Preparation method of nano-micelle for reducing responsive sensitization tumor immunochemotherapy

Abstract

The invention provides a preparation method of nano-micelles for reducing responsive sensitization tumor immunochemotherapy, and belongs to the technical field of biomedicines.The preparation method comprises the steps that under the inert atmosphere, floxuridine is dissolved in an organic solvent, then 4-dimethylaminopyridine and triphosgene are added, and a mixed solution is obtained; 2-hydroxyethyl disulfide dissolved in an organic solvent is dropped into the mixed solution in an inert atmosphere, the solvent is removed after the reaction is finished, and a disulfide bond-containing floxuridine intermediate FUDR-SS is obtained through silica gel column chromatography; the preparation method comprises the following steps: dissolving lauric acid in an organic solvent, then adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine, and stirring and activating to obtain a reaction solution; dissolving the FUDR-SS in an organic solvent, dropwise adding the obtained solution into the reaction liquid, carrying out a reaction in a dark place, and carrying out silica gel column chromatography purification to obtain a reduction responsive drug-drug conjugate FL; and dissolving the FL and niclosamide in an organic solvent together, uniformly dispersing the obtained mixed solution in water, and dialyzing to obtain the nano-micelle FL (at) NIC. The nano-micelle prepared by the method integrates antibacterial and anti-tumor functions, can reverse the chemotherapy drug resistance of tumor cells, and enhances the tumor immunochemotherapy curative effect by jointly inhibiting the interburial effect of macrophages.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of a nano-micelle for reducing-responsive sensitized tumor immunochemotherapy. Background Art

[0002] In China, the incidence of colorectal cancer (CRC) has been on the rise year by year, and most patients have metastases at the time of diagnosis, with a very serious prevention and control situation. Although various treatment methods such as immunotherapy and targeted therapy have emerged in recent years, adjuvant chemotherapy based on fluorouracil drugs still has an unshakable position in the treatment of CRC. Unfortunately, due to multi-drug resistance and immune deterioration, more than 40% of CRC patients will experience tumor recurrence and ultimately lead to patient death. Therefore, there is an urgent need to develop new strategies to improve the clinical chemotherapy efficacy of CRC.

[0003] Recently, more and more studies have demonstrated a strong correlation between CRC chemotherapy recurrence and specific gut microbiota. Among them, Fusobacterium nucleatum (F. nucleatum, Fn) is specifically enriched in colon adenomas and colorectal cancers and has become the focus of CRC diagnosis and treatment. F. nucleatum is a highly adherent and invasive anaerobic bacterium that usually exists as a major member of the subgingival flora in the oral cavity. However, after hematogenous dissemination, F. nucleatum localizes in CRC by recognizing Gal-GalNAc overexpressed in colorectal cancer, can regulate multiple cell signaling pathways and activate the autophagy pathway, improve CRC chemotherapy resistance and promote the proliferation of CRC cells. Currently, antibiotics are mainly used to inhibit the growth of F. nucleatum, but they will disrupt the balance of the gut microbiota. In addition, high doses of antibiotics are required to effectively eliminate bacteria, leading to the emergence of antibiotic-resistant bacteria. Therefore, there is an urgent need to introduce non-antibiotic antibacterial agents with antibacterial effects against F. nucleatum to solve the CRC drug resistance problem. Summary of the Invention

[0004] To solve the problem of CRC chemotherapy drug resistance, the present invention provides a preparation method of a nano-micelle for reducing-responsive sensitized tumor immunochemotherapy. The nano-micelle prepared by this method integrates antibacterial and anti-tumor functions, can reverse the chemotherapy drug resistance of tumor cells, and jointly inhibit the efferocytosis of macrophages to enhance the efficacy of tumor immunochemotherapy.

[0005] The present invention also provides a nano-micelle for reducing-responsive sensitized tumor immunochemotherapy and its application.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a preparation method of a nano-micelle for reducing-responsive sensitized tumor immunochemotherapy, and the preparation method includes:

[0008] Under an inert atmosphere, fluorouracil is dissolved in an organic solvent, and then 4-dimethylaminopyridine and triphosgene are added. After stirring for activation, a mixed solution is obtained.

[0009] Under an inert atmosphere, 2-hydroxyethyl disulfide dissolved in an organic solvent is dropped into the mixed solution. After the reaction is completed, the solvent is removed, and through silica gel column chromatography, a fluorouracil intermediate FUDR-SS containing a disulfide bond is obtained.

[0010] Lauric acid is dissolved in an organic solvent, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine are added. After stirring for activation, a reaction solution is obtained.

[0011] The FUDR-SS is dissolved in an organic solvent and then dropped into the reaction solution. The reaction is carried out in the dark. After purification by silica gel column chromatography, a reduction-responsive drug-drug conjugate FL is obtained.

