Dendrimer prodrugs and nanoassemblies thereof

Abstract

The application provides a dendrimer prodrug and a nano-assembly thereof, and belongs to the technical field of antitumor drugs. The application constructs a dendrimer prodrug Epi-DBP shown in formula I by bridging PEG and Epi dendrimer conjugate through a borate ester bond. An IDO inhibitor NLG919 is loaded in the hydrophobic cavity of the Epi-DBP to form a nano-assembly NLG919@Epi-DBP. The nano-assembly NLG919@Epi-DBP has a stable nano structure and can be enriched at a tumor tissue. In an acidic environment of a tumor cell, a hydrazone bond is broken to cause the release of Epi and NLG919. Experimental results show that the NLG919 in the NLG919@Epi-DBP has excellent synergistic antitumor effect with the Epi-DBP. The application constructs an IDO inhibition combined with chemotherapy nano platform based on a dendrimer, realizes the remodeling of a tumor immune microenvironment while synergistically performing chemotherapy to enhance the antitumor effect, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a dendritic macromolecular prodrug and its nanoassembly. Background Technology

[0002] Immunotherapy, which stimulates the body's immune response to kill tumor cells, has emerged as a novel treatment approach in clinical practice. Immune checkpoint blockade (ICB)-based immunotherapy can modulate the tumor immune microenvironment and slow tumor progression to some extent. However, due to its weak immunogenicity and the tumor immunosuppressive microenvironment (TIME), the clinical response rate to ICB treatment is low, less than 20%. Furthermore, systemic administration of ICB often leads to immune-related side effects, hindering its application. Therefore, developing a novel strategy to improve ICB response rates by inducing a strong anti-tumor immune response is essential.

[0003] In immune regulation, immunosuppressive enzymes and immunosuppressive cells play crucial roles in maintaining tumor self-tolerance. Indoleamine 2,3-dioxgenase 1 (IDO1) is a cytoplasmic enzyme that metabolizes the essential amino acid L-tryptophan (Trp) into kynurenine (Kyn), promoting the activation of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs promote the polarization of tumor-associated macrophages into immunosuppressive M2-type macrophages. Simultaneously, Kyn inhibits the cytotoxic CD8+ of tumor cells. + The antitumor activity of T cells and natural killer (NK) cells is significant. Therefore, IDO1 plays a crucial role in suppressing the immune response. Blocking the IDO pathway to reprogram the tumor immune microenvironment is essential. Furthermore, achieving effective induction of immunogenic cell death (ICD) to enhance the immunogenicity of tumor cells is a necessary step in cancer treatment. During ICD, damage-associated molecular patterns (DAMPs) are released as natural adjuvants to initiate a series of immune responses, including promoting dendritic cell maturation, tumor-associated antigen delivery, and the initiation of adaptive antitumor immunity. Commonly used chemotherapeutic agents, such as anthracyclines, are often used as ICD inducers. Therefore, chemotherapy combined with IDO inhibition shows great potential in enhancing the immune response and remodeling the tumor microenvironment.

[0004] Despite the development of numerous drug delivery systems in cancer treatment, multiple physiological barriers in the body still limit drug accumulation, cellular internalization, and penetration into deeper tumor tissues, thus limiting therapeutic efficacy. The introduction of polyethylene glycol (PEG) can improve the stability of nanodelivery systems and prolong drug circulation time, but it can reduce drug uptake by tumor cells. Furthermore, targeted modification of the delivery system can improve tumor cell uptake efficiency, but may increase unnecessary immune recognition and clearance. Therefore, developing a stealthy nanodelivery system that shields the target group during circulation but exposes it in tumor tissue is of great significance for improving drug pharmacokinetics, enhancing tumor accumulation and tumor cell internalization, and improving the efficacy of cancer treatment. Summary of the Invention

[0005] One object of the present invention is to provide a dendritic macromolecular prodrug, and another object of the present invention is to provide a nanoassembly formed by the self-assembly of the above-mentioned dendritic macromolecular prodrug and an IDO inhibitor, as well as the preparation method and use thereof.

[0006] This invention provides a dendritic prodrug of epirubicin, the structure of which is shown in Formula I:

[0007]

[0008] Where n is 10-30;

[0009] The epirubicin dendritic macromolecular prodrug contains 3-10 wt% epirubicin.

[0010] Furthermore, the epirubicin content in the epirubicin dendritic macromolecular prodrug is 6.3 wt%.

[0011] The present invention also provides a nano-assembly formed by the self-assembly of the above-mentioned epirubicin dendritic macromolecular prodrug and an IDO inhibitor.

[0012] Furthermore, the IDO inhibitor is an IDO1 inhibitor.

[0013] Furthermore, the IDO1 inhibitor is NLG919.

[0014] Furthermore, in the nano-assembly, the mass ratio of epirubicin to IDO inhibitor is 1:(0.5-10).

[0015] Furthermore, in the nano-assembly, the mass ratio of epirubicin to IDO inhibitor is 1:(4-5).

[0016] The present invention also provides a method for preparing the above-mentioned nano-assemblies, the method comprising the following steps: mixing epirubicin dendritic macromolecular prodrug and IDO inhibitor uniformly in an organic solvent, dropping the mixture into water, and self-assembling to obtain nano-assemblies.

[0017] The present invention also provides the use of the above-mentioned nanoassemblies in the preparation of antitumor drugs.

[0018] Furthermore, the tumor is either colon cancer or breast cancer.

[0019] The present invention has achieved the following beneficial effects:

[0020] This invention constructs a phenylboronic acid (PBA)-shielded dendritic nanomedicine that integrates an immunogenic cell death (ICD) inducer (epirorubicin, Epi) and an indoleamine 2,3-dioxgenase 1 (IDO1) inhibitor (NLG919) for chemoimmunotherapy of tumors. The boronic acid ester-bridged polyethylene glycol-covalently linked Epi dendritic molecule (named Epi-DBP) loaded with NLG919 forms the assembly NLG919@Epi-DBP, which maintains long-term nanostructural stability during cycling.

[0021] This invention tested the killing effect of NLG919@Epi-DBP on CT26 (colon cancer) cells at different Epi to NLG919 mass ratios (1:0.5 to 1:10). It found that when the mass ratio of Epi to NLG919 was 1:4 and 1:5, NLG919 and Epi-DBP had excellent anti-tumor synergistic effects, with the strongest anti-tumor synergistic effect observed when the mass ratio of Epi to NLG919 was 1:5.

