A gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate, prodrug nanomicelle, preparation method and application

Abstract

The present application relates to the technical field of biological medicine, in particular to a gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate, a prodrug nanomicelle, a preparation method and an application. The conjugate covalently connects gemcitabine and NLG919 through a connecting arm containing a triazole structure, forms an amphiphilic molecule with a hydrophilic-hydrophobic structure, and can self-assemble into stable small-size nanomicelles in an aqueous solution. The nanomicelle has a pH and enzyme dual-responsive drug release property, can improve the stability and delivery efficiency of gemcitabine, promote cell uptake, and enhance the synergistic antitumor effect of chemotherapy and immunomodulation. The present application also provides an application of the nanomicelle in the preparation of an antitumor drug.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate, a prodrug nanomicelle, its preparation method, and its application. Background Technology

[0002] Cancer is a disease caused by abnormal cell proliferation, and its harm is mainly manifested in the following aspects: First, cancer destroys the structure and function of normal cells, leading to organ failure; second, the invasion and metastasis of cancer can affect multiple organs, increasing the difficulty of treatment.

[0003] Currently, drug therapy is one of the main treatment methods for cancer, with chemotherapy and immunotherapy being common approaches. Each has its own advantages and limitations. Chemotherapy works by using chemical drugs to inhibit or kill tumor cells. Chemotherapy drugs are used at various stages, including preventing cancer recurrence, inhibiting cancer cell metastasis, and accelerating tumor cell death. The advantages of chemotherapy are its rapid onset of action and wide applicability. However, because chemotherapy drugs lack selectivity in their distribution within the body, they easily cause various toxic side effects such as inflammation, hair loss, and vomiting, causing patients considerable suffering, while the therapeutic effect is often unsatisfactory. Furthermore, the drug resistance of tumor cells to chemotherapy drugs is also a significant factor limiting its effectiveness.

[0004] Given that pancreatic cancer is one of the deadliest malignant tumors, with a five-year survival rate of only about 10% and a median survival of less than six months, GEM has been a first-line and gold standard drug for treating pancreatic cancer for many years. However, its widespread drug resistance, chemotherapy side effects, and metabolic stability still need improvement. Therefore, there is an urgent need to develop more effective anti-pancreatic cancer drugs.

[0005] In recent years, the combined use of chemotherapy and immunotherapy has been considered to produce synergistic anti-tumor effects. However, due to significant differences in the physicochemical properties, in vivo distribution, and pharmacokinetic behavior of chemotherapeutic drugs and immunomodulatory drugs, achieving efficient co-delivery of the two remains challenging. While existing nanocarrier systems can achieve multi-drug co-loading, they typically suffer from problems such as complex carriers, unstable drug loading, and difficulty in precisely controlling release. Therefore, there is an urgent need to develop a prodrug system with a controllable structure that can self-assemble and achieve synergistic delivery of chemotherapeutic and immunomodulatory drugs to improve the efficacy of anti-tumor therapy. Summary of the Invention

[0006] To address the problem of efficient synergistic delivery of chemotherapy drugs and immunomodulatory drugs in existing technologies, this invention provides a gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate, prodrug nanomicelles, preparation method, and application.

[0007] This invention is achieved through the following technical solution: A gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate, wherein the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate is formed by covalently linking gemcitabine and indoleamine 2,3-dioxygenase inhibitor through a linker arm containing a 1,2,3-triazole ring.

[0008] The linker arm comprises: a 1,2,3-triazole ring formed by a click chemical reaction of an alkynyl group and an azide group; and an alkyl chain connected to both sides of the triazole structure.

[0009] The gemcitabine is connected to the linker arm via an amide bond, and the indoleamine 2,3-dioxygenase inhibitor is connected to the linker arm via an ester bond.

[0010] The conjugate comprises a hydrophilic gemcitabine fragment and a hydrophobic indoleamine 2,3-dioxygenase inhibitor fragment, and is amphiphilic and capable of self-assembling into a nanostructure in aqueous solution.

[0011] Preferably, the general structural formula of the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate is as follows: .

[0012] Where n is an integer between 0 and 10; m is an integer between 0 and 10.

[0013] Preferably, when n=0 and m=0, the structure of the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate is shown in Formula I.

[0014] Formula I.

[0015] The method for preparing the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate as described in Formula I includes the following steps: (1) NLG919, N,N-dicyclohexylcarbodiimide, azidopentanoic acid and DMAP are acylated in an organic solvent to obtain NLG919 azidopentanoic acid; the molar ratio of NLG919, N,N-dicyclohexylcarbodiimide, azidopentanoic acid and 4-dimethylaminopyridine is 1:2:2:0.01~1.

[0016] (2) Gemcitabine, tert-butyldimethylchlorosilane and imidazole are chemically reacted in an organic solvent and N2 atmosphere to obtain target compound I-1; the molar ratio of gemcitabine, tert-butyldimethylchlorosilane and imidazole is 1:2~5:2~5.

[0017] (3) 4-pentyneic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and target compound I-1 are reacted in an organic solvent to obtain target compound I-2; the molar ratio of 4-pentyneic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and target compound I-1 is 1:1~5:1~5:1~5.

[0018] (4) The target compound I-2 is reacted with tetrabutylammonium fluoride in an organic solvent to obtain the target compound I-3; the molar ratio of the target compound I-2 to tetrabutylammonium fluoride is 1:1~8.

[0019] (5) Using copper sulfate pentahydrate and sodium ascorbate as catalysts, the target compound I-3 and NLG919 azide are chemically reacted to obtain an amphiphilic GEM-NLG919 conjugate; the molar amounts of copper sulfate pentahydrate, VcNa, target compound I-3 and NLG919 azide are 0.01~0.1:0.05~0.2:1:1; the solvent used in the reaction is a mixed solvent of tetrahydrofuran and water with a volume ratio of 4:1; the reaction temperature is 40℃~60℃.

[0020] Preferably, the organic solvent is dichloromethane, N,N-dimethylformamide, or tetrahydrofuran.

[0021] A prodrug nanomicelle, wherein the prodrug nanomicelle is prepared by self-assembly of the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate.