[0012] The FL and niclosamide are co-dissolved in an organic solvent, and the resulting mixed solution is uniformly dispersed in water. After dialysis, a nanomicelle FL@NIC is obtained.

[0013] Furthermore, the step of under an inert atmosphere, dissolving fluorouracil in an organic solvent, then adding 4-dimethylaminopyridine and triphosgene, and obtaining a mixed solution through stirring activation specifically includes:

[0014] Under an inert atmosphere, fluorouracil is dissolved in tetrahydrofuran, then 4-dimethylaminopyridine and triphosgene are added, and stirring activation is carried out for 1 ± 0.5 h under light-shielded conditions to obtain a mixed solution.

[0015] Among them, the molar ratio of fluorouracil, 4-dimethylaminopyridine, and triphosgene is 1:(2 - 3):1 / 3.

[0016] Furthermore, the step of under an inert atmosphere, dropping 2-hydroxyethyl disulfide dissolved in an organic solvent into the mixed solution, removing the solvent after the reaction is completed, and obtaining a fluorouracil intermediate FUDR-SS containing a disulfide bond through silica gel column chromatography specifically includes:

[0017] Under an inert atmosphere, 2-hydroxyethyl disulfide dissolved in tetrahydrofuran is dropped into the mixed solution, and stirring reaction is carried out for 12 ± 2 h. Then the solvent is removed by rotary evaporation. Using a mixed solvent of dichloromethane and methanol with a volume ratio of 10:1 as the eluent, separation and purification are carried out through silica gel column chromatography, and then the solvent is evaporated to dryness to obtain a fluorouracil intermediate FUDR-SS containing a disulfide bond.

[0018] Among them, the molar ratio of fluorouracil to 2-hydroxyethyl disulfide is 1:3.

[0019] Further, dissolving lauric acid in an organic solvent, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, and stirring and activating to obtain a reaction solution, specifically including:

[0020] Dissolve lauric acid in dimethyl sulfoxide, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, and stir and activate at room temperature for 30 ± 10 min to obtain a reaction solution;

[0021] Among them, the molar ratio of lauric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine is 1:1.5:1.5.

[0022] Further, dissolving the FUDR-SS in an organic solvent and then dropping it into the reaction solution, reacting in the dark, and purifying by silica gel column chromatography to obtain the reduction-responsive drug-drug conjugate FL, specifically including:

[0023] Dissolve the FUDR-SS in dimethyl sulfoxide and then drop it into the reaction solution, stir in the dark at room temperature for 24 ± 3 h, and purify by silica gel column chromatography to obtain the reduction-responsive drug-drug conjugate FL;

[0024] Among them, the molar ratio of the FUDR-SS to lauric acid in the reaction solution is 1:1.

[0025] Further, dissolving the FL and niclosamide together in an organic solvent, uniformly dispersing the obtained mixed solution in water, and performing dialysis to obtain the nanomicelle FL@NIC, specifically including:

[0026] Dissolve the FL and niclosamide together in dimethyl sulfoxide, slowly add the obtained mixed solution to water, then stir and perform ultrasonic treatment, and then perform dialysis with a 1000 Da dialysis bag to obtain the nanomicelle FL@NIC;

[0027] Among them, the mass ratio of the FL to the niclosamide is (3 - 5):1.

[0028] Based on the same inventive concept, the present invention provides a nanomicelle for reducing-responsive sensitized tumor immunochemotherapy, and the nanomicelle is prepared by the preparation method of the nanomicelle for reducing-responsive sensitized tumor immunochemotherapy as described above.

[0029] Alternatively, the present invention provides a nanomicelle for reducing-responsive sensitized tumor immunochemotherapy, the nanomicelle includes an amphiphilic molecule and niclosamide, the amphiphilic molecule encapsulates the niclosamide inside by self-assembly, the hydrophilic end of the amphiphilic molecule is fluorouracil, the hydrophobic end is lauric acid, and the hydrophilic end and the hydrophobic end are connected by a disulfide bond.

[0030] Based on the same inventive concept, the present invention provides an application of a reduction-responsive tumor immunochemotherapy-sensitizing nanomicelle in the preparation of a therapeutic drug for Fusobacterium nucleatum-infected colorectal cancer or its related diseases.

[0031] Based on the same inventive concept, the present invention provides an application of a reduction-responsive tumor immunochemotherapy-sensitizing nanomicelle in the preparation of an anti-Fusobacterium nucleatum drug and / or a drug for inhibiting efferocytosis.