[0022] When NLG919@Epi-DBP reaches tumor tissue, the slightly acidic environment exposes the PBA group, which binds to sialic acid residues on the surface of tumor cells, promoting internalization and penetration into deeper tumor tissue. In the acidic environment within tumor cells, Epi-DBP rapidly disassembles, releasing the physically loaded NLG919 and covalently linked Epi. Epi induces a strong ICD effect in tumor cells and evokes a powerful immune response. Simultaneously, NLG919 inhibits IDO1 activity, reducing tryptophan (Trp) metabolism to kynurenine (Kyn), thereby reducing tumor cell recruitment of immunosuppressive cells and reshaping the tumor immune microenvironment. This invention constructs a dendritic molecular-based nanoplatform for IDO inhibition combined with chemotherapy, achieving tumor immune microenvironment reshaping while synergistically enhancing anti-tumor efficacy with chemotherapy, demonstrating broad application prospects.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 1H NMR spectrum of compound 7 in deuterated DMSO.

[0026] Figure 2 1H NMR spectrum of compound 8 in deuterated chloroform.

[0027] Figure 3 Mass spectrum of compound 8.

[0028] Figure 4 1H NMR spectrum of compound 10 in deuterated chloroform.

[0029] Figure 5 Mass spectrum of compound 10.

[0030] Figure 6 1H NMR spectrum of compound 11 in deuterated DMSO.

[0031] Figure 7 1H NMR spectrum of compound 16 in deuterated DMSO.

[0032] Figure 8UV absorption spectra and standard curves of DMSO solutions with different concentrations of Epi.

[0033] Figure 9 Stability of Epi-DP and Epi-DBP in aqueous solutions.

[0034] Figure 10 Particle size responsiveness of Epi-DP and Epi-DBP in PBS at different pH values.

[0035] Figure 11 Stability of .NLG919@Epi-DBP in aqueous solution.

[0036] Figure 12 Stability of .NLG919@Epi-DP in aqueous solution.

[0037] Figure 13 Drug release behavior in vitro in .NLG919@Epi-DBP.

[0038] Figure 14 Drug release behavior in vitro in .NLG919@Epi-DP.

[0039] Figure 15 Particle size responsiveness of NLG919@Epi-DP and NLG919@Epi-DBP in PBS at different pH values.

[0040] Figure 16 Cytotoxicity of CT26 by Epi.HCl, Epi-DP and Epi-DBP.

[0041] Figure 17 .NLG919 cytotoxicity against CT26.

[0042] Figure 18 Joint exponential curve.

[0043] Figure 19 Anti-tumor curve.

[0044] Figure 20 Tumor inhibition rate.

[0045] Figure 21 Changes in mouse body weight.

[0046] Figure 22 Mouse lifespan.

[0047] Figure 23 The maturation status of DCs in mouse lymph nodes after treatment.

[0048] Figure 24 The maturation status of DCs in the spleen of mice after treatment.

[0049] Figure 25 DC maturation status of mouse tumor tissue after treatment.

[0050] Figure 26 Interleukin-2 levels in tumor tissue of CT26 tumor-bearing mice after treatment.

[0051] Figure 27 Interleukin-6 levels in tumor tissue of CT26 tumor-bearing mice after treatment.

[0052] Figure 28 Interleukin-12 levels in tumor tissue of CT26 tumor-bearing mice after treatment.

[0053] Figure 29 .Interferon gamma levels in tumor tissue of CT26 tumor-bearing mice after treatment.

[0054] Figure 30 Tumor necrosis factor levels in tumor tissue of CT26 tumor-bearing mice after treatment.

[0055] Figure 31 CD4 in tumor tissue after treatment in CT26 tumor-bearing mice + T cell count.

[0056] Figure 32 CD8+ in tumor tissue of CT26 tumor-bearing mice after treatment + T cell count.

[0057] Figure 33 Water content of natural killer (NK) cells in tumor tissue after treatment in CT26 tumor-bearing mice.

[0058] Figure 34 Content of myeloid-derived suppressor cells (MDSCs) in tumor tissue after treatment in CT26 tumor-bearing mice.

[0059] Figure 35 Macrophage typing in tumor tissue of CT26 tumor-bearing mice after treatment.

[0060] Figure 36 The content of regulatory T cells (Treg cells) in tumor tissue after treatment in CT26 tumor-bearing mice. Detailed Implementation

[0061] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0062] Example 1: Synthesis of Epi-DBP

[0063] 1. Synthesis of phenylboronic acid-modified prodrug

[0064] The phenylboronic acid-modified prodrug, namely compound 7, was synthesized according to the following route:

[0065]

[0066] (1) Synthesis of second-generation (G2) lysine dendritic macromolecules

[0067] H-Lys-OMe.2HCl (5.0 g, 21.5 mmol), Boc-lys(Boc)-OH (22.3 g, 64.4 mmol), EDCI (12.3 g, 64.4 mmol), and 1-hydroxybenzotriazole (8.7 g, 64.4 mmol) were dissolved in anhydrous dichloromethane under a nitrogen atmosphere. DIEA (28.4 mL, 171.6 mmol) was slowly added to the mixture, and the mixture was stirred in an ice bath for 30 minutes. The reaction was allowed to continue at room temperature for 24 h. The reaction was monitored by thin-layer chromatography (TLC). The mixture was washed three times consecutively with saturated sodium chloride solution, hydrochloric acid (1 M), and saturated sodium bicarbonate solution. The organic phase was collected and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (ethyl acetate:dichloromethane = 1:1) after rotary evaporation to give compound 1 as a white solid in 88.6% yield. Compound 1 (5.0 g, 6.1 mmol) was dissolved in 10 mL of dichloromethane under a nitrogen atmosphere. Trifluoroacetic acid (18.3 mL, 244.9 mmol) was added, and the reaction was carried out for 12 h to remove the Boc group. The solvent was removed in diethyl ether (EE) to give compound 2.

[0068] (2) Functionalization of G2 lysine dendritic macromolecules

[0069] Compound 2 (2.0 g, 4.8 mmol), pinacol 4-carboxyphenylboronic acid (7.2 g, 29.0 mmol), EDCI (5.5 g, 28.7 mmol), and 1-hydroxybenzotriazole (3.9 g, 28.9 mmol) were dissolved in anhydrous N,N-dimethylformamide (DMF) under a nitrogen atmosphere. DIEA (12.7 mL, 76.6 mmol) was slowly added to the mixture, and the mixture was stirred in an ice bath for 30 min and reacted at room temperature for 36 h. The reaction was monitored by thin-layer chromatography. After the addition of dichloromethane, the mixture was washed three times successively with saturated sodium chloride solution, hydrochloric acid (1 M), and saturated sodium bicarbonate solution. The organic phase was collected and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (dichloromethane:methanol = 15:1) after rotary evaporation to give compound 3 as a white solid in a yield of 65.4%. Compound 3 (5.0 g, 6.1 mmol) was dissolved in sodium hydroxide / methanol (1 M, 30 mL) and stirred at room temperature for 4 h. The methoxy deprotection reaction was monitored by thin-layer chromatography. After removing methanol under vacuum, dichloromethane was added to dissolve the residue. Hydrochloric acid (1 M) was added to adjust the pH to 3. The organic phase was collected and dried over anhydrous sodium sulfate to give compound 4 as a white solid (yield: 88.4%).