[0022] Preferably, the method includes the following steps: The gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate was added to dimethyl sulfoxide to prepare a GEM-NLG919 stock solution with a concentration of 30.0 mg / mL to 200.0 mg / mL.

[0023] The GEM-NLG919 stock solution was added directly or after being mixed with a mixed solvent to a rotating PBS to obtain prodrug nanomicelles.

[0024] Preferably, the mixed solvent is composed of PEG300 and Tween 80; the volume ratio of PEG300 to Tween 80 is 8:1.

[0025] Preferably, the pH of the PBS is 7.4.

[0026] Preferably, the rotation speed is 1000rpm~3000rpm.

[0027] The application of the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate or the prodrug nanomicelles in the preparation of antitumor drugs is characterized in that the antitumor effect includes the synergistic effect of chemotherapy and immunomodulation.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention presents the first design and synthesis of a novel amphiphilic GEM-NLG919 conjugate. This conjugate simultaneously incorporates a hydrophilic gemcitabine (GEM) fragment and a hydrophobic IDO inhibitor NLG919 fragment into its molecular structure, covalently coupled via a cleavable responsive linker to form a clearly defined amphiphilic structure. Based on this unique design, the conjugate can spontaneously self-assemble in aqueous solution to form structurally stable and uniformly sized nanomicelles without the need for additional carriers, thus simplifying the formulation process. Furthermore, the responsive linkers in the conjugate can cleave under the acidic conditions of the tumor microenvironment and the action of specific enzymes, enabling time-controlled release of GEM and NLG919, improving targeted drug release efficiency at the tumor site and reducing systemic toxicity. By covalently modifying GEM into this prodrug form, its metabolic stability is significantly enhanced, particularly its resistance to cytidine deaminase is improved, which is beneficial for prolonging in vivo circulation time and improving bioavailability. This structure integrates chemotherapy and immunomodulatory functions within a single molecule, ensuring co-delivery and synergistic release of the two in vivo, providing a new drug design basis for chemotherapy-immunotherapy combination therapy for malignant tumors such as pancreatic cancer.

[0029] Furthermore, this invention utilizes the amphiphilic GEM-NLG919 conjugate to prepare pH / enzyme dual-responsive self-assembled nanoprodrugs GNNMs. A systematic evaluation of the self-assembly performance, stability, release performance, in vitro anticancer activity, delivery efficiency, hemolysis, and in vivo tumor-suppressive activity of these nanomicelles confirmed that this amphiphilic molecule-based self-assembled nanoprodrug system significantly improves the metabolic stability and targeting of the parent drug GEM, enabling rapid cellular uptake, promoting DC maturation, and achieving targeted accumulation at tumor sites. It also enhances its immunogenic cell death-inducing effect, ultimately exhibiting significantly enhanced antitumor activity. Compared to free GEM and free NLG919, at the same dosage, the prodrug nanoformulation GNNMs showed more effective anticancer activity in vivo and effectively reduced the toxic side effects of GEM. This invention provides a new drug candidate for cancer treatment and demonstrates the potential application value of nanotechnology-based self-assembled prodrugs in the field of cancer therapy. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a diagram illustrating the synthesis process of the azide NLG919 of this invention.

[0032] Figure 2 This is a diagram illustrating the synthesis process of compound I of the present invention.

[0033] Figure 3 This is the proton NMR spectrum of compound I of the present invention.

[0034] Figure 4 This is the carbon NMR spectrum of compound I of the present invention.

[0035] Figure 5 This is the mass spectrum of compound I of the present invention.

[0036] Figure 6 This is a graph showing the critical micelle concentration of compound I determined by the pyrene fluorescent probe spectrometry method of this invention.

[0037] Figure 7 The images show the self-assembly of the amphiphilic compound I of the present invention into small-sized nanomicelles (GNNMs); wherein, A) is a size diagram of GNNMs; B) is a morphology diagram of GNNMs; and C) is a size diagram of GNNMs after being placed at room temperature for seven days.

[0038] Figure 8 This is a graph showing the in vitro hemolytic toxicity detection of the GNNMs of this invention (n=3).

[0039] Figure 9 The graph shows the biochemical index results of mice after treatment with GNNMs according to this invention.

[0040] Figure 10 This is a drug release diagram of the GNNMs of the present invention under pH 5.0 and pH 7.4 and papain conditions.

[0041] Figure 11 This is a graph showing the metabolic stability of the GNNMs of this invention in vitro.

[0042] Figure 12 The diagram shows the CCK8 cell proliferation experiment of this invention; wherein, A) the in vitro antitumor proliferation activity of GNNMs on SW1990 cells; B) the in vitro antitumor proliferation activity of GNNMs on 4T1 cells; C) the IC50 assay of GNNMs on SW1990 cells. 50Value graph.

[0043] Figure 13 This is a diagram of the cellular uptake behavior of GNNMs of the present invention; wherein, A) flow cytometry images during 4h co-incubation of free DOX and GNNMs / DOX; B) cell positivity rate during 4h co-incubation of free DOX and GNNMs / DOX; C) intracellular fluorescence intensity results.

[0044] Figure 14 The figures show the in vivo antitumor effects of the nanomicelles GNNMs of this invention on tumor-bearing mice; where A) tumor volume changes after treatment of SW1990 cell subcutaneous tumor model with GNNMs; B) representative tumor images; and C) mouse weight changes.

[0045] Figure 15 This figure shows the in vivo antitumor effect of the GNNM nanomicelles of this invention on tumor-bearing mice.

[0046] Figure 16 This is a diagram showing the results of tumor cell apoptosis induced by GNNMs of this invention.

