulfoxide; in steps 1) and 2), the catalyst used includes at least one of catalysts such as 1 - ethyl - 3(3 - dimethylpropylamine)carbodiimide, 4 - dimethylaminopyridine, N,N - diisopropylethylamine, 2-(7 - azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, carbodiimide hydrochloride and triethylamine; in step 3), the acid used is at least one of trifluoroacetic acid, hydrochloric acid, acetic acid, sulfuric acid and phosphoric acid.

[0028] The third object of the present invention is to provide a method for preparing a nano-micelle for reducing-responsive chemo-immunotherapy prodrug: dissolving the above-mentioned reducing-responsive chemo-immunotherapy prodrug and a diblock copolymer in an organic solvent as the oil phase, dropping it into the stirred aqueous phase, and rapidly stirring to disperse it evenly, and then removing the organic solvent by rotary evaporation to obtain a reducing-responsive chemo-immunotherapy prodrug micelle.

[0029] The present invention uses a synthesized reducing-responsive chemo-immunotherapy prodrug (OID) to prepare nano-micelles by the emulsion-solvent evaporation method. The hydrodynamic particle size of the micelles is between 20 and 300 nm, and the drug loading is between 1% and 5%. The nano-micelles have a suitable particle size, a high encapsulation efficiency, good stability and biosafety; they have good particle size potential, dispersibility and in vitro release behavior.

[0030] Further, the organic solvent is any one or a mixture of dichloromethane, absolute ethanol, tetrahydrofuran, acetone, N,N-dimethylformamide, dimethyl sulfoxide.

[0031] Further, the diblock copolymer is any one of poly(lactic-co-glycolic acid), hyaluronic acid-thiol copolymer, gelatin-thiol copolymer, polyethylene glycol-polylactide copolymer, polycaprolactone-polyethylene glycol copolymer.

[0032] Further, the volume ratio of the oil phase to the water phase is 1:(1 - 10).

[0033] Further, the concentration of the reducing-responsive chemo-immunotherapy prodrug in the oil phase is 0.1 - 10 mg / ml.

[0034] Further, the stirring speed is 300 - 600 rpm.

[0035] Further, the evaporation temperature of the organic solvent is 35 - 45 °C.

[0036] The fourth object of the present invention is to provide a reducing-responsive chemo-immunotherapy prodrug nano-micelle (OID nano-micelle) prepared by the above preparation method.

[0037] Further, the reducing-responsive chemo-immunotherapy prodrug micelle is mainly used for the treatment of cancer, and the cancer types include colorectal cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, liver cancer, head and neck cancer, gastric cancer, etc.

[0038] The beneficial effects of the present invention are as follows:

[0039] The OID nanomicelles prepared from the synthetic reduction-responsive chemotherapy-immunotherapy prodrug of the present invention have an appropriate particle size, a high encapsulation rate, good stability and biological safety; they can be effectively taken up by cells and enter the cell interior, have an obvious cytotoxic effect on mouse colon cancer cells (HT29), and can effectively inhibit the growth of HT29 tumors; in different release media, most of the drugs are released from the OID nanomicelles after 48 h, and the drug release is more complete in an acidic environment.

[0040] The OID nanomicelles of the present invention can spontaneously aggregate and retain for a long time in tumor tissues through the EPR effect to achieve long circulation. The kinetic inertness and lipophilicity of the tetravalent platinum prodrug micelles in the blood circulation promote the accumulation of oxaliplatin in tumor cells, improving the anti-tumor effect of the drug and the selectivity for the lesion site.