[0070] Compound 4 (3.0 g, 2.3 mmol), tert-butyloxycarbazide (0.6 g, 4.5 mmol), EDCI (0.9 g, 4.7 mmol), and 1-hydroxybenzotriazole (0.6 g, 4.4 mmol) were dissolved in anhydrous DMF under a nitrogen atmosphere. DIEA (1.5 mL, 9.1 mmol) was slowly added to the mixture, and the mixture was stirred in an ice bath for 30 min and reacted at room temperature for 24 h. The reaction was monitored by thin-layer chromatography. After adding dichloromethane to the mixture, it was washed three times successively with saturated sodium chloride solution, hydrochloric acid (1 M), and saturated sodium bicarbonate solution. The organic phase was collected and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (dichloromethane:methanol = 10:1) after rotary evaporation to give compound 5 as a white solid in a yield of 76.7%. Compound 5 (2.0 g, 1.4 mmol) was dissolved in 5 mL of dichloromethane under a nitrogen atmosphere. Trifluoroacetic acid (5.2 mL, 69.8 mmol) was added and reacted for 12 h to remove the Boc and pinacol groups. The solvent was removed in an EE to precipitate compound 6.

[0071] (3) Synthesis of phenylboronic acid-modified dendritic prodrugs

[0072] Compound 6 (500.0 mg, 49.6 μmol) and Epi.HCl (345.1 mg, 59.5 μmol) were dissolved in methanol, and a catalytic amount of glacial acetic acid was added. The reaction was carried out at room temperature for 24 h. The organic solvent was removed by rotary evaporation, and unreacted Epi.HCl was removed by dialyzing in deionized water (molecular weight cutoff of 1000 Da) for 48 h. Compound 7 was lyophilized to obtain a red powder. The 1H NMR spectrum of compound 7 is shown below. Figure 1 As shown.

[0073] 2. Synthesis of dopamine-modified methoxylated polyethylene glycol compounds

[0074] The dopamine-modified methoxy polyethylene glycol compound, namely compound 10, was synthesized according to the following route:

[0075]

[0076] Methoxylated polyethylene glycol (4.0 g, 3.6 mmol), succinic anhydride (0.8 g, 7.5 mmol), DMAP (0.6 g, 5.0 mmol), and TEA (347 μL, 2.5 mmol) were dissolved in anhydrous DMF at 40 °C for 24 h. After adding dichloromethane, the solution was washed twice with hydrochloric acid (1 M). The organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was washed with acetone to give compound 8, a white waxy solid. The 1H NMR spectrum of compound 8 is shown below. Figure 2 As shown, the mass spectrum of compound 8 is as follows: Figure 3 As shown.

[0077] Compound 8 (4.0 g, 3.6 mmol), NHS (0.6 g, 5.5 mmol), and DCC (1.1 g, 5.5 mmol) were dissolved in tetrahydrofuran (THF) and reacted at room temperature for 12 h. After filtering the byproduct N,N'-bicyclohexaneurea (DCU), the solvent in the filtrate was removed under vacuum. The crude product was precipitated multiple times in EE to give compound 9 (yield: 80.5%). Compound 9 (3.0 g, 2.5 mmol) and DA were also reacted. Hydrochloric acid (0.6 g, 3.2 mmol) was dissolved in anhydrous DMF under a nitrogen atmosphere. TEA (521 μL, 3.8 mmol) was added, and the reaction was carried out at room temperature for 12 h. Dichloromethane was added, and the mixture was washed twice with hydrochloric acid (1 M). The organic phase was dried over anhydrous sodium sulfate. After removing the solvent, compound 10 was given as a white waxy solid with a yield of 77.7%. The 1H NMR spectrum of compound 10 is shown below. Figure 4 As shown, the mass spectrum of compound 10 is as follows: Figure 5 As shown. According to Figure 3 and Figure 5 It can be seen that n on polyethylene glycol in the molecule is 10-26.

[0078] 3. Synthesis of Epi-DBP

[0079] Epi-DBP, i.e., compound 11, was synthesized according to the following route:

[0080]

[0081] Compound 7 (300.0 mg, 195.7 μmol) and compound 10 (1103.4 mg, 880.6 μmol) were dissolved separately in deionized water to obtain aqueous solutions. The aqueous solution of compound 10 was added dropwise to the aqueous solution of compound 7 while stirring. The mixture was reacted in the dark for 24 hours. The mixture was transferred to a dialysis tube (Spectra / PorMWCO = 2000 Da) and dialyzed against deionized water for 48 hours. Compound 11 was lyophilized to obtain a red powder. The 1H NMR spectrum of compound 11 is shown below. Figure 6 As shown.

[0082] Example 2: Preparation of nanoassemblies NLG919@Epi-DBP

[0083] 20 mg of Epi-DBP and 6.3 mg of NLG919 were mixed in DMSO. The mixture was then sonicated and 1 mL of deionized water was added dropwise. After 30 min of self-assembly, a nano-assembly loaded with NLG919 was prepared and named NLG919@Epi-DBP. The mass ratio of Epi to NLG919 in the obtained NLG919@Epi-DBP was 1:5.

[0084] The following is the method for preparing the control sample.

[0085] Comparison with Example 1: Synthesis of Epi-DP

[0086] Epi-DP, or compound 16, was synthesized according to the following route:

[0087]

[0088] Compound 2 (0.5 g, 1.2 mmol), methoxy polyethylene glycol acetic acid (7.2 g, 7.2 mmol), EDCI (1.4 g, 7.3 mmol), and 1-hydroxybenzotriazole (1.0 g, 7.4 mmol) were dissolved in anhydrous dichloromethane under a nitrogen atmosphere. DIEA (3.2 mL, 19.3 mmol) was slowly added to the mixture, and the mixture was stirred in an ice bath for 30 minutes. The mixture was allowed to react at room temperature for 36 hours. The reaction was monitored by thin-layer chromatography. The mixture was washed three times consecutively with saturated sodium chloride solution, hydrochloric acid (1 M), and saturated sodium bicarbonate solution. The organic phase was collected and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (dichloromethane:methanol = 15:1) after rotary evaporation to give compound 12 as a white solid in a yield of 46.0%. Compound 12 (2.0 g, 460.4 μmol) was dissolved in sodium hydroxide / methanol (1 M, 4.6 mL) and stirred at room temperature for 4 hours. The methoxy deprotection reaction was monitored by thin-layer chromatography. Methanol was removed under vacuum, and dichloromethane was added to dissolve the residue. Hydrochloric acid (1 M) was added to adjust the pH to 3. The organic phase was collected and dried over anhydrous sodium sulfate to give compound 13 as a white solid (yield: 80.4%).