[0047] Figure 17 This is a distribution diagram of the GNNMs of this invention in mice. Detailed Implementation

[0048] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0049] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0050] The synthetic route for the amphiphilic gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate of this invention uses gemcitabine (GEM) as the starting material. First, the 2′,3′-hydroxyl group of GEM is protected with tert-butyldimethylchlorosilane (TBSCl) in the presence of imidazole to obtain a double-TBS-protected intermediate I-1, thus avoiding side reactions of the hydroxyl group in the sugar ring during subsequent reactions. Subsequently, intermediate I-1 undergoes amide coupling with a carboxylic acid compound containing a terminal alkyne group under EDCI / HOBt mediation, introducing an alkyne functional side chain into the N4 position of gemcitabine to obtain intermediate I-2. Then, the TBS protecting group is removed using TBAF to restore the free hydroxyl group in the sugar ring structure, generating the click-reactive alkylated gemcitabine intermediate I-3. On the other hand, using the immune checkpoint inhibitor NLG919 as the parent nucleus, an esterification reaction is carried out between the hydroxyl group in its structure and a carboxylic acid derivative with an azide terminal to construct a functionalized ligand containing an azide group. Finally, under Cu catalysis, the terminal alkyne group on I-3 undergoes a Cu(I)-catalyzed azido-alkyne cycloaddition reaction with the NLG919-derived azide functional ligand, forming a stable 1,2,3-triazole linkage structure, ultimately yielding gemcitabine-NLG919 conjugated dual prodrugs. This entire route, through strategies such as hydroxyl protection, side-chain coupling, deprotection, and click chemistry splicing, achieves precise covalent integration of chemotherapeutic drugs and IDO inhibitors, providing an effective chemical construction method for building bifunctional prodrug systems with both self-assembly properties and synergistic chemotherapy-immunotherapy functions.

[0051] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0052] Gemcitabine (GEM) was purchased from Shanghai Aladdin Chemical Reagent Co., Ltd.

[0053] The indoleamine 2,3-dioxygenase inhibitor NLG919 was purchased from Shanghai Myriel Co., Ltd.

[0054] Example 1: A conjugate of an amphiphilic gemcitabine-indoleamine 2,3-dioxygenase inhibitor (1) Synthesis of NLG919 azide A clean, dried 25 mL flask was used as the reaction flask. 28.2 mg of NLG919 was accurately weighed and dissolved in 3 mL of dichloromethane. Then, N,N-dicyclohexylcarbodiimide (DCC, 41.3 mg) and azidovallic acid (28.6 mg) were added sequentially to the flask. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 4-dimethylaminopyridine (DMAP, 6.2 mg). The flask was sealed and kept in the dark at room temperature for 3 hours. The reaction was then stopped. The mixture was extracted twice with 10.0 mL of saturated sodium bicarbonate and 20.0 mL of dichloromethane, respectively. The organic phase was collected and extracted again with 10.0 mL of brine. The organic phase was collected, dried with anhydrous sodium sulfate for at least 1 hour, filtered, and then evaporated to dryness. 2.0 mL of ethyl acetate and 10.0 mL of petroleum ether were added, and the mixture was allowed to stand for 6 hours to remove byproducts. The mixture was then filtered again and evaporated to dryness. Further purification was performed using silica gel column chromatography with DCM / CH3OH (50:1) as the eluent, yielding a pale yellow oily product, namely NLG919 azide, in 75.8% yield. The synthesis process is as follows: Figure 1 As shown.

[0055] (2) Take a 25.0 mL round-bottom flask, add a suitable magnetic flask, Gem (263.0 mg), and 4.0 mL of N,N-dimethylformamide (DMF), and slowly add tert-butyldimethylchlorosilane (TBSCl, 602.4 mg) and imidazole (273.0 mg). Seal the flask and insert a balloon filled with N2. Let it react at room temperature until the reaction is complete as detected by TLC. After stopping the reaction, add 30.0 mL of distilled water and 20.0 mL of ethyl acetate for extraction. Collect the upper organic phase and extract the lower aqueous phase twice with ethyl acetate. After collecting all the organic phase, dry it with anhydrous sodium sulfate for one hour, filter it, and concentrate it under vacuum. Finally, the crude organic phase was loaded onto a silica gel column using a wet method. Silica gel was soaked in 100.0 mL of dichloromethane with 1.0 mL of triethylamine (used to neutralize the acidic silica gel). The triethylamine was extracted using the same amount of dichloromethane. A dichloromethane:methanol (100:3) solution was used as the eluent, and a dichloromethane:methanol (10:1) solution was used as the developing solvent. Color development was performed using a UV spectrophotometer. After purification, a white solid was obtained, which was the target compound I-1, with a yield of 63%.

[0056] (3) Take a dry 25.0 mL round-bottom flask and place a magnetic stir bar inside. Under ice bath conditions, add tetrahydrofuran (THF, 1.5 mL), 4-pentyneic acid (58.8 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 191.6 mg), and 1-hydroxybenzotriazole (HOBt, 135.0 mg) in sequence and react for 5 minutes. Then transfer to room temperature and add I-1 (49.1 mg) dissolved in 2.0 mL THF. Stir the mixture at room temperature for 24 hours, remove the solvent, take a small amount of ethyl acetate to wash the magnetic stir bar, clean the organic phase, add 15.0 mL of ethyl acetate to dissolve, then add 15.0 mL of water, transfer to a separatory funnel, shake slowly and vent repeatedly, take the upper layer of the organic phase, extract the aqueous phase twice with ethyl acetate (10.0 mL each time), dry the collected organic phase with anhydrous Na2SO4, filter the solid, and concentrate under vacuum. The target compound I-2 was obtained by dry loading onto a silica gel column using petroleum ether:ethyl acetate = 2:1 as the eluent, followed by color development under a UV spectrophotometer. After purification, a white solid was obtained, with a yield of 33.0%.

[0057] (4) In a clean 25.0 mL round-bottom flask, add a suitable magnetic stir bar, then add compound I-2 (113.0 mg), THF (3.0 mL), and tetrabutylammonium fluoride (TBAF, 0.5 mL) to obtain an orange solution. Stir the orange solution at room temperature to allow it to react fully for 40 minutes; then wash the magnetic stir bar with dichloromethane, concentrate the mixture under vacuum, and retain the organic phase; by using thin-layer column chromatography, dry loading, pure dichloromethane soaked in silica gel, and using dichloromethane:methanol (10:1) as the eluent, a white solid is obtained, which is the functional target compound I-3, with a yield of 58.0%.