[0032] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0033] 1. The preparation method of a reduction-responsive tumor immunochemotherapy-sensitizing nanomicelle of the present invention connects lauric acid with antibacterial function and the anticancer drug fluorouracil through a disulfide bond. The obtained amphiphilic molecule self-assembles in water by hydrophilic-hydrophobic interaction and encapsulates the hydrophobic TMEM 16F inhibitor niclosamide in the hydrophobic cavity to prepare the nanomicelle FL@NIC. FL@NIC can break the disulfide bond under the stimulation of high glutathione (GSH) in the tumor microenvironment, and at the same time release fluorouracil, lauric acid, and niclosamide. The preparation method of the nanomicelle of the present invention is simple and easy to implement, provides a method for chemotherapy resistance treatment, and further provides new ideas for the clinical application of tumor immunity.

[0034] 2. The reduction-responsive tumor immunochemotherapy-sensitizing nanomicelle of the present invention is an amphiphilic molecule constructed with the anticancer drug fluorouracil (FUDR) as the hydrophilic block, the disulfide bond as the redox-sensitive fragment, and the antibacterial drug lauric acid (LA) as the hydrophobic block, and encapsulates the hydrophobic TMEM 16F inhibitor niclosamide (NIC) to form the reduction-responsive micelle FL@NIC. FL@NIC releases fluorouracil, lauric acid, and niclosamide in the tumor microenvironment. Among them, LA can change the membrane permeability by interacting with the bacterial cell membrane, resulting in the leakage of intracellular substances and ultimately leading to the death of F. nucleatum, overcoming CRC chemotherapy resistance. At the same time, NIC can inhibit the externalization of tumor cell PS, block the efferocytosis of TAMs (macrophages), convert tumor cell apoptosis into secondary necrosis, and relieve the immune deterioration caused by tumor cell apoptosis, thereby improving the CRC chemotherapy immune efficiency in a two-pronged manner.

[0035] 3. The nano - micelles for reducing - responsiveness - enhanced tumor immunochemotherapy of the present invention, the FL@NIC micelles have a uniform particle size of 176.4 nm, which can reduce the toxic and side effects of FUDR on normal cells and achieve the chemotherapeutic effect of reducing toxicity and increasing efficacy. FL@NIC has good stability and can be stored for a long time at 4°C. The reducing - responsiveness micelles FL@NIC prepared by the present invention integrate antibacterial and anti - tumor functions. Through the enhanced permeability and retention (EPR) effect of tumor tissues, the nano - drugs are enriched in tumor sites. Under the action of high glutathione, the micelles dissociate, and the simultaneous release of FUDR, LA, and NIC can be achieved. The synergistic effect of FUDR, LA, and NIC can reverse the chemoresistance of tumor cells and jointly inhibit the efferocytosis of macrophages, synergistically enhancing the efficacy of tumor immunochemotherapy. Brief Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following - described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0037] Figure 1 It is the synthetic process route of the nano - micelles FL prepared by the present invention.

[0038] Figure 2 It is the 1H - NMR spectra of FUDR, LA, and FL.

[0039] Figure 3 It is the particle size and zeta potential diagram of FL@NIC.

[0040] Figure 4 It is the transmission electron microscopy (TEM) image of FL@NIC.

[0041] Figure 5 It is the in vitro simulated drug - release curve of FL@NIC under different concentrations of dithiothreitol (DTT).

[0042] Figure 6 It is the detection result of the antibacterial activity of FL@NIC.

[0043] Figure 7 It is the in vitro cytotoxicity of each substance against CT26 at different concentrations.

[0044] Figure 8 It is the ability of each substance to inhibit phosphatidylserine (PS) externalization.

[0045] Figure 9 It is the ability of each substance to inhibit the efferocytosis of macrophages. Detailed Embodiments

[0046] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are for illustrating the present invention rather than limiting the present invention.

[0047] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any conflict, this specification shall prevail.

[0048] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0049] Unless otherwise specifically stated, the experimental methods used in the following examples are all conventional methods.

[0050] The overall idea of the present invention is as follows:

[0051] The main mechanism by which chemotherapy kills tumor cells is to induce tumor cell apoptosis. During the process of tumor cell apoptosis, "phagocytic" signals will be released, that is, membrane phosphatidylserine (PS) flips from the inner side of the lipid membrane to the outer side, and then triggers macrophage efferocytosis. Specifically, macrophages recognize the externalized "phagocytic" signal PS on apoptotic cells through the MerTK phagocytic receptor and phagocytose and clear apoptotic tumor cells. The inventors found that: a large number of immunosuppressive cytokines will be released during the above phagocytic clearance process, deteriorating the immunosuppression of the tumor microenvironment and severely weakening the efficacy of chemotherapy; interestingly, if apoptotic tumor cells cannot be quickly recognized and cleared by macrophages in a timely manner, they will lose cell membrane integrity and turn into secondary necrosis. Contrary to apoptosis, secondary necrosis will produce various tumor-associated antigens (TAEs) and damage-associated molecular patterns (DAMPs), thereby recruiting immune cells to absorb and process TAEs and receive further stimulation by DAMPs, inducing an anti-tumor immune response to synergistically inhibit tumor growth with chemotherapy. Therefore, the inventors believe that blocking macrophage efferocytosis, making apoptotic cells unable to be quickly recognized and cleared by macrophages, and transforming apoptotic cells into necrotic cells may be a more attractive strategy to relieve the immune deterioration caused by chemotherapy and thus improve the efficacy of chemotherapy.