[0041] After entering the body, the OID nanomicelles of the present invention can also release the IDO inhibitor with immunomodulatory effects, synergize chemotherapy and immune regulation, and play a combined therapeutic effect, providing new strategies and ideas for the treatment of cancer and immune escape, and having broad application prospects in the field of medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the synthetic route diagram of OID in Example 1 of the present invention;

[0043] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum diagram of OID prepared in Example 1 of the present invention;

[0044] Figure 3 It is the mass spectrum diagram of OID prepared in Example 1 of the present invention;

[0045] Figure 4 It is the particle size characterization diagram of the OID nanomicelles prepared in Example 2 of the present invention;

[0046] Figure 5 It is the critical micelle concentration diagram of the OID nanomicelles of the present invention;

[0047] Figure 6 It is the hemolysis rate of rabbit blood by different concentrations of the OID nanomicelles of the present invention;

[0048] Figure 7 It is the cytotoxicity diagram of different concentrations of oxaliplatin, OID, OID nanomicelles, 1-MT, and oxaliplatin + 1-MT on colorectal cancer HT29 cells of the present invention;

[0049] Figure 8 It is the flow cytometry diagram of HT29 cells taking up the OID nanomicelles loaded with coumarin-6 of the present invention;

[0050] Figure 9 This is the average blood drug concentration-time curve of SD rats after administration of oxaliplatin and OID nano-micelles of the present invention; DETAILED DESCRIPTION

[0051] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0052] Materials and equipment:

[0053] Material:

[0054] PEG2K-PLGA4K was purchased from Jinan Daigang Bioengineering Co., Ltd.;

[0055] 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.;

[0056] N-tert-Butyloxycarbonyl-D-tryptophan was purchased from SINO High Goal Chemical Technology Co., Ltd.;

[0057] N,N-Diisopropylethylamine, trifluoroacetic acid, and coumarin-6 were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0058] Ethanolamine and 1-ethyl-3 (3-dimethylpropylamine) carbodiimide were purchased from Shanghai Bailingwei Technology Co., Ltd.;

[0059] Thiazolyl blue (MTT) and Nile red were purchased from Sigma-Aldrich (China);

[0060] Oxaliplatin was purchased from Shandong Platinum Source Company;

[0061] Pancreatin was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0062] HT29 human colorectal adenocarcinoma cells were purchased from the ATCC cell bank in the United States, and DMEM medium and fetal bovine serum for cell culture were purchased from Gibco;

[0063] PBS was purchased from Shanghai Titan Technology Co., Ltd.;

[0064] Unless otherwise specified, the other reagents and solvents used in the present invention were purchased from Sinopharm Group (Shanghai) Chemical Reagent Co., Ltd.

[0065] equipment:

[0066] JNM-ECZ400S / L1 NMR spectrometer;

[0067] MALVERN NANO SIZER laser particle size analyzer;

[0068] BD FACSCelesta flow cytometer.

[0069] Unless otherwise specified, the equipment and testing methods used in the present invention are all conventional equipment and methods in the art.

[0070] Example 1:

[0071] Synthesis of a reduction-responsive chemo-immunotherapeutic prodrug:

[0072] Monocarboxylated and alkylated oxaliplatin (Cm-OXA-COOH) was prepared according to "PEGylated Oxaliplatin Prodrug and Its Preparation Method and Use" disclosed in Patent CN106074379B.

[0073] As Figure 1 As shown in the synthesis route diagram, 200 mg of monocarboxylated and alkylated oxaliplatin was weighed and dissolved in 10 mL of anhydrous dichloromethane. Then, 15 mg of ethanolamine, 142 mg of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 97 mg of N,N-diisopropylethylamine were added, and the mixture was stirred at 25 °C for 4 hours. After the reaction, it was concentrated by rotary evaporation, redissolved in methanol, and then purified by preparative chromatography and freeze-dried to obtain amidated and alkylated oxaliplatin.

[0074] 80 mg of amidated and alkylated oxaliplatin was weighed and dissolved in 5 mL of anhydrous dichloromethane. Then, 30.2 mg of N-Boc-D-tryptophan and 23.1 mg of 4-dimethylaminopyridine were added, and the mixture was stirred at 25 °C for 5 hours. After the reaction, it was concentrated by rotary evaporation, redissolved in methanol, and then purified by preparative chromatography and freeze-dried to obtain tert-butoxycarbonyl-protected 1-methyl-tryptophan-alkylated oxaliplatin.