[0058] (5) Take a dry Schlenk tube, place a clean small magnetic plunger inside, apply a small amount of Vaseline to the stopcock, add I-3 (41.2 mg) to the Schlenk tube, seal it, turn on the water pump, and vent the air three times. Dissolve NLG919 (40.7 mg) in 3.2 mL of THF and inject it into the Schlenk tube. Dissolve copper sulfate pentahydrate (1.3 mg) in 0.8 mL of water and sodium ascorbate (VcNa, 2.0 mg) in the Schlenk tube using a syringe. Seal the tube and protect it from light. React in an oil bath at 60 degrees Celsius for 3 hours, then stop the reaction and evaporate to dryness. Further purify the tube using silica gel column chromatography with DCM / CH3OH (30:1) as the eluent. The white solid obtained is compound I, namely the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate (GEM-NLG919 conjugate), with a yield of 51.4%. The synthesis process of compound I is as follows: Figure 2 As shown, the proton NMR spectrum is as follows: Figure 3 As shown, the carbon NMR spectrum is as follows: Figure 4 As shown, the mass spectrum is as follows Figure 5 As shown.

[0059] Example 2: Preparation method of prodrug nanomicelles GNNMs Prodrug nanomicelles (GNNMs) were prepared using the amphiphilic GEM-NLG919 conjugate obtained in Example 1 via a coprecipitation method. The specific preparation method is as follows: An amphiphilic GEM-NLG919 conjugate was added to dimethyl sulfoxide (DMSO) to prepare a GEM-NLG919 stock solution with a concentration of 38.0 mg / mL. 2.0 mL of PBS (pH 7.4) was added to a centrifuge tube. Then, the GEM-NLG919 stock solution (2.0 μL) was mixed with a 0 μL mixture of PEG300 and Tween 80, and slowly added dropwise to the PBS being centrifuged at 1000 rpm. The mixture was filtered to obtain prodrug nanomicelles, labeled GNNMs. The mixed solvent consisted of PEG300 and Tween 80 in a volume ratio of 8:1.

[0060] Example 3: Preparation method of prodrug nanomicelles GNNMs Prodrug nanomicelles (GNNMs) were prepared using the amphiphilic GEM-NLG919 conjugate obtained in Example 1 via a coprecipitation method. The specific preparation method is as follows: An amphiphilic GEM-NLG919 conjugate was added to dimethyl sulfoxide (DMSO) to prepare a GEM-NLG919 stock solution with a concentration of 200.0 mg / mL. 1060.0 µL of pH 7.4 PBS was added to a centrifuge tube. Then, 40.0 µL of the GEM-NLG919 stock solution was mixed with 900.0 µL of a mixed solvent of PEG300 and Tween 80, and slowly added dropwise to the PBS in the centrifuge tube while it was being spun at high speed (3000 rpm). The mixture was filtered to obtain prodrug nanomicelles, labeled GNNMs. The mixed solvent consisted of PEG300 and Tween 80 in a volume ratio of 8:1.

[0061] Experimental Example 1 I. Size and Morphology Characterization of Prodrug Nanomicelles (GNNMs) 1. Determination of Critical Micelle Concentration (CMC) The CMC of the amphiphilic GEM-NLG919 conjugate in Example 1 was determined using pyrene fluorescent probe spectroscopy. First, aqueous solutions of the amphiphilic GEM-NLG919 conjugate at different concentrations were prepared and added to a container containing the fluorescent probe pyrene. The mixture was then sonicated at room temperature for 30 min to promote the formation of nanomicelles, followed by standing for two hours to ensure the micelles completed their self-assembly. The fluorescence emission spectrum at an excitation wavelength of 335 nm was then measured using a fluorescence spectrophotometer. The ratio I of the fluorescence intensity at emission wavelengths of 373 nm and 384 nm was calculated. 373 / I 384 and with I 373 / I 384 A graph was plotted against the logarithm of the concentration of the amphiphilic GEM-NLG919 conjugate solution to obtain a curve of the critical micelle concentration of the amphiphilic GEM-NLG919 conjugate. The critical micelle concentration of the amphiphilic GEM-NLG919 conjugate was then calculated from this curve. The results are as follows: Figure 6 As shown, the amphiphilic GEM-NLG919 conjugated molecules can form stable nanomicelles with a critical micelle concentration (CMC) of 0.8 μM.

[0062] 2. Particle size and surface potential determination The hydrated particle size and Zeta potential of the GNNMs nanomicelles obtained in Example 2 were measured. The results showed that the amphiphilic GEM-NLG919 conjugate could self-assemble in aqueous solution to form spherical GNNMs nanomicelles with a hydrated particle size of 6.5 nm and a surface potential of approximately -7.4 mV. Transmission electron microscopy results were consistent with this, clearly showing the formation of small particles within the nanomicelle range, such as... Figure 7 As shown in A and B in the figure. Studies have shown that smaller particles can more easily penetrate into the deep layers of tumor tissue. The negative charge of the nanomicelles and the negatively charged cell membrane and serum proteins exhibit a repulsive effect, which can generate electrostatic repulsion, which helps to prevent nanoparticle aggregation and thus shows good stability.

[0063] These results demonstrate that the amphiphilic GEM-NLG919 conjugate can spontaneously self-assemble into stable small-sized nanomicelles, effectively improving the solubility of hydrophobic therapeutic drugs in aqueous solutions. Furthermore, after storing the GNNM nanomicelles at room temperature for one week, no significant change in size potential was observed. Figure 7 As shown in C, this demonstrates the good stability of GNNMs nanomicelles.

[0064] The following method was used for TEM sample preparation: a 25.0 μM aqueous solution of GNNMs nanomicelles was prepared, and 5.0 μL of the sample was dropped onto a carbon-coated copper grid. The sample was dried in a constant temperature desiccator at 42 °C. Then, 1.0% uranium acetate solution was dropped onto the carbon-coated copper grid for staining for 30 s. Excess staining solution was blotted with filter paper and the sample was dried at room temperature. The morphology and size of the dried sample were detected using a transmission electron microscope.

[0065] II. Safety Study of Prodrug Nanomicelles (GNNMs) The following experiments were conducted using the GNNMs prepared in Example 2 as an example.