[0089] Compound 13 (1.5 g, 346.4 μmol), tert-butyloxycarbazide (68.6 mg, 519.3 μmol), EDCI (99.6 mg, 519.6 μmol), and 1-hydroxybenzotriazole (70.2 mg, 519.6 μmol) were dissolved in anhydrous dichloromethane under a nitrogen atmosphere. DIEA (229 μL, 1.4 mmol) was slowly added in an ice bath and stirred for 30 minutes. The mixture was allowed to react at room temperature for 24 hours. The reaction was monitored by thin-layer chromatography. The mixture was washed three times consecutively with saturated sodium chloride solution, hydrochloric acid (1 M), and saturated sodium bicarbonate solution. The organic phase was collected and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (dichloromethane:methanol = 15:1) after rotary evaporation to give compound 14 as a white solid, in a yield of 74.7%. Compound 14 (500.0 mg, 112.5 μmol) was dissolved in 84 μL of dichloromethane. Trifluoroacetic acid (84 μL, 1.1 mmol) was added, and the reaction was carried out for 12 hours to remove the Boc group. The solvent was removed in EE to precipitate compound 15.

[0090] Compound 15 (500.0 mg, 115.1 μmol) and Epi.HCl (100.1 mg, 172.6 μmol) were dissolved in methanol, and a catalytic amount of glacial acetic acid was added. The reaction was carried out at room temperature for 24 hours. The organic solvent was removed by rotary evaporation, and unreacted Epi.HCl was removed by dialyzing in deionized water (molecular weight cutoff of 1000 Da) for 48 hours. Compound 16 was lyophilized to give a red powder. The 1H NMR spectrum of compound 16 is shown below. Figure 7 As shown.

[0091] Comparative Example 2: Preparation of nanoassemblies NLG919@Epi-DP

[0092] 20 mg Epi-DP and 6.3 mg NLG919 were mixed in DMSO, and 1 mL of deionized water was added dropwise to the mixture under sonication. The mixture was then self-assembled for 30 minutes to prepare a nano-assembly loaded with NLG919, which was named NLG919@Epi-DP.

[0093] Comparative Example 3: Preparation of Empty Nanoassemblies

[0094] 2 mg of Epi-DP was dissolved in 20 μL of DMSO, and 1 mL of deionized water was added dropwise under ultrasonication. Empty nanoassemblies were prepared by self-assembly for 30 minutes.

[0095] The following specific experiments demonstrate the beneficial effects of the present invention. Unless otherwise specified, the NLG919@Epi-DBP sample used in the following experiments is the one obtained in Example 2.

[0096] I. Experimental Methods

[0097] 1. Physicochemical characterization

[0098] (1) Measurement of Epi loading

[0099] The Epi loading in Epi-DP or Epi-DBP was determined using UV-Vis spectrophotometry. Simply put, Epi-DP or Epi-DBP was weighed and dissolved in DMSO. The Epi content in Epi-DP or Epi-DBP was calculated using a standard curve plotted from the absorbance values ​​of Epi at 480 nm.

[0100] (2) Particle size distribution and morphology

[0101] At 25℃, the hydrated particle size of Epi-DP, Epi-DBP assemblies and NLG919-loaded assemblies (NLG919@Epi-DP, NLG919@Epi-DBP) at a concentration of 100 μg / mL was observed using a light scattering instrument. Each sample was measured three times.

[0102] An aqueous solution of Epi-DP, Epi-DBP assembly and NLG919-loaded assembly with a concentration of 1 mg / mL was prepared. The solution was dropped onto a copper grid and dried at room temperature. The microstructure of Epi-DP, Epi-DBP assembly and NLG919-loaded assembly was observed by transmission electron microscopy.

[0103] (3) Stability and responsiveness of the assembly in aqueous solution

[0104] Epi-DP, Epi-DBP, NLG919@Epi-DP, and NLG919@Epi-DBP were dispersed in deionized water or phosphate buffered saline (PBS, pH 7.4), respectively. The hydrated particle size was measured at different time points at 25°C to investigate the stability of each assembly in aqueous solution.

[0105] Epi-DP, Epi-DBP, NLG919@Epi-DP, and NLG919@Epi-DBP were dispersed in PBS at pH 7.4, 6.5, and 5.0, respectively. The hydrated particle size was measured at different time points at 25°C to investigate the particle size response of each assembly in different pH environments.

[0106] (4) In vitro drug release

[0107] NLG919@Epi-DP and NLG919@Epi-DBP were dissolved in 1 mL of PBS at different pH values ​​(7.4, 6.5, and 5.0), respectively. These solutions were then transferred to dialysis bags with a molecular weight cutoff of 1000 Da and immersed in centrifuge tubes containing 20 mL of PBS. The solutions were incubated at 37°C in a shaker. At each set time point, 1 mL of the solution was collected in an EP tube, and 1 mL of buffer solution at the corresponding pH was added. The fluorescence of the collected solution was measured using a multi-mode microplate reader, and the amount of Epi released was calculated using a standard curve method. The released NLG919 was determined by high-performance liquid chromatography (HPLC) using methanol / water containing 1‰ trifluoroacetic acid (v / v = 95 / 5) as the mobile phase at a flow rate of 0.5 mL / min. The amount of NLG919 released was calculated using a standard curve method. A drug release curve was plotted with incubation time on the x-axis and the cumulative release amount of Epi or NLG919 on the y-axis.

[0108] 2. Cell experiments

[0109] (1) Cell Culture

[0110] Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing antibiotics (100 U / mL penicillin and 100 U / mL streptomycin) and 10% fetal bovine serum and grown in a humid environment at 37°C with 5% CO2.

[0111] (2) Infiltrates into multicellular spheres

[0112] Multicellular spheroids (MCSs) were constructed to simulate tumor tissue, and the permeability of Epi-DP or Epi-DBP was evaluated. CT26 cells were cultured at 1 × 10⁶ cells per well. 5 Three-dimensional MCSs were constructed by seeding cells at a density of cells / well in 6-well ultra-low adsorption plates and culturing for one week.