[0052] Based on this, the present invention provides a reducing-responsive and sensitizing nano micelle for tumor immuno-chemotherapy and a preparation method thereof. The nano micelle is an amphiphilic molecule constructed with the anti-cancer drug FUDR as the hydrophilic block, disulfide bond as the redox-sensitive fragment, and the antibacterial drug LA as the hydrophobic block, and encapsulates the hydrophobic TMEM 16F inhibitor NIC to form the reducing-responsive micelle FL@NIC. FL@NIC can release FUDR, LA, and NIC in the tumor microenvironment. Among them, LA can change the membrane permeability through its interaction with the bacterial cell membrane, leading to the leakage of intracellular substances and ultimately the death of F. nucleatum, overcoming CRC chemotherapy resistance. At the same time, NIC can inhibit the externalization of tumor cell PS, block the efferocytosis of TAMs, convert tumor cell apoptosis into secondary necrosis, and relieve the immune deterioration caused by tumor cell apoptosis. Both approaches significantly improve the chemo-immunotherapy efficiency of CRC.

[0053] Specifically, the present invention provides a preparation method of a reducing-responsive and sensitizing nano micelle for tumor immuno-chemotherapy, and the preparation method includes:

[0054] S1. Under an inert atmosphere, dissolve fluorouracil in an organic solvent, then add 4-dimethylaminopyridine and triphosgene, and stir for activation to obtain a mixed solution;

[0055] S2. Under an inert atmosphere, drop 2-hydroxyethyl disulfide dissolved in an organic solvent into the mixed solution. After the reaction is completed, remove the solvent and perform silica gel column chromatography to obtain the fluorouracil intermediate FUDR-SS containing a disulfide bond;

[0056] S3. Dissolve lauric acid in an organic solvent, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, and stir for activation to obtain a reaction solution;

[0057] S4. Dissolve the FUDR-SS in an organic solvent and then drop it into the reaction solution, react under light avoidance conditions, and perform silica gel column chromatography purification to obtain the reducing-responsive drug-drug conjugate FL;

[0058] S5. Dissolve the FL and niclosamide in an organic solvent together, uniformly disperse the obtained mixed solution in water, and perform dialysis to obtain the nano micelle FL@NIC.

[0059] Step S1 specifically includes:

[0060] Under an inert atmosphere, dissolve fluorouracil in tetrahydrofuran, then add 4-dimethylaminopyridine and triphosgene, and stir for activation for 1 ± 0.5 h under light avoidance conditions to obtain a mixed solution;

[0061] Among them, the molar ratio of fluorouracil, 4-dimethylaminopyridine, and triphosgene is 1:(2 - 3):1 / 3.

[0062] In the present invention, triphosgene is mainly used to introduce a carbonyl functional group to form a carbonate intermediate, and 4-dimethylaminopyridine is mainly used as a catalyst to increase the reaction rate. Stirring the mixed solution for 1 ± 0.5 h under light-shielded conditions can effectively improve the yield and selectivity of the subsequent reaction.

[0063] In the present invention, the molar ratio of fluorouracil, 4-dimethylaminopyridine, and triphosgene being 1:(2 - 3):1 / 3 has the advantage that triphosgene is the reactive reagent of the reaction, and its appropriate use can ensure sufficient reaction with the hydroxyl group in fluorouracil to generate the desired intermediate. As a catalyst, the excessive use of 4-dimethylaminopyridine can ensure that there is always enough catalyst in the reaction system to promote the reaction, thereby improving the reaction efficiency, reducing side reactions, increasing the product purity, and enhancing the controllability of the reaction.

[0064] Step S2 specifically includes:

[0065] Under an inert atmosphere, 2-hydroxyethyl disulfide dissolved in tetrahydrofuran is dropped into the mixed solution, and the mixture is stirred and reacted for 12 ± 2 h. Then, the solvent is removed by rotary evaporation. Using a mixed solvent of dichloromethane and methanol with a volume ratio of 10:1 as the eluent, it is separated and purified by silica gel column chromatography, and then the solvent is evaporated to dryness to obtain the fluorouracil intermediate FUDR-SS containing a disulfide bond.

[0066] Among them, the molar ratio of fluorouracil to 2-hydroxyethyl disulfide is 1:3.