[0066] 1. Hemolysis test (1) Take 1.0 mL of fresh whole blood (EDTA-K2 anticoagulated blood) from healthy mice, collect red blood cells by centrifugation at 3500 rpm for 5 min, wash and centrifuge repeatedly with 1×PBS buffer at pH 5 until the supernatant is clear and colorless, and then resuspend the red blood cells in 15.0 mL of fresh PBS buffer. (2) 0.5 mL of red blood cell suspension was mixed with 0.5 mL of GNNMs micelle solution of different concentrations (1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0 μM) (dissolved in 1×PBS buffer) to obtain red blood cell suspensions with final concentrations of 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, or 50.0 μM. The above samples were placed at room temperature for 2 hours and then centrifuged at 3500 rpm for 5 min. The sample after mixing 0.5 mL of red blood cell suspension with 0.5 mL of PBS buffer was used as the negative control group; the sample after mixing 0.5 mL of red blood cell suspension with 0.5 mL of deionized water was used as the positive control group.

[0067] (3) Finally, 100 μL of the supernatant of each sample was transferred into a 96-well plate, and the absorbance of heme in the supernatant was detected by an enzyme-linked immunosorbent assay (ELISA) reader. The absorption wavelength was set to 540 nm.

[0068] The percentage of hemolysis is calculated using the following formula: Hemolysis percentage = [Sample absorbance - Negative control absorbance) / (Positive control absorbance - Negative control absorbance)] × 100%.

[0069] The results are as follows Figure 8 As shown, GNNMs did not exhibit significant hemolysis from low to high concentrations, indicating that GNNMs nanomicelles can be safely administered via intravenous injection without causing hemolysis.

[0070] III. Biosafety Experiments - In vivo toxicity testing of nanocarriers To assess the in vivo toxicity of GNNMs nanocarriers, 5-week-old C57BL / 6 mice were intravenously injected with either PBS buffer or GNNMs nanocarriers (20 mg / kg, i.e., 100 µL of 4 mg / mL GNNMs nanocarrier solution per mouse in the experimental group; and 100 µL of blank solvent in the solvent control group), with 3 mice in each group. Three hours later, the mice were sacrificed and their blood was collected. Serum samples were collected after centrifugation. Various biochemical indicators in the blood were detected using an automated biochemical analyzer, including alanine transferase (ALT) and gamma-glutamyl transferase (γ-GT) reflecting liver function, creatinine (CRE) and blood urea nitrogen (BUN) reflecting kidney function, and changes in blood cholesterol levels. Results are as follows: Figure 9 As shown, none of these parameters changed significantly compared to the control group, indicating that the formulation of the present invention was well tolerated at the doses used and did not exhibit significant liver or kidney damage.

[0071] IV. In vitro release study of prodrug nanomicelles GNNMs Controllable in vitro release of drugs is a crucial indicator for evaluating nanoprodrug nanoparticles. Ultraviolet spectrophotometry was used to detect the release of Gem from GNNMs nanomicelles prepared from the amphiphilic GEM-NLG919 conjugate under different pH conditions and in the presence and absence of papain. Cathepsin B (CTSB) is an important hydrolytic enzyme located in lysosomes and is often used as a model enzyme to study enzyme-responsive drug release. Papain has similar activity to CTSB. Compared to CTSB, papain is more readily available, and several studies have used it as a substitute for CTSB in studying enzyme-responsive drug release. Based on this, this invention uses papain to simulate CTSB to study the enzyme responsiveness of nanoprodrug nanoparticles.

[0072] First, the nano-prodrug was prepared according to the aforementioned method, and then the original content of the two drugs was calculated using the methanol demulsification method. Next, the enzyme was prepared: 1.5 mg of papain was added to 60.0 mL of PBS solution at pH 7.4 and pH 5.0, respectively. Two 20 mL tubes of each solution were then used as the external solution, and the remaining solution was used as the replenishment solution.

[0073] Soak the dialysis bags in boiling water for 30 minutes. Add 400.0 μL of nano-prodrug to each dialysis bag. Place the resulting solution in a shaker at 37°C for a total of 48 hours. Take 2.0 mL of the solution at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, and 48 h to detect UV radiation. At the same time, add 2.0 mL of replenishing solution. Calculate the cumulative release rate based on the obtained data and the standard curve.

[0074] The results are as follows Figure 10As shown, the drug release was greater at pH 5.0 than at pH 7.4; and the drug release was even greater in the presence of papain compared to the absence of papain, with more Gem cells being released continuously over time. This confirms that GNNMs micelles possess dual pH and papain-responsive drug release properties, and that this process is effective and sustainable. Therefore, it is speculated that GNNMs will exhibit stronger tumor permeability in the acidic and high-tissue-protease environment of tumor sites, and the micelles will show better stability in the circulatory system, thus enhancing their therapeutic effect.

[0075] V. Metabolic Stability Experiment of Prodrug Nanomicelles (GNNMs) The metabolic stability of GNNMs in vitro was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0076] (1) Dilute the deoxycytidine enzyme CDA kit to 0.25 μg / 50 μL with 50 mM Tris-HCl buffer (pH 7.4).

[0077] (2) Prepare 100.0 μM gemcitabine solution and 100.0 μM GNNMs solution using 50 mM Tris-HCl (pH 7.4) buffer.

[0078] (3) Use a pipette to pipette 150.0 μL of gemcitabine solution or GNNMs solution and add it to a 96-well plate. Then add 50.0 μL of the 0.3 μg / 50 μL CDA enzyme reagent prepared in (1) to each well. Set the absorption wavelength of the microplate reader to 280 nm, shake the multi-well plate for 2 minutes, and measure the absorbance of each well at 1 min, 2 min, 3 min, 4 min, 5 min, 20 min, 45 min, and 60 min.

[0079] The results are as follows Figure 11 As shown, the absorbance of the GEM group sample decreased over time in the presence of CDA, indicating that Gem underwent continuous enzymatic reduction. In contrast, the absorbance of the GNNMs group solution showed no significant difference between the presence and absence of CDA enzymes, and even increased over time, indicating that GNNMs possess sustained stability against CDA and suggesting that GEM was gradually released during the test. Therefore, the CDA metabolic stability of GNNMs is far superior to that of free GEM, which is beneficial for GNNMs to exert greater efficacy in vivo.