[0113] After incubating MCSs with Epi-DP or Epi-DBP at a concentration of 10 μg / mL for 3 hours, the cells were washed three times with PBS, and the cell spheres were resuspended in a glass dish. Fluorescence signals of the multicellular spheres were collected every 5 μm using a laser confocal microscope to compare the permeability of Epi-DP and Epi-DBP in the multicellular spheres.

[0114] (3) Blocking of multicellular spheroid permeation and cell internalization

[0115] CT26 cells or CT26 multicellular spheroids were pre-incubated with phenylboronic acid (4 mM) for 0.5 hours, followed by incubation with Epi-DP or Epi-DBP for 3 hours, respectively. Fluorescence signals in CT26 cells and multicellular spheroids were observed using a laser confocal microscope, and the fluorescence signal intensity in CT26 cells was detected using flow cytometry. Alternatively, Epi-DP or Epi-DBP were pretreated with sialic acid (0.5 mM) for 0.5 hours, followed by incubation with CT26 cells or multicellular spheroids. Fluorescence signals in CT26 cells and multicellular spheroids were then observed using a laser confocal microscope, and the fluorescence signal intensity in CT26 cells was detected using flow cytometry.

[0116] CT26 multicellular spheres were incubated with Epi-DP or Epi-DBP in RPMI 1640 medium at pH 7.4 and pH 6.5, respectively, and the differences in fluorescence signals in the multicellular spheres were observed using a laser confocal microscope.

[0117] (4) Cellular internalization and intracellular transport

[0118] CT26 cells were seeded in glass dishes and incubated with Epi-DP and Epi-DBP for different times. The nuclei or lysosomes were stained with Hoechst 33342 and LysoBlue, respectively. After washing twice with PBS, the distribution of the delivery system and the nuclei or lysosomes was observed using laser confocal microscopy. Pearson's correlation coefficient and Mander's coefficient were calculated to examine the colocalization level of the delivery system and lysosomes.

[0119] (5) Cellular internalization pathway

[0120] CT26 cells were seeded in 12-well plates and incubated for 1 hour with 2-DG (20 mM), sodium azide (10 mM), genistein (400 μM), chlorpromazine (10 μg / mL), or amiloride (50 μM) in serum-free medium, respectively. Then, they were incubated for 3 hours with Epi-DP or Epi-DBP at a concentration of 10 μg / mL. Another group of cells was cultured at 4°C and incubated for 3 hours with Epi-DP or Epi-DBP at a concentration of 10 μg / mL. Cells were collected, and intracellular fluorescence intensity was detected by flow cytometry. CT26 cells incubated with Epi-DP or Epi-DBP at 37°C served as a positive control.

[0121] (6) Cytotoxicity test

[0122] CT26 cells were distributed at a rate of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated for 24 hours with different concentrations of Epi-DP or Epi-DBP or different concentrations of NLG919. The culture medium was discarded, and the cells were gently washed twice with PBS. Serum-free medium containing 10% CCK-8 was added, and the absorbance (OD) of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: (OD...) sample -OD background ) / (OD control -OD background (7) In vitro synergistic cytotoxicity of combined therapy

[0123] Following the method described in Example 2, NLG919@Epi-DBP nanoassemblies with Epi to NLG919 mass ratios of 1:0.5, 1:1, 1:2, 1:4, 1:5, and 1:10 were prepared. The feed amounts are shown in Table 1.

[0124] Table 1. Feed amount of each NLG919@Epi-DBP nanoassembly

[0125]

[0126]

[0127] Note: The mass ratio of Epi to NLG919 in NLG919@Epi-DBP is calculated based on the drug loading of Epi in Epi-DBP and HPLC results.

[0128] CT26 cells were distributed at a rate of 5 × 10⁶ cells per well. 3Cells were seeded at a density of cells / well in 96-well plates and incubated with NLG919@Epi-DBP at mass ratios of 1:0.5, 1:1, 1:2, 1:4, 1:5, and 1:10 for 24 hours. The culture medium was discarded, and the cells were gently washed twice with PBS. Serum-free medium containing 10% CCK-8 was added, and the absorbance (OD) of each well at 450 nm was measured using a microplate reader to calculate cell viability. The combination index (CI) of Epi-DBP and NLG919 was calculated using the following formula: CI = (D)1 / (Dx)1 + (D)2 / (Dx)2. Where (D)1 and (D)2 are the concentrations of drug 1 and drug 2 in the combination formulation that inhibit x% of cells; (Dx)1 and (Dx)2 are the concentrations of drug 1 and drug 2, respectively, when administered as single drugs to inhibit the same x% of cells. A CI value greater than 1, equal to 1, or less than 1 indicates an antagonistic effect, an additive effect, or a synergistic effect, respectively.

[0129] (8) Apoptosis assay

[0130] CT26 cells were seeded in 12-well plates and treated for 24 hours with Epi.HCl, NLG919, Epi-DP, Epi-DBP, NLG919@Epi-DP, or NLG919@Epi-DBP, respectively, with Epi concentrations of 0.5 μg / mL and NLG919 concentrations of 2.5 μg / mL. Cells were collected, stained with Annexin V-FITC and 7-AAD, and apoptosis was assessed by flow cytometry.

[0131] (9) IDO expression inhibition

[0132] 4T1 cells were loaded at 5 × 10 3 Recombinant mouse interferon-γ (IFN-γ) was seeded at a density of 100 ng / mL per well in 96-well plates, and incubated for 24 hours to stimulate IDO expression. IDO expression was assessed by detecting metabolic differences between tryptophan (Trp) and kynurenine (Kyn). After 24 hours, the supernatant from each well was transferred to a new 96-well plate, and the Kyn content was analyzed by HPLC using acetonitrile / water (v / v = 90 / 10, pH 4.0 sodium acetate aqueous solution) at a flow rate of 0.5 mL / min.

[0133] (10) Measurement of CRT, HMGB1 and extracellular ATP

[0134] In vitro CRT exposure and HMGB1 release induced by Epi-DBP, Epi-DP, or NLG919, or combinations thereof, were assessed using CLSM. CT26 cells were seeded in glass dishes and incubated for 24 h with different formulations of Epi at 1 μg / mL and NLG919 at 5 μg / mL, after which the supernatant was discarded. Cells were washed twice with cold PBS, fixed with 4% paraformaldehyde for 10 min, and blocked with 5% FBS for 30 min. They were then incubated with anti-CRT primary antibody for 1 h, washed three times with PBS, and incubated with anti-rabbit secondary antibody. Incubate for 1 hour. After DAPI staining, observe cell nuclei and CRT expression under CLSM at 405 nm and 561 nm, respectively. To release HMGB1, infiltrate fixed cells with 0.1% Triton-100 for 10 minutes. After blocking with 5% FBS, incubate with anti-HMGB1 primary antibody for 1 hour, wash 3 times with PBS, and then incubate with anti-rabbit secondary antibody. Incubate for 1 hour. Observe the cell nuclei after staining with DAPI.