[0067] In the present invention, using a mixed solvent of dichloromethane and methanol with a volume ratio of 10:1 as the eluent has the advantage of having a good separation effect on the FUDR-SS intermediate. The molar ratio of fluorouracil to 2-hydroxyethyl disulfide being 1:3 has the advantage that the excessive 2-hydroxyethyl disulfide can react fully with fluorouracil, reducing the residue of unreacted fluorouracil, thereby increasing the product purity.

[0068] Step S3 specifically includes:

[0069] Dissolve lauric acid in dimethyl sulfoxide, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, and stir and activate at room temperature for 30 ± 10 min to obtain a reaction solution.

[0070] Among them, the molar ratio of lauric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 4-dimethylaminopyridine is 1:1.5:1.5.

[0071] In the present invention, 1-ethyl-(3-dimethylaminopropyl)carbodiimide can increase the reaction activity of carboxylic acids, and with the catalytic effect of 4-dimethylaminopyridine, it can reduce the reagent dosage and reaction time required for the reaction, thereby increasing the reaction rate and yield.

[0072] In the present invention, the molar ratio of lauric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine is 1:1.5:1.5, which can ensure that the carboxylic acid group is effectively activated and has sufficient catalytic activity to promote the reaction.

[0073] Step S4 specifically includes:

[0074] Dissolve the FUDR-SS in dimethyl sulfoxide and then drop it into the reaction solution, stir in the dark at room temperature for 24 ± 3 h, and purify by silica gel column chromatography to obtain the reduction-responsive drug-drug conjugate FL;

[0075] Among them, the molar ratio of the FUDR-SS to lauric acid in the reaction solution is 1:1.

[0076] In the present invention, the advantage of the molar ratio of the FUDR-SS to lauric acid in the reaction solution being 1:1 is that it can ensure that the alcohol and acid in the reactants can react completely, avoiding side reactions or incomplete reactions caused by excess or deficiency.

[0077] Step S5 specifically includes:

[0078] Dissolve the FL and niclosamide in dimethyl sulfoxide, slowly add the obtained mixed solution to water, then stir and perform ultrasonic treatment, and then dialyze with a 1000 Da dialysis bag to obtain the nanomicelles FL@NIC;

[0079] Among them, the mass ratio of the FL to the niclosamide is (3 - 5):1.

[0080] In the present invention, the purpose of slowly adding the mixed solution to water and then performing stirring and ultrasonic treatment is to accelerate the self-assembly process and promote drug encapsulation. Dialyzing with a 1000 Da dialysis bag can remove the unencapsulated free drugs and residual organic solvents, further improving the purity of the drug-loaded micelles.

[0081] In the present invention, the mass ratio of the FL to the niclosamide being (3 - 5):1 has a high encapsulation efficiency and drug loading capacity, and can reduce the loss of raw materials.

[0082] Next, the preparation method of a nanomicelle for reducing-responsive sensitized tumor immunochemotherapy of the present invention will be described in detail in combination with examples and experimental data.

[0083] Example 1

[0084] As Figure 1 shown, this example provides a preparation method of a nanomicelle for reducing-responsive sensitized tumor immunochemotherapy, which is specifically as follows:

[0085] (1) Preparation of fluorouracil intermediate (FUDR-SS) containing a reduction-responsive bond: Under an atmosphere of nitrogen, first dissolve 100 mg of fluorouracil in 10 mL of tetrahydrofuran solvent. Then, add 124.1 mg of DMAP (4-dimethylaminopyridine) and 40.17 mg of solid triphosgene respectively. After stirring for 1 h in the dark, slowly drip 188 mg of 2-hydroxyethyl disulfide dissolved in anhydrous tetrahydrofuran into the above solution. After stirring overnight, remove the solvent by rotary evaporation. Then, using dichloromethane:methanol (10:1) as the developing agent, perform silica gel column chromatography to obtain the fluorouracil intermediate (FUDR-SS).

[0086] (2) Preparation of amphiphilic drug-drug conjugate: Dissolve 31.47 mg of lauric acid in DMSO, add 45.17 mg of EDC·HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and 28.79 mg of DMAP respectively, and stir at room temperature for 30 min for activation. Then, weigh 67 mg of the disulfide-containing fluorouracil intermediate (FUDR-SS) prepared in step (1) and dissolve it in dimethyl sulfoxide (DMSO). Dropwise add it to the above system and stir in the dark at room temperature for 24 h. After purification by silica gel column chromatography, obtain the reduction-responsive drug-drug conjugate (FL).