[0080] In summary, this invention prepared amphiphilic prodrug nanomicelles (GNNMs) based on nanotechnology using a co-precipitation method. These molecules can self-assemble into stable and uniformly sized near-spherical nanomicelles in aqueous solution. In vitro release assays showed that these nanomicelles exhibited good pH and papain-responsive drug release, indicating their efficient targeted release to tumor sites, thereby reducing toxic side effects on normal human tissues. In vitro CDA enzyme stability experiments showed that the construction of this molecule significantly improved the metabolic stability of GEM, suggesting that GNNMs may also exhibit enhanced stability and release more GEM in vivo, thus enhancing its antitumor effect. In vitro hemolysis assays and detection of liver and kidney-related biochemical indicators showed that this formulation did not cause hemolysis and did not exhibit toxic side effects to liver and kidney tissues, making it a relatively safe biological agent.

[0081] Experimental Example 2: In vitro activity evaluation of prodrug nanomicelles GNNMs The following experiments were conducted using GNNMs prepared in Example 2 as an example. The cells used in this experiment were human pancreatic cancer SW1990 cells and mouse breast cancer 4T1 cells, both of which were cryopreserved in liquid nitrogen in our laboratory.

[0082] I. Evaluation of the in vitro antiproliferative activity of prodrug nanomicelles GNNMs To detect the in vitro antiproliferative activity of prodrug nanomicelles GNNMs, the inhibitory effect of GNNMs on the proliferation of SW1990 and 4T1 cells was determined by the CCK8 assay.

[0083] 1. When the cells to be used in the experiment are in good condition as observed under a microscope, wash the cells and add trypsin to digest them to prepare a single-cell suspension. Then, accurately count the cells and seed them at 3500 cells / well, 100 μL / well in a 96-well cell culture plate. Each group has 5 replicates and two control groups: a cell-seeded group without drug (the other wells are simultaneously replaced with 200 μL / well DMEM on the second day when the drug is added), and a blank group without cells (no drugs are added initially, and CCK8 reagent is added simultaneously at the end). After the cells are seeded, they are placed in a 37°C constant temperature cell culture incubator with 5% CO2 for incubation.

[0084] 2. On the second day, drug / DMEM culture medium suspensions were prepared. Drug dilution was achieved using DMEM + 1% bispecific antibody SP. Four drugs were used (Gem, NLG919, a simple mixture of Gem and NLG919, and GNNM nanomicelles), and all drugs were prepared at concentrations ranging from 50 µM to 0.05 µM. In the Gem and NLG919 simple mixture, the concentrations of both drugs were identical, ranging from 50 µM to 0.05 µM. The culture medium in the 96-well plates was replaced with the drug-containing medium, 200 μL / well, and incubated for 72 hours.

[0085] 3. After the appropriate time for drug action, prepare a CCK8 detection solution containing 10% CCK8 solution and 90% complete culture medium. Discard the supernatant using a pipette (change pipette tips between different drug zones), and add the CCK8 detection solution (100 μL / well). Add the CCK8 detection solution immediately after aspirating the culture medium from each drug zone to prevent cell drying and death.

[0086] 4. After incubation at 37℃ for 1 hour, read the absorbance (A value) at 450 nm using a microplate reader. The formula for calculating cell proliferation activity is as follows:

[0087] Cell proliferation activity (%) = [A (drug-treated) - A (blank)] / [A (0-drug-treated) - A (blank)] × 100 Note: A (dosage added): Absorbance of wells containing cells, drug solution, and CCK8 solution; A (0 dosage added): Absorbance of wells containing cells and CCK8 solution but no drug solution; A (blank): Absorbance of wells containing culture medium and CCK8 solution but no cells or drug.

[0088] The results are as follows Figure 12 As shown in Figures A, B, and C, at the same dosage, GNNM nanomicelles exhibited tumor cell inhibition comparable to or even stronger than free GEM and the GEM / NLG919 physical mixture in SW1990 and 4T1 cells. This may be attributed to the rapid cellular uptake of nano-sized drugs. Since they are small-molecule prodrug nanoparticles, prodrugs are typically inactive in vitro and require chemical transformation in vivo to release their activity. Therefore, in in vivo experiments, GNNM prodrugs show promise in demonstrating stronger anti-tumor proliferation capabilities than the parent drug.

[0089] II. Evaluation of prodrug nanomicelle GNNMs cellular uptake rate Flow cytometry was used to evaluate the rate at which prodrug nanomicelles GNNMs were taken up by SW1990 cells.

[0090] 1. After preparing a single-cell suspension from SW1990 cells in good growth condition, accurately count the cells according to a ratio of 3 × 10⁻⁶ cells / cells. 5 Seeds were placed in 96-well cell culture plates at 2.0 mL / well and cultured overnight in a 37°C incubator containing 5% CO2.

[0091] 2. On the second day, drug / DMEM culture medium suspensions were prepared, containing two drugs (fluorescently labeled nanosystems GNNMs / DOX, and free fluorescently labeled DOX as a positive control), with a final DOX concentration of 11 µM in both groups. The culture medium in the six-well plates was replaced with drug-containing medium at a rate of 1.5 mL / well for incubation periods of 10 min, 20 min, 30 min, 1 h, 2 h, and 4 h. A negative control group (pure cells without drug) and a positive control group (DOX incubation for 4 h) were also established.

[0092] 3. After 10 minutes or the corresponding time of drug treatment, discard the supernatant and wash the cells three times with pre-cooled PBS. After digesting the cells with trypsin, collect the cells and transfer them to 15 mL centrifuge tubes, protecting them from light during the process. Collect the cells after centrifuging at 1000 rpm for 5 minutes.

[0093] 4. Add 0.5 mL / tube of 4% PFA to fix the cells, resuspend the cells thoroughly with a pipette, and incubate at room temperature for 15 min, avoiding light. After centrifugation at 1000 rpm for 5 min, discard the supernatant, add 0.5 mL / tube of PBS, resuspend the cells thoroughly, transfer the cell-PBS suspension to FACS tubes, and perform flow cytometry analysis.