[0075] 50 mg of tert-butoxycarbonyl-protected 1-methyl-tryptophan-alkylated oxaliplatin was weighed and dissolved in 8 mL of anhydrous dichloromethane. Then, 250 μL of trifluoroacetic acid was added, and the mixture was stirred at 25 °C for 2 hours. After the reaction, it was concentrated by rotary evaporation, redissolved in methanol, and then purified by preparative chromatography and freeze-dried to obtain the reduction-responsive chemo-immunotherapeutic prodrug OID. The obtained substance was characterized by 1H NMR and mass spectrometry, and the results are as Figure 2 、 Figure 3 shown.

[0076] The results showed that 1 1.20 - 1.35 ppm and 0.88 ppm in the 1H NMR spectrum correspond to C 16-CH2- (b) and -CH3 (a) in the chain; 3.74 ppm corresponds to -CH3 (c) connected to N on 1-MT; 4.16 ppm corresponds to H (d) where OXA is connected to 1-MT. The benzene ring H in the 1-MT structure appears at 7.00 - 7.50 ppm (e), and 4.16 ppm corresponds to H on the methylene group connected to -NH2 in the 1-MT structure. As shown in the mass spectrometry diagram, the molecular weight of the synthesized product is 1042, which is consistent with the predicted result, further proving the successful preparation of the reduction-responsive chemo-immunotherapy prodrug, that is, the successful synthesis of OID.

[0077] Example 2:

[0078] Preparation of reduction-responsive chemo-immunotherapy prodrug nanomicelles:

[0079] Take 10 mg of the OID prodrug prepared in Example 1 and dissolve it together with 40 mg of PEG2K-PLGA4K in 2 mL of chloroform. Then slowly drip this solution into 4 mL of stirred pure water and stir at 500 rpm at room temperature for 20 min; after the reaction, slowly rotary evaporate to remove the organic solvent at 42 °C and filter through a 0.22 μm microporous filter membrane to obtain OID nanomicelles.

[0080] The micelle particle size was measured to be 91.94 ± 0.02, PDI was 0.211, and Zeta potential was 1.49 ± 0.26 using a Malvern particle size analyzer. The results are as Figure 4 shown in Table 1.

[0081] Table 1 Characterization results of nanomicelles (n = 3)

[0082]

[0083] Result test:

[0084] 1. Evaluation of the encapsulation efficiency and drug loading of OID nanomicelles

[0085] Measure 100 μL of the nanomicelles prepared in Example 2, add 900 μL of acetonitrile and ultrasonicate for 5 min to demulsify, and measure its total content. Measure 2 mL of the nanomicelle solution, place it in an ultrafiltration tube (10k) and centrifuge at 6000 rpm for 10 min. Take 100 μL of the upper-layer nanomicelle solution in the ultrafiltration tube, add 900 μL of acetonitrile to measure the concentration of the upper-layer solution, and then measure 1.0 mL of the lower-layer solution to determine the free drug content. Measure its absorbance value at 255 nm using HPLC. Calculate the drug concentration in the drug-loaded micelles through the OID standard concentration curve, and the drug loading (LC) and encapsulation efficiency (EE) can be calculated by formula (1) and formula (2).

[0086]

[0087]

[0088] The measured encapsulation efficiency was 99.16% and the drug loading was 2.90%.