[0087] (3) Preparation of FL@NIC nanomicelles: Dissolve 4.25 mg of FL and 1.06 mg of NIC in DMSO. Slowly add the resulting mixed solution to 5 mL of deionized water, stir at room temperature for 30 min, perform ultrasonic treatment at 100 W for 10 min, and then transfer this solution to a dialysis bag (MWCO = 1000 Da) and dialyze with deionized water for 24 h to obtain the reduction-responsive micelles FL@NIC. The encapsulation efficiency of NIC is 71.44%, and the drug loading rate is 14.29%.

[0088] Example 2

[0089] In this example, the micelles obtained in Example 1 were subjected to a functional test, which is specifically as follows:

[0090] (1) Proton nuclear magnetic resonance (1H NMR)

[0091] 1H NMR was measured on a Bruker AVANCE III 400 MHz nuclear magnetic resonance spectrometer using DMSO-d6 as the solvent and trimethylsilane (TMS) as the internal standard.

[0092] The measurement results are as Figure 2, in the 1H NMR spectrum, it can be found by comparison that the signal peak of the primary hydroxyl group on the original FUDR (5.15 ppm) completely disappears in the product. Compared with the spectrum of LA, the hydrogen on the methylene group connected to -COOH shows a slight shift. Compared with the 1H NMR of FUDR, the proton peak on the hydroxyl group disappears, and at the same time, the methylene group connected to the primary hydroxyl group shows a displacement, which proves the successful synthesis of FL.

[0093] (2) Determination of particle size, zeta potential and polydispersity index (PDI)

[0094] The nanomicelles FL@NIC with a concentration of 1 mg / mL were aspirated into the particle size cell and zeta potential cell, and the particle size, zeta potential and PDI of the samples were investigated by a Malvern particle size analyzer.

[0095] The results are as Figure 3 shown. The particle size of the FL@NIC nanomicelles is 176.4 ± 3.174 nm, and the PDI is 0.130 ± 0.028, which can passively target the tumor site through the enhanced permeability and retention (EPR) effect. In addition, the zeta potential of the micelles is -29.3 ± 1.243 mV, indicating that it can stably exist in the solution.

[0096] (3) Transmission electron microscopy (TEM) images

[0097] The prepared nanomicelles were diluted by an appropriate multiple, dropped onto a 200-mesh copper grid. After the water was dried, an appropriate amount of 2% phosphotungstic acid was added and stained for 1 min. The negative staining solution was aspirated, and it was dried at room temperature. Then, the morphological characteristics of the micelles were observed by TEM.

[0098] The results are as Figure 4 shown. The FL@NIC micelles are regular spherical in shape, with uniform size, and the particle size is about 170 nm, which is basically consistent with the results of the hydrodynamic diameter.

[0099] (4) In vitro release of nanomicelles

[0100] First, a standard curve of FUDR was established. 10 mg of FUDR was accurately weighed and dissolved in ultrapure water to prepare a stock solution of 1 mg / mL. Then, it was successively diluted to standard solutions with concentrations of 100, 80, 50, 40, 20, 10, and 1 μg / mL. The absorbance of FUDR at a wavelength of 269 nm was measured. With the absorbance value as the ordinate and the concentration as the abscissa, the FUDR standard curve equation was obtained: y = 0.0168x + 0.0179, R 2 = 0.9994.

[0101] The standard curve of NIC was established by first preparing a stock solution at 1 mg / mL, and then successively diluting it with ethanol to standard solutions at concentrations of 30, 25, 20, 15, 10, 5, 2, and 1 μg / mL. The absorbance of NIC at a wavelength of 335 nm was measured. With the absorbance value as the ordinate and the concentration as the abscissa, the standard curve equation of NIC was obtained: y = 0.0782x - 0.0094, R 2 = 0.9998.

[0102] The in vitro drug release behavior was measured by dialysis. PBS solution containing 2% Tween 80 was used as the release medium. 1 mg / mL of the nanomicelles was placed in a dialysis bag (cut-off molecular weight Mw = 1000 Da), and then the dialysis bag was placed in the release medium containing different concentrations (0, 5, 10 mM) of DTT. It was shaken at a constant temperature of 37°C. At predetermined time points, 2 mL of the release medium was taken and an equal volume of the release medium was added. The cumulative drug release rates of FUDR and NIC were calculated by converting to concentration using the standard curve.

[0103] The results are as Figure 5 shown. At a DTT concentration of 10 mM, the release amounts of both FUDR and NIC were significantly higher than those at low DTT concentrations. The cumulative release amount of FUDR reached 51.83% at 72 h, and the cumulative release amount of NIC was 84.51% at 72 h.