[0094] Qualitative results of flow cytometry as follows Figure 13 As shown in Figure A, compared with the free DOX-treated tumor cell group, the GNNMs / DOX-treated group exhibited a stronger fluorescence signal, and the fluorescence intensity increased significantly over time, indicating that GNNMs have a higher internalization efficiency. Quantitative results from FCM confirmed this (e.g., Figure 13 As shown in B and C, compared to free DOX, GNNMs / DOX were taken up by cells faster than free DOX within the first 60 minutes. Figure 13 As shown in C, under the same conditions, it can be observed that the amount of GN / DOX taken up by tumor cells is significantly greater than the amount of free DOX taken up, further indicating that GNNMs loaded with DOX can be taken up by cells more efficiently than free DOX.

[0095] Experimental Example 3: In vivo activity study of prodrug nanomicelles GNNMs The following experiments were conducted using the GNNMs prepared in Example 2 as an example. The cells used in this experiment included human pancreatic cancer SW1990 cells that were cryopreserved in liquid nitrogen in the laboratory.

[0096] The experimental animals used in this experiment were 4-week-old female Balb / c nude mice purchased from Vital River Laboratories (Beijing, China), and were SPF grade.

[0097] All animal experimental protocols in this study were conducted in accordance with the guidelines provided by the Laboratory Animal Management and Use Committee of Xinxiang Medical University and approved by the Ethics Committee of Xinxiang Medical University (2019 / 11 / 20, XYLL-2019 S 017). The mice in this experiment were housed in an SPF (specific pathogen-free) animal facility authorized by Xinxiang Medical University, where temperature and humidity were maintained under normal conditions, and the mice had free access to water and food.

[0098] Construction of tumor-bearing mouse models: Pancreatic cancer cells SW1990 were subcutaneously inoculated into 5-week-old female BALB / c nude mice, and the tumors were allowed to grow to 100 mm². 3 At the time of administration, mice were injected via tail vein with gemcitabine, NLG919, a physical mixture of gemcitabine and NLG919, and GNNM nanomicelles, respectively. The dosage of each formulation was calculated based on the amount of gemcitabine or NLG919, both at 5 mg / kg. Five mice were administered per group. A control group was prepared by injecting only a mixture of dimethyl sulfoxide, PEG300, Tween 80, and PBS (volume ratio 1:20:2.5:26.5) containing the drugs.

[0099] Plotting tumor growth curves and mouse body weight change curves: Two days after drug administration, the mice's body weight was measured and changes were recorded to assess the toxicity of the five drugs. Simultaneously, tumor volume was measured and recorded: V = length × width. 2 / 2 to evaluate tumor proliferation. After 15 days, the experiment was stopped, mice were euthanized, and tumors (weighed and photographed), along with liver, kidneys, and other organs, were collected. After washing with PBS, half of the tumors were fixed in 4% paraformaldehyde solution for histopathological analysis. Graphad software was used to analyze and plot changes in mouse body weight and tumor volume.

[0100] 1. Prodrug nanomicelles (GNNMs) exhibit significantly enhanced antitumor activity. The in vivo antitumor activity of GNNM nanomicelles was evaluated using a tumor-bearing mouse model. SW1990 tumor-bearing mice were used as the model, with a solvent blank as a control. Mice were intravenously injected with the solvent blank, GEM, NLG919, a physical mixture of GEM / NLG919, and GNNMs, once every 4 days for a total of four injections. The dosage of each formulation was calculated based on the amount of gemcitabine or NLG919, both at 5 mg / kg. Tumor volume was measured every two days. The relative tumor volume changes in each group are shown in the figure below. Figure 16As shown in Figure A. Compared to the solvent-free group, immunotherapy with the IDO inhibitor NLG919 showed a weak tumor growth inhibition effect, while the chemotherapy drug GEM showed a moderate tumor-suppressing effect. The physical mixture of GEM and NLG919 was more effective than GEM or NLG919 alone. Notably, the nanomicelle GNNMs treatment group showed the strongest tumor-suppressing effect. During the experiment, changes in mouse body weight could indicate the presence of systemic toxicity. Figure 14 As shown in Figure C, compared with the control group, the mouse body weight did not change significantly, indicating that the GNNM nanomicelles have good safety. After the experiment, the mice were sacrificed, the tumors were removed, weighed, and photographed. The final tumor weight and volume of each group after drug treatment further verified that the GNNM nanomicelle treatment group had a more significant tumor-suppressing effect than other treatment groups (e.g., Figure 14 (B)

[0101] 2. Detection of liver and kidney tissue damage by GNNMs To further evaluate the relevant toxic side effects in different treatment groups, histological studies were performed using hematoxylin and eosin (H&E) staining. Figure 15 As shown, significant tissue damage was observed in both the liver and kidneys in the free Gem treatment group, indicating that GEM exhibits both hepatotoxicity and nephrotoxicity. Conversely, no significant tissue damage was observed in the kidneys or liver in the GNNMs treatment group compared to the control group. These results confirm that GNNMs offer enhanced safety compared to free Gem, possibly due to the EPR effect of the nanomedicine promoting tumor-specific uptake of GEM.

[0102] 3. GNNMs induce tumor cell apoptosis GNNM micelles exhibited significant inhibitory activity against tumor growth in tumor-bearing mice, suggesting that the drug's tumor-targeting aggregation and cytotoxic effects may induce tumor cell apoptosis. To verify the cytotoxicity of GNNM micelles, the terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) assay was used to determine apoptosis in tumor tissues after drug treatment. The TUNEL assay is a commonly used technique that reflects apoptosis by detecting DNA molecule breaks. Figure 16 As shown in Figure A, compared with the control group (NS and GEM groups), significantly enhanced TUNEL fluorescence signal was observed in the tumor tissue of the GNNMs-treated group. Semi-quantitative analysis results of fluorescence signal intensity (e.g., Figure 16 B) shows a significant difference in fluorescence signal intensity between the GNNMs-treated group and the GEM-treated group, indicating that GNNMs can induce a greater degree of tumor cell apoptosis.