[0089] 2. Evaluation of the critical micelle concentration of OID nanomicelles

[0090] Take 10 parts of the nanomicelles prepared in Example 2, and dilute the micelle solution into a series of sample solutions with concentrations of 0.0001 mg·mL -1 , 0.0005 mg·mL -1 , 0.001 mg·mL -1 , 0.005 mg·mL -1 , 0.01 mg·mL -1 , 0.05 mg·mL -1 , 0.1 mg·mL -1 , 0.5 mg·mL -1 , 1.0 mg·mL -1 , 2.0 mg·mL -1 . Respectively measure 10 parts of 20 μL of Nile red solution with a concentration of 0.08 mg·mL -1 , place each in a centrifuge tube, blow dry the dichloromethane (DCM) solution after nitrogen blowing, and then add the above series of micelle solutions to the centrifuge tubes respectively. After ultrasonic treatment for 40 min, let it stand in the dark at room temperature for 3 h. Transfer the above sample solutions to a 96-well plate in the dark, and use a multifunctional microplate reader to measure the fluorescence intensity of the micelle solutions with different concentrations at an excitation wavelength of 543 nm and an emission wavelength of 660 nm. Plot two tangents with the sample concentration as the abscissa and the fluorescence intensity as the ordinate, and the concentration at the intersection of the two tangents is the critical micelle concentration (CMC) of the prodrug micelles. The results are as Figure 5 shown.

[0091] The results show that using Nile red as a fluorescence probe, the CMC of OID nanomicelles measured by measuring the change in fluorescence intensity in micelle solutions with different concentrations is 0.049 mg·mL -1 , which is very close to the CMC of common surfactants and NPs, indicating that the OID nanomicelles have good stability and are easy to form a nano state.

[0092] 3. Safety test of OID nanomicelles

[0093] Experimental group: Take 6 parts of the nanomicelles prepared in Example 2, and dilute the micelle solution into a series of sample solutions with concentrations of 500, 250, 125, 62.5, 31.25, and 15.625 μM with normal saline respectively. Then measure 0.3 mL and place it in a 10 mL centrifuge tube, and then add 2.5 mL of 2% red blood cell suspension and 2.2 mL of normal saline to the tube.

[0094] Positive control group: Add 2.5 mL of 2% red blood cell suspension into a 10 mL centrifuge tube, and then measure 2.5 mL of purified water and place it into the centrifuge tube.

[0095] Negative control group: Add 2.5 mL of 2% red blood cell suspension into a 10 mL centrifuge tube, and then add 2.5 mL of normal saline into the centrifuge tube.

[0096] Respectively place the above experimental groups and control groups in an incubator at 37 °C for 4 h and then centrifuge (4 °C, 3000 r / min, 10 min), take pictures to record the experimental phenomena, use an ultraviolet spectrophotometer to measure the absorbance value at 540 nm of the supernatant after centrifugation of the experimental groups and control groups, and the hemolysis rate of the drug-loaded micelles can be calculated through formula (3). The results are as Figure 6 shown.

[0097]

[0098] Among them, Abs experimental group in the formula represents the absorbance of the sample, Abs blank control group represents the absorbance of the negative control group, and Abs positive control group represents the absorbance of the positive control group.

[0099] The results show that in the concentration range of 15 - 500 μM, the hemolysis rates are 0.66%, 0.94%, 0.20%, 1.14%, 3.56% and 4.52% in sequence. The above values are all less than 5.0%. Therefore, the OID nano-micelles have good blood compatibility and the safety of intravenous administration.

[0100] 4. Cytotoxicity evaluation of OID nano-micelles

[0101] Prepare the OID nano-micelles prepared in Example 2 and OID, 1-MT, OXA + 1-MT, and oxaliplatin raw material drug at an equimolar concentration of 500 mol / L, and then prepare 6 gradient concentrations in sequence by the three-fold dilution method. Inoculate HT29 human colorectal adenocarcinoma cells into a 96-well cell culture plate (4000 cells / well), and add 0.1 mL of DMEM medium (containing 10% serum) to each well. After culturing for 48 h, change the medium, add 100 μL of drug-containing medium solutions with different concentrations into the cell culture plate, and co-incubate with the cancer cells for 48 h, and then use the standard MTT method to measure the cell metabolic activity. The results are as Figure 7 shown.