[0135] CT26 cells were seeded in 96-well plates with Epi concentration of 1 μg / mL and NLG919 concentration of 5 μg / mL. The cells were incubated with Epi-DP, Epi-DBP, or NLG919 or combinations thereof for 24 h. The secretion of ATP in the supernatant was detected by an ATP luminescent cell viability assay kit.

[0136] (11) In vitro DC maturation and cytokine secretion

[0137] To evaluate Epi-DP or Epi-DBP-mediated dendritic cell (DC) maturation in vitro, bone marrow-derived dendritic cells (BMDCs) were generated from the bone marrow of 4-week-old Balb / c mice. Typically, BMDCs were obtained by washing the femoral and tibial medullary cavities with cold RPMI 1640 medium. After centrifugation, the cells were dispersed in erythrocyte lysis buffer and centrifuged again at 1500 rpm for 5 min. Cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, 100 U / mL streptomycin, and 20 ng / mL GM-CSF, and resuspended for 7 days to obtain immature BMDCs.

[0138] To investigate the stimulatory effect on BMDC maturation, CT26 cells were treated for 24 h with different formulations at an equivalent Epi concentration of 1 μg / mL and an NLG919 concentration of 5 μg / mL, respectively. Then, immature DCs (1 × 10⁻⁶) were... 6(Number of cells / sample) were co-cultured with pretreated CT26 cell supernatant for 24 hours. After staining with anti-CD11c, anti-MHC II, anti-CD80, and anti-CD86 antibodies according to standard protocol, mature dendritic cells (DCs) were detected by flow cytometry. Cytokines, including IL-6, IFN-γ, and TNF-α, in the supernatant were detected using an ELISA kit.

[0139] 3. Animal experiments

[0140] All animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of West China Hospital, Sichuan University. (5×10) 5 One CT26 cell was resuspended in 50 μL PBS and subcutaneously injected into the right side of a 6-week-old male Balb / c mouse. When the tumor grew to 100 mm... 3 At that time, mice can be used for fluorescence imaging and tumor treatment experiments.

[0141] (1) In vivo fluorescence imaging

[0142] To map the in vivo biodistribution of Epi-DP or Epi-DBP, we encapsulated the hydrophobic fluorescent dye Cy5 into the hydrophobic core of Epi-DP and Epi-DBP. Cy5-loaded Epi-DP or Cy5-loaded Epi-DBP was intravenously injected at a dose of 1 mg / kg. Mice were anesthetized with isoflurane at predetermined time points, followed by imaging with excitation at 640 nm and emission at 680 nm. Forty-eight hours post-injection, tumor-bearing mice were sacrificed, and their major organs and tumors were harvested for ex vivo imaging. Tumor tissues were collected for frozen sections, stained with anti-CD31 antibody, and the distribution of Epi-DP or Epi-DBP was observed using CLSM.

[0143] (2) Pharmacokinetic analysis

[0144] Male Balb / c mice (n=4) were randomly divided into 3 groups. All mice were intravenously injected with NLG919+Epi, HCl, NLG919@Epi-DP, or NLG919@Epi-DBP at an equivalent Epi dose of 5 mg / kg and an NLG919 dose of 25 mg / kg. Orbital venous plexus blood samples were collected at 0.25, 0.5, 1, 3, 6, 9, 12, 24, and 48 hours post-injection.

[0145] Epi assay: Blood samples were collected and centrifuged at 5000 rpm for 5 min. The supernatant was collected and treated with 5M hydrochloric acid at 50°C for 90 min. After cooling to room temperature, 1M sodium hydroxide was added, followed by chloroform / isopropanol (v / v, 4:1), and the mixture was vortexed for 90 seconds. After centrifugation at 10000g for 5 min, the organic phase was collected and dried overnight at room temperature. 100 μL LDMSO was added to dissolve the Epi, and the Epi concentration was determined using a microplate reader.

[0146] For NLG919 analysis, blood samples were centrifuged at 1000g for 15 min, and the supernatant was extracted twice with dichloromethane. The organic layer was collected and dried overnight at room temperature. The residue was dissolved in methanol, centrifuged at 10000g for 8 min, and then analyzed by HPLC.

[0147] (3) Anti-tumor effect

[0148] The antitumor effect was evaluated in CT26 tumor-bearing Balb / c mice. The tumor volume reached 100 mm². 3 At that time, mice were randomly divided into 7 groups (n=5), and were intravenously injected with NLG919 (G2 group), Epi.HCl (G3 group), NLG919+Epi.HCl (G4 group), NLG919@Epi-DP (G5 group), Epi-DBP (G6 group), and NLG919@Epi-DBP (G7 group), respectively. The drugs were administered every 3 days for a total of 4 doses. The Epi concentration was 5 mg / kg, and the NLG919 concentration was 25 mg / kg. CT26 tumor-bearing mice treated with physiological saline served as controls (G1 group). Tumor volume and body weight were recorded every 3 days for a total of 21 days. The tumor volume was calculated using the formula: V = (Length × Width) / (Length × Width). 2 ) / 2. When the tumor volume reaches 2000 mm 3 Mice were considered dead at a certain point. At the end of the experiment, the tumors were removed, weighed, and tumor growth inhibition (TGI) was calculated.

[0149] (4) Histological analysis

[0150] Major organs and tumors were harvested and fixed with 4% paraformaldehyde. Hematoxylin-eosin (H&E), CD31, Ki67, IDO, fibronectin, α-SMA, Masson, and TUNEL staining were performed. The stained sections were observed under a fluorescence microscope or CLSM.

[0151] (5) In vivo DC maturation

[0152] To assess the in vivo maturation of dendritic cells (DCs) mediated by Epi-DP or Epi-DBP, CT26 tumor-bearing mice were intravenously injected with saline, NLG919, Epi.HCl, NLG919+Epi.HCl, NLG919@Epi-DP, Epi-DBP, or NLG919@Epi-DBP. The equivalent dose of Epi was 5 mg / kg, and the equivalent dose of NLG919 was 25 mg / kg, administered every 3 days for a total of 4 doses. Two days after the last administration, the mice were sacrificed, and lymph nodes, spleen, and tumors were collected. The collected tissues were gently ground to prepare a single-cell suspension. After staining with anti-CD45, anti-CD11c, anti-MHC II, anti-CD80, and anti-CD86 antibodies, mature DCs were detected by flow cytometry.