[0103] 4. GNNMs enhance the drug targeting and specific accumulation in tumors. The distribution of GNNM nanomicelles in mice was investigated using a small animal in vivo imaging system. SW1990 cells were diluted to a concentration of 10. 7 100 μl of cell suspension was injected subcutaneously into the right foreleg of mice at a rate of 100 cells per milliliter. The mice's condition and tumor growth were monitored regularly. When the tumor reached 100 mm², [further action was taken]. 3 Mice were randomly divided into three groups. Since GNNMs nanomicelles are non-fluorescent, the in vivo imaging fluorescent dye DIR was used to label them. The three groups were: a negative control (IV injection of PBS without DIR via tail vein); a positive control (IV injection of DIR solution via tail vein); and an experimental group (IV injection of GNNMs / DIR solution via tail vein). The DIR dose was the same for both the positive control and experimental groups, at 40 µg / kg. Fluorescence signal intensity at different sites in the mice was detected and photographed at 2 h, 4 h, 8 h, 24 h, and 48 h after administration.

[0104] Using SW1990 tumor-bearing mice, the in vivo tumor-targeting drug delivery capability of GNNMs / DiR was observed using small animal in vivo imaging technology. Figure 17 As can be seen, both DiR and GNNMs / DiR exhibited strong fluorescent signals in the liver, which is because the liver is the main organ for drug metabolism. Furthermore, compared to free DiR, stronger fluorescent signals were observed at the tumor sites in the GNNMs / DiR group mice, indicating that the drug accumulated significantly and effectively at these sites, suggesting that GNNMs possess a stronger tumor-targeting effect.

[0105] It should be noted that the same experiment was performed using the nanomicelles GNNMs in Example 3, and the results were the same, so no further details will be provided.

[0106] In summary, this invention, through the in vivo antitumor activity study of GNNM nanomicelles in tumor-bearing mice, demonstrates that this prodrug molecule can significantly inhibit tumor cell growth in vivo without significant toxic side effects on normal tissues and organs. The enhanced in vivo antitumor activity may be related to the significantly improved tumor targeting, intratumoral aggregation, and tumor cell apoptosis-inducing ability of GNNMs. Combined with previous studies, another important reason for the highly efficient antitumor activity of GNNM nanomicelles may be attributed to the improved metabolic stability of GEM, preventing its rapid metabolism by CDA enzymes and increasing its circulation time in vivo. Furthermore, the amphiphilic prodrug nanomicelles GNNMs possess pH- and enzyme-responsive drug release properties, which facilitates the specific release of GEM and NLG919 from the micelles at the tumor site, thereby improving drug delivery efficiency and significantly enhancing the antitumor effect.

[0107] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate, characterized in that, The gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate is formed by covalently linking gemcitabine and indoleamine 2,3-dioxygenase inhibitor through a linker arm containing a 1,2,3-triazole ring. The linker arm comprises: a 1,2,3-triazole ring formed by a click chemical reaction of an alkynyl group and an azide group; and alkyl chains connected to both sides of the triazole structure; The gemcitabine is connected to the linker arm via an amide bond, and the indoleamine 2,3-dioxygenase inhibitor is connected to the linker arm via an ester bond. The conjugate comprises a hydrophilic gemcitabine fragment and a hydrophobic indoleamine 2,3-dioxygenase inhibitor fragment, and is amphiphilic and capable of self-assembling into a nanostructure in aqueous solution.

2. The gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate as described in claim 1, characterized in that, The general structural formula is as follows: Where n is an integer between 0 and 10; m is an integer between 0 and 10.

3. The method for preparing the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate according to any one of claims 1 to 2, characterized in that, When n=0 and m=0, the preparation of the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate includes the following steps: (1) NLG919, N,N-dicyclohexylcarbodiimide, azidopentanoic acid and DMAP are acylated in an organic solvent to obtain NLG919 azidopentanoic acid; the molar ratio of NLG919, N,N-dicyclohexylcarbodiimide, azidopentanoic acid and 4-dimethylaminopyridine is 1:2:2:0.01~1; (2) Gemcitabine, tert-butyldimethylchlorosilane and imidazole are chemically reacted in an organic solvent and under a N2 atmosphere to obtain the target compound I-1; the molar ratio of gemcitabine, tert-butyldimethylchlorosilane and imidazole is 1:2~5:2~5; (3) 4-pentyneic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and target compound I-1 are reacted in an organic solvent to obtain target compound I-2; the molar ratio of 4-pentyneic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and target compound I-1 is 1:1~5:1~5:1~5; (4) The target compound I-2 is reacted with tetrabutylammonium fluoride in an organic solvent to obtain the target compound I-3; the molar ratio of the target compound I-2 to tetrabutylammonium fluoride is 1:1~8; (5) Using copper sulfate pentahydrate and sodium ascorbate as catalysts, the target compound I-3 and NLG919 azide are chemically reacted to obtain an amphiphilic GEM-NLG919 conjugate; the molar amounts of copper sulfate pentahydrate, VcNa, target compound I-3 and NLG919 azide are 0.01~0.1:0.05~0.2:1:1; the solvent used in the reaction is a mixed solvent of tetrahydrofuran and water with a volume ratio of 4:1; the reaction temperature is 40℃~60℃.

4. The preparation method according to claim 3, characterized in that, The organic solvent is dichloromethane, N,N-dimethylformamide, or tetrahydrofuran.

5. A prodrug nanomicelle, characterized in that, The prodrug nanomicelles were prepared by self-assembly of the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate as described in claim 1.

6. The method for preparing prodrug nanomicelles as described in claim 5, characterized in that, Includes the following steps: The gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate was added to dimethyl sulfoxide to prepare a GEM-NLG919 stock solution with a concentration of 30.0 mg / mL to 200.0 mg / mL. The GEM-NLG919 stock solution was added directly or after being mixed with a mixed solvent to a rotating PBS to obtain prodrug nanomicelles.

7. The method for preparing prodrug nanomicelles as described in claim 6, characterized in that, The mixed solvent consists of PEG300 and Tween 80; the volume ratio of PEG300 to Tween 80 is 8:

1.

8. The method for preparing prodrug nanomicelles as described in claim 6, characterized in that, The rotation speed is 1000rpm~3000rpm.

9. The use of the gemcitabine-indoleamine 2,3-dioxygenase inhibitor conjugate as described in claim 1 or the prodrug nanomicelles as described in claim 5 in the preparation of therapeutic antitumor drugs, characterized in that, The anti-tumor drug is one that works synergistically with chemotherapy and immunomodulation.

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