[0102] The results showed that for HT29 cells, there was no significant difference in the change of cell viability between the combination of oxaliplatin and 1-MT and the group using oxaliplatin alone. However, the reduction-responsive chemo-immunotherapy prodrug OID significantly enhanced the killing toxicity to HT29 cells. The OID nanomicelles at a low concentration had an obvious killing effect on cells, showing a significant difference compared with the oxaliplatin group.

[0103] 5. Cellular uptake experiment of OID nanomicelles

[0104] Referring to Example 2, 20 μL of 1 mg / mL coumarin-6 solution was added to the organic phase. The organic phase was slowly dropped into 2 mL of rapidly stirred aqueous phase. After stirring for 20 min to mix well, the organic phase was removed by rotary evaporation. The micellar solution loaded with coumarin-6 was obtained by passing through a 0.22 μm aqueous filter membrane and placed in an ultrafiltration tube (1000k) and centrifuged at 8000 rpm for 10 min to remove free coumarin-6.

[0105] HT29 cells in the logarithmic growth phase were seeded in a 24-well plate and incubated overnight at 37 °C and 5% CO2 to allow the cells to adhere. The micellar solution loaded with coumarin-6 was diluted with DMEM basal medium and administered at a final concentration of coumarin-6 of 500 ng / mL. After incubation with the cells for 6 h, the cells were digested with 0.3 mL of trypsin, transferred to a centrifuge tube, washed three times with PBS, and the cells were collected by centrifugation. The cells were resuspended in 500 μL of PBS and passed through a sieve for detection by flow cytometry.

[0106] The results showed that after incubation of the micellar solution loaded with coumarin-6 with HT29 cells for 6 h, there was a significant difference in the fluorescence intensity inside the cells compared with the non-administered cells. Therefore, the OID nanomicelles could be well phagocytosed by HT29 cells to release the dye, and the cellular uptake rate was relatively fast.

[0107] 6. Pharmacokinetic evaluation of OID nanomicelles

[0108] Eight SD rats were randomly divided into 2 groups, with 4 rats in each group. They were respectively injected with OXA (5 mg·kg -1 ) and OID nanomicelles (13.1 mg·kg-1) via the tail vein, and the administration volume was 1.0 mL. The administered rats were bled from the orbital cavity at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 h, about 0.3 mL of blood each time, and placed in heparinized centrifuge tubes. The blood was centrifuged at 3000 rpm / min for 3 min, and the upper serum was taken as the sample to be measured.

[0109] Accurately weigh about 0.10 g of serum into a digestion tank, add 3 mL of nitric acid and 5 mL of hydrogen peroxide, and then carry out digestion. After digestion, transfer it to an acid-evaporation plate, slowly evaporate and drive off the acid at about 60 °C, and then make up the volume to 25 mL in a volumetric flask. The concentration of Pt was determined by ICP-MS. The results are shown in Table 2 andFigure 9 as shown

[0110] Table 2 Pharmacokinetic parameters of rats after tail vein injection of oxaliplatin and OID nanomicelles

[0111]

[0112] The results showed that the AUC (area under the plasma concentration-time curve) of the prodrug micelles was much larger than that of the free drug, and compared with OXA, the OID nanomicelles had a longer residence time in vivo, a longer half-life, and a higher bioavailability.

[0113] Unless otherwise specified, the test methods used in the present invention are all conventional equipment and methods in the art.

[0114] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A reductive-responsive chemo-immunotherapy prodrug, characterized in that, Its structural formula is shown in Formula 1: Formula 1 The drug molecular unit of Formula 1 is an oxaliplatin derivative, wherein m is an integer with a range of 6 - 30.