[0153] (6) Secretion of cytokines in tumor tissue and serum

[0154] Following treatment, tumors and blood were collected from mice to detect the levels of cytokines IL-2, IL-6, IL-12, IFN-γ, and TNF-α. Tumors were collected, homogenized, and lysed with RIPA buffer containing 1 mM PMSF. After centrifugation, cytokines in the supernatant were detected using an ELISA kit. To quantify serum cytokine levels, blood was collected, centrifuged, and serum was obtained. Serum cytokine levels were detected using an ELISA kit.

[0155] (7) In vivo evaluation of tumor-infiltrating immune cells

[0156] Flow cytometry was used to analyze immune cells in tumor tissue. After the experimental treatment, mouse tumors were taken, cut into small pieces, and digested at 37°C for 30 minutes with collagenase IV (1 mg / mL), hyaluronidase (1 mg / mL), and DNase I (7.5 μg / mL). After enzymatic digestion, the suspension was filtered through a nylon mesh to obtain a single-cell suspension. CD45 was used to analyze the immune cells. + Positive intracellular analysis of cytotoxic T lymphocytes (CD3) + CD8 + ), T helper cells (CD3) + CD4 + ), regulatory T cells (Treg, CD3) + CD4 + CD25 + FoxP3 + Myeloid-derived suppressor cells (MDSCs, CD11b) + Gr-1 + ), natural killer cells (CD3) - CD49b + ) and tumor-associated macrophages (TAMs, M1-type TAMs are CD11b + F4 / 80 + CD80 + M2 type TAMs are CD11b + F4 / 80 + CD206 + The immune infiltration status of tumor tissue.

[0157] II. Experimental Results

[0158] Epi-DBP was prepared via a condensation reaction, in which Epi and PEG are covalently linked to the same dendritic molecule via acid-sensitive hydrazone bonds and borate ester bonds, respectively. As a control, a molecule was constructed in which Epi is linked by hydrazone bonds, but PEG is linked to the same dendritic molecule via stable covalent bonds; this molecule was named Epi-DP. A standard curve was constructed using UV spectroscopy. Figure 8 The Epi content in Epi-DP and Epi-DBP was calculated to be 11.8 wt% and 6.3 wt%, respectively. After assembly in water, they yielded particles with hydrated particle sizes of 133.0 ± 17.3 nm and 150.8 ± 1.4 nm, respectively, both with negative surface charges. When dissolved in deionized water and phosphate-buffered saline (PBS, pH 7.4), the particle size of Epi-DP varied significantly in the range of 130 nm to 400 nm in both aqueous solutions. Epi-DBP, however, maintained a relatively stable hydrated particle size for 20 weeks in both aqueous solutions, and its polydispersity index (PDI) remained below 0.3. Figure 9 When both Epi-DP and Epi-DBP were dissolved in PBS at different pH values ​​(pH 7.4, 6.5, 5.0), the hydrated particle size of both Epi-DP and Epi-DBP changed significantly within 3 hours at pH 5.0, indicating that they exhibit acid-sensitive response characteristics. Figure 10 ).

[0159] The only difference in the chemical structure between Epi-DP and Epi-DBP lies in the covalent bonding of the PEG chains. Using sialic acid-rich mouse colon cancer cells (CT26) as a cell model, CT26 cells were pre-incubated with PBA for 0.5 hours and then co-cultured with the delivery system for 3 hours; alternatively, the delivery system was pre-incubated with sialic acid and then co-cultured with CT26 cells for 3 hours. Co-culturing the delivery system with CT26 cells for 3 hours served as a control. Cells were collected, and the fluorescence intensity of CT26 cells was detected by flow cytometry. The results showed that the average fluorescence intensity of cells in the Epi-DP group did not change significantly, while the average fluorescence intensity of cells treated with Epi-DBP significantly decreased after pre-incubation with PBA or sialic acid. Three-dimensional multicellular tumor spheres were constructed using CT26 cells, and similar phenomena were observed using laser confocal microscopy (CLSM) with the same treatment method, indicating that PBA in Epi-DBP can bind to sialic acid, promoting the uptake of CT26 cells and the penetration of the three-dimensional cell spheres. To further verify the weakly acid-responsiveness of Epi-DBP, CT26 multicellular tumor spheres were incubated with Epi-DP and Epi-DBP for 3 hours at pH 7.4 and pH 6.5, respectively. The fluorescence intensity and distribution in the multicellular tumor spheres were then observed using a CLSM. Under different pH conditions, the fluorescence intensity of Epi-DP was very weak and showed no significant change. However, at pH 6.5, Epi-DBP showed brighter fluorescence and a more uniform fluorescence distribution in the tumor spheres, indicating that in a weakly acidic environment, the borate ester bonds are weakened, promoting the interaction between PBA and sialic acid on the cell surface, thus facilitating the deep penetration of the delivery system into the tumor spheres. Forty-eight hours after CT26 tumor-bearing mice were injected intravenously with Epi-DP and Epi-DBP, respectively, the tumors were harvested, frozen sections were cut, and the blood vessels were stained. Bright fluorescence signals were observed in the Epi-DBP-treated group, spreading to areas far from the blood vessels, indicating that Epi-DBP can penetrate deeply into the tumor.

[0160] The amphiphilic structures of Epi-DP and Epi-DBP endow them with the ability to load hydrophobic small molecules. After encapsulating NLG919, NLG919@Epi-DP and NLG919@Epi-DBP were obtained, respectively, with their hydrated particle sizes significantly reduced to approximately 95 nm and zeta potentials around -25 mV. Furthermore, after encapsulation with NLG919, NLG919@Epi-DBP also exhibited excellent stability in deionized water and PBS (pH 7.4). Figure 11 ), while NLG919@Epi-DP still exhibits poor stability in both aqueous solutions. Figure 12 ).

[0161] The in vitro release behavior of NLG919@Epi-DP and NLG919@Epi-DBP was investigated in PBS at different pH values ​​(pH 7.4, 6.5, 5.0) to simulate different in vivo microenvironments. In pH 7.4 and pH 6.5 environments, less than 6% and 24% of Epi were released from NLG919@Epi-DBP, respectively, within 96 hours; while in pH 5.0, approximately 75% of Epi was released within 24 hours, and the release rate reached as high as 89% at 96 hours. Similarly, in pH 7.4 and pH 6.5 environments, less than 6% and 28% of NLG919 were released from NLG919@Epi-DBP, respectively, within 96 hours; while in pH 5.0, approximately 65% ​​of Epi was released within 24 hours, and the release rate reached as high as 85% at 96 hours. Figure 13 The release of the drug in NLG919@Epi-DP showed a similar pattern, but the release rate was slightly slower. Figure 14 The hydration particle size of NLG919@Epi-DP and NLG919@Epi-DBP in PBS (pH 5.0) also showed significant changes over 24 hours. Figure 15 The above experiments show that Epi-DBP can be loaded with NLG919 to obtain NLG919@Epi-DBP with a stable nanostructure during cycling, and under acidic conditions, it will degrade to release Epi and NLG919 and exert its biological effects.