[0104] (5) Detection of the antibacterial activity of nanomicelles

[0105] The antibacterial activity of the nanomicelles was detected by the spread plate method. The bacteria F. nucleatum grown on a solid culture plate were transferred to a liquid medium and cultured under anaerobic conditions at 37°C for 24 h. The absorbance of OD 600 was measured with an enzyme-linked immunosorbent assay (ELISA) reader and diluted to 1×10 8 CFU / mL with physiological saline. Then, different concentrations of LA and FL@NIC were added to the above bacterial solution, and it was continuously cultured overnight under anaerobic conditions at 37°C. Subsequently, the bacterial concentration was measured, diluted with an appropriate dilution factor, and 50 μL of the diluted bacterial suspension was spread on a solid agar plate. After culturing at 37°C for 16 h, the number of colonies was observed and the survival rate was calculated.

[0106] The results are as Figure 6 shown. As the concentration of the antibacterial molecule LA increased, the survival rate of F. nucleatum gradually decreased and the antibacterial effect gradually enhanced. At the same time, FL@NIC also showed antibacterial activity similar to that of LA. As the concentration of LA increased, the inhibitory effect of FL@NIC on F. nucleatum gradually enhanced. When the concentration of LA was 90 μg / mL, the antibacterial rate reached 90%.

[0107] (6) In vitro cytotoxicity of nanomicelles

[0108] The CCK8 method was used to evaluate the toxicity of FUDR, FL, and FL@NIC to CT26 cells in the presence and absence of Fusobacterium nucleatum infection.

[0109] CT26 cells were seeded in 96-well plates at a density of 7×10 3 / well and cultured. When the cell density reached over 80%, the culture medium in the wells was removed and the cells were washed with 1×PBS. Then, Fusobacterium nucleatum suspension was added to make the MOI 50:1, and the cells were cultured with medium without antibiotics for 6 h. Next, different concentrations of the samples to be tested were added and co-cultured with CT26 cells for 24 h. The blank control group was added with fresh medium. After continuing to culture in the incubator for 24 h, the CCK8 method was used to determine the cell viability.

[0110] The results were as Figure 7 shown. After Fusobacterium nucleatum infection, the anti-tumor effect of the FUDR group weakened, while the FL and FL@NIC groups still had good anti-tumor effects after Fusobacterium nucleatum infection.

[0111] (7) Ability of nanomicelles to inhibit PS externalization

[0112] CT26 cells were seeded in six-well plates at a density of 3×10 5 / well and cultured for 24 h. The culture medium in the wells was removed and the cells were washed with 1×PBS. Then, FUDR, FL, FUDR+NIC, and FL@NIC were added respectively and incubated for 24 h. After that, the cells were collected, incubated with AnnexinV-FITC for 10 minutes to label the exposed PS, loaded onto a flow cytometer, and the PS externalization of each group was measured and analyzed by Flowjo software.

[0113] The results were as Figure 8 shown. The percentage of AnnexinV-FITC positive cells in the FL@NIC group was significantly inhibited, effectively reducing the exposure of PS on the outer surface.

[0114] (8) Ability of nanomicelles to inhibit macrophage phagocytosis of apoptotic tumor cells

[0115] BMDMs were extracted from mouse bone marrow and plated in 12-well plates on the sixth day of induced culture. The cell density was 1×10 5 / The wells were seeded with cells and incubated overnight. After that, apoptotic tumor cells induced by different formulations were stained with DiI and then added to BMDMs, and they were co-cultured for 4 h under specific conditions. The cells were collected, and macrophages were stained and labeled with FITC-CD11b antibody. Then the cells were resuspended in 1×PBS solution, and the phagocytosis of macrophages was analyzed by flow cytometry. The strength of phagocytosis was determined by the proportion of double-positive macrophages of DiI and CD11b in macrophages.

[0116] The results are as Figure 9 shown. After treatment with nanoparticles FL@NIC containing PS scramblase inhibitor, the phagocytic ability of BMDMs was blocked, indicating that FL@NIC can effectively inhibit the phagocytosis of apoptotic tumor cells by BMDMs while inducing apoptosis of tumor cells.

[0117] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0118] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0119] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing reduction-responsive nanomicelles for enhancing tumor immunochemotherapy, characterized in that: The preparation method comprises: Under an inert atmosphere, fluorouridine is dissolved in an organic solvent, and then 4-dimethylaminopyridine and triphosgene are added, and the mixture is activated by stirring to obtain a mixed solution; Under an inert atmosphere, 2-hydroxyethyl disulfide dissolved in an organic solvent is dropped into the mixed solution, and after the reaction is completed, the solvent is removed and subjected to silica gel column chromatography to obtain a disulfide-bonded fluorouridine intermediate FUDR-SS; Dissolving lauric acid in an organic solvent, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, and stirring to activate the mixture to obtain a reaction solution; The FUDR-SS is dissolved in an organic solvent and then added dropwise to the reaction solution, reacted in the dark, and purified by silica gel column chromatography to obtain a reduction-responsive drug-drug conjugate FL; The FL and niclosamide are dissolved in an organic solvent, and the obtained mixed solution is uniformly dispersed in water, and dialyzed to obtain nanomicelles FL@NIC.