2. A method for preparing a reductive-responsive chemo-immunotherapeutic prodrug as described in claim 1, characterized in that, It includes the following steps: 1) Oxaliplatin undergoes monocarboxylation and alkylation, and then an amide reaction with ethanolamine to obtain amidated and alkylated oxaliplatin; 2) Using the free hydroxyl group as a free arm, and then a condensation reaction with N - tert - butoxycarbonyl - 1 - methyl - D - tryptophan to obtain tert - butyloxycarbonyl - protected 1 - methyl - tryptophan and alkylated oxaliplatin; 3) After removing the tert - butyloxycarbonyl group from tert - butyloxycarbonyl - protected 1 - methyl - tryptophan and alkylated oxaliplatin with an acid, alkylated oxaliplatin conjugated with 1 - methyl - tryptophan is obtained, which is the reduction - responsive chemo - immunotherapy prodrug.

3. The preparation method of the reductive-responsive chemo-immunotherapeutic prodrug according to claim 2, characterized in that, In the step 1), the molar ratio of ethanolamine to the monocarboxylated and alkylated oxaliplatin is (1 - 10):1; in the step 2), the molar ratio of N - tert - butoxycarbonyl - 1 - methyl - D - tryptophan to the amidated and alkylated oxaliplatin is (1 - 10):

1.

4. The preparation method of the reductive-responsive chemo-immunotherapeutic prodrug according to claim 2, wherein, The reaction temperature of the step 1) and step 2) is 20 - 60 °C, and the reaction time is 6 - 48 h; the reaction temperature of the step 3) is 0 - 40 °C, and the light - avoiding reaction time is 1 - 12 h.

5. The preparation method of the reductive-responsive chemo-immunotherapeutic prodrug according to claim 2, wherein In the step 3), the amount of the acid added is 1 - 10 wt% of the solvent mass; the acid is at least one of trifluoroacetic acid, hydrochloric acid, acetic acid, sulfuric acid, and phosphoric acid.

6. The preparation method of the reductive-responsive chemo-immunotherapeutic prodrug according to claim 2, wherein, In the steps 1), 2) and 3), the organic solvent used is at least one of dichloromethane, acetonitrile, tetrahydrofuran, acetone, N,N - dimethylformamide, N,N - dimethylacetamide, and dimethyl sulfoxide; the catalyst used is at least one of 1 - ethyl - 3(3 - dimethylpropylamine)carbodiimide, 4 - dimethylaminopyridine, N,N - diisopropylethylamine, 2-(7 - azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, carbodiimide hydrochloride, and triethylamine.

7. A method for preparing the nanomicelles of the reductive-responsive chemo-immunotherapeutic prodrug as described in claim 1, characterized in that, Dissolve the reduction - responsive chemo - immunotherapy prodrug and the diblock copolymer in an organic solvent as the oil phase, and drop it into the stirred aqueous phase to make it disperse evenly, and then remove the organic solvent by rotary evaporation to obtain the reduction - responsive chemo - immunotherapy prodrug nanomicelles.

8. The preparation method of the nano micelles according to claim 7, characterized in that, The organic solvent is any one or a mixture of dichloromethane, absolute ethanol, tetrahydrofuran, acetone, N,N - dimethylformamide, and dimethyl sulfoxide; the diblock copolymer is any one of poly(lactic - co - glycolic acid), hyaluronic acid - thiol copolymer, gelatin - thiol copolymer, polyethylene glycol - poly(lactide) copolymer, and poly(caprolactone)-poly(ethylene glycol) copolymer.

9. The preparation method of the nano micelles according to claim 7, characterized in that, The volume ratio of the oil phase to the aqueous phase is 1:(1 - 10); the concentration of the reduction - responsive chemo - immunotherapy prodrug in the oil phase is 0.1 - 10 mg / ml; the stirring speed is 300 - 600 rpm; the evaporation temperature of the organic solvent is 35 - 45 °C.

10. A reduction - responsive chemo - immunotherapy prodrug nanomicelle prepared by the preparation method of the reduction - responsive chemo - immunotherapy prodrug nanomicelle according to any one of claims 7 - 9.

Images