2. The method for preparing a reduction-responsive nanomicelle for enhancing tumor immunochemotherapy according to claim 1, characterized in that: The method comprises dissolving fluorouridine in an organic solvent under an inert atmosphere, adding 4-dimethylaminopyridine and triphosgene, and stirring to activate the mixture to obtain a mixed solution, which specifically comprises: Under an inert atmosphere, fluorouridine was dissolved in tetrahydrofuran, and then 4-dimethylaminopyridine and triphosgene were added, and the mixture was stirred and activated for 1±0.5h under light-proof conditions to obtain a mixed solution; The molar ratio of fluorouridine, 4-dimethylaminopyridine and triphosgene is 1:(2-3):1 / 3.

3. The method for preparing a reduction-responsive nanomicelle for enhancing tumor immunochemotherapy according to claim 1, characterized in that: The method comprises the following steps: adding 2-hydroxyethyl disulfide dissolved in an organic solvent to the mixed solution under an inert atmosphere, removing the solvent after the reaction is completed, and performing silica gel column chromatography to obtain a disulfide-bonded fluorouridine intermediate FUDR-SS, which specifically comprises: Under an inert atmosphere, 2-hydroxyethyl disulfide dissolved in tetrahydrofuran is dropped into the mixed solution, and the mixture is stirred to react for 12±2 hours, and then the solvent is removed by rotary evaporation, and a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1 is used as a developing solvent, and the mixture is separated and purified by silica gel column chromatography, and then the solvent is evaporated to obtain a disulfide bond-containing fluorouridine intermediate FUDR-SS; The molar ratio of fluorouridine to 2-hydroxyethyl disulfide is 1:

3.

4. The method for preparing a reduction-responsive nanomicelle for enhancing tumor immunochemotherapy according to claim 1, characterized in that: The method comprises dissolving lauric acid in an organic solvent, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, and stirring and activating the mixture to obtain a reaction solution, which specifically comprises: Dissolve lauric acid in dimethyl sulfoxide, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, and stir and activate at room temperature for 30±10 minutes to obtain a reaction solution; The molar ratio of lauric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine is 1:1.5:1.

5.

5. The method for preparing a reduction-responsive nanomicelle for enhancing tumor immunochemotherapy according to claim 1, characterized in that: The FUDR-SS is dissolved in an organic solvent and then added dropwise to the reaction solution, reacted in the dark, and purified by silica gel column chromatography to obtain a reduction-responsive drug-drug conjugate FL, which specifically includes: The FUDR-SS is dissolved in dimethyl sulfoxide and then added dropwise to the reaction solution, stirred at room temperature in the dark for 24±3 hours, and purified by silica gel column chromatography to obtain a reduction-responsive drug-drug conjugate FL; Wherein, the molar ratio of the FUDR-SS to the lauric acid in the reaction solution is 1:

1.

6. The method for preparing a reduction-responsive nanomicelle for enhancing tumor immunochemotherapy according to claim 1, characterized in that: The FL and niclosamide are dissolved in an organic solvent, the obtained mixed solution is uniformly dispersed in water, and the nano-micelles FL@NIC are obtained by dialysis, which specifically comprises: The FL and niclosamide were dissolved in dimethyl sulfoxide, and the resulting mixed solution was slowly added into water, followed by stirring and ultrasonic treatment, and then dialyzed with a 1000Da dialysis bag to obtain nanomicelles FL@NIC; Wherein, the mass ratio of the FL to the niclosamide is (3-5):

1.

7. A reduction-responsive nanomicelle for enhancing tumor immunochemotherapy, characterized in that: The nanomicelles are prepared by the method for preparing reduction-responsive tumor immunochemotherapy-enhancing nanomicelles according to any one of claims 1 to 6.

8. A reduction-responsive nanomicelle for enhancing tumor immunochemotherapy, characterized in that: The nano micelles contain amphiphilic molecules and niclosamide, wherein the amphiphilic molecules encapsulate the niclosamide inside by self-assembly, the hydrophilic end of the amphiphilic molecules is fluorouridine, the hydrophobic end is lauric acid, and the hydrophilic end and the hydrophobic end are connected by a disulfide bond.

9. Use of the reduction-responsive tumor immunochemotherapy-enhancing nanomicelles as claimed in claim 7 or 8 in the preparation of therapeutic drugs for colorectal cancer infected with Fusobacterium nucleatum or related diseases thereof.

10. Use of the reduction-responsive tumor immunochemotherapy-enhancing nanomicelle according to claim 7 or 8 in the preparation of anti-Fusobacterium nucleatum drugs and / or drugs for inhibiting cell thrombosis.

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