[0162] The in vitro antitumor effect of NLG919@Epi-DBP on CT26 cells was studied using the CCK-8 assay. CT26 cells were used as a control without any treatment. The IC50 values ​​of Epi.HCl and Epi-DBP after 24 hours were calculated. 50 The values ​​were 1.1 μg / mL and 2.3 μg / mL, respectively, while the IC50 of Epi-DP was... 50 The value was 56.1 μg / mL. Figure 16 NLG919 treatment for 24 hours on CT26 cells' IC50 50 The value was 34.3 μg / mL ( Figure 17This invention also tested the cytotoxic effect of NLG919@Epi-DBP on CT26 cells at different Epi to NLG919 mass ratios (1:0.5 to 1:10) and calculated the combination index (CI). The results showed that when the Epi to NLG919 mass ratio was 1:4 and 1:5, NLG919 and Epi-DBP had excellent anti-tumor synergistic effects. Specifically, when the Epi to NLG919 mass ratio was 1:5, the CI value was less than 0.5 at all effect sizes, indicating that NLG919 and Epi-DBP had a strong anti-tumor synergistic effect at this ratio. Figure 18 ).

[0163] Cells were stained with Annexin V-FITC and 7-aminoactinomycin D (7-AAD), and the cell death pattern of CT26 cells was investigated by flow cytometry. The results showed that after 24 hours of treatment with Epi-DP (1 μg / mL) and NLG919 (5 μg / mL), 15.0% and 25.8% of CT26 cells, respectively, entered late apoptosis compared to untreated cells. The combined use of NLG919 and Epi-DBP treatment increased the late apoptosis rate to 29.1%, demonstrating a synergistic effect between the two treatments.

[0164] The anthracycline drug Epi can induce a strong ICD effect in cells. Therefore, the effect of Epi-DBP treatment on the release of DAMPs, including calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP), was further investigated. The results showed that, compared with other groups, NLG919@Epi-DBP induced a significant CRT exposure level in CT26 cells. Epi-DBP induced significant extracellular release of HMGB1 and ATP secretion, and the combined use of NLG919 further enhanced these effects.

[0165] Tumor cells release DAMPs, which send a "eat me" signal to external cells, stimulating the maturation of dendritic cells (DCs) and enabling antigen presentation. Mouse bone marrow-derived dendritic cells (BMDCs) were extracted to further verify the effect of NLG919@Epi-DBP on DC maturation. CT26 cells were co-cultured with different delivery systems for 24 hours, and then the cell supernatant was co-cultured with BMDCs for another 24 hours. BMDCs were then subjected to fluorescent antibody treatment, and flow cytometry was used to examine BMDC maturation. The results showed that the Epi-DBP group induced the maturation of 33.3% of BMDCs, and after NLG919@Epi-DBP treatment, the BMDC maturation rate significantly increased to 52.7%, indicating that NLG919@Epi-DBP induced the strongest ICD effect. Cytokines secreted by BMDCs were detected by enzyme-linked immunosorbent assay (ELISA). The results showed that, compared with the free drug group, BMDCs treated with NLG919@Epi-DBP secreted more interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In addition, the level of interferon-gamma (IFN-γ) secretion in BMDCs treated with NLG919@Epi-DBP was significantly higher than that in other groups.

[0166] The in vivo biological behavior of each group was investigated at the animal level. First, hydrophobic Cy5 was encapsulated in the hydrophobic lumens of Epi-DP and Epi-DBP, with physiological saline and a physical mixture of Epi.HCl and Cy5 as controls. After tail vein injection, the distribution and changes of fluorescence signals in mice at different time points were examined. At 24 hours, significant fluorescence signals were observed at the tumor sites in both the Cy5@Epi-DP and Cy5@Epi-DBP groups, with the Cy5@Epi-DBP group showing stronger fluorescence signals. In contrast, the fluorescence signal in the Epi.HCl and Cy5 physical mixture group decreased continuously after tail vein injection, almost disappearing after 24 hours. Forty-eight hours later, the mice were euthanized, and in vitro fluorescence imaging of the major organs and tumors was performed on the Epi and Cy5 channels, respectively. The results showed that Cy5@Epi-DBP exhibited the strongest fluorescence signal at the tumor site in both different fluorescence channels.

[0167] Table 2. Pharmacokinetic parameters of Epi in vivo after administration via different delivery systems (Epi dosage: 5 mg / kg)

[0168] Unit Epi. Hydrochloric acid NLG919@Epi-DP NLG919@Epi-DBP <![CDATA[C max ]]> μg / mL 14.32 19.67 24.43 AUC μg / mL*h 15.36 91.02 127.28 <![CDATA[t 1 / 2 ]]> h 1.04 6.85 7.89

[0169] C maxMaximum Epidural plasma concentration; AUC; Area under the curve; t 1 / 2 ,half life.

[0170] Table 3. Pharmacokinetic parameters of NLG919 in vivo after administration via different delivery systems (NLG919 dosage: 25 mg / kg)

[0171] Unit NLG919 NLG919@Epi-DP NLG919@Epi-DBP <![CDATA[C max ]]> μg / mL 31.18 47.54 65.73 AUC μg / mL*h 50.59 100.77 211.58 <![CDATA[t 1 / 2 ]]> h 1.67 5.25 6.28

[0172] C max Maximum Epidural plasma concentration; AUC; Area under the curve; t 1 / 2 ,half life.

[0173] Normal mice were intravenously injected with a physical mixture of Epi.HCl and NL919, NLG919@Epi-DP, and NLG919@Epi-DBP, respectively. Pharmacokinetic analysis was performed on the blood levels of Epi and NLG919 at different time points. The results showed that both Epi-DP and Epi-DBP prolonged the blood half-life of Epi by 6.6-fold and 7.6-fold, respectively (Table 2); the area under the curve (AUC) increased by 5.9-fold and 8.3-fold, respectively. NLG919@Epi-DBP and NLG919@Epi-DP also effectively prolonged the blood half-life of NLG919 and significantly increased the AUC (Table 3).

[0174] In vivo antitumor effects of each group were evaluated in a CT26 tumor-bearing mouse model, with tumor volume approaching 100 mmHg. 3 Mice were randomly divided into 7 groups of 5 mice each. Each mouse was administered 5 mg / kg Epi and / or 25 mg / kg NLG919 via tail vein every 3 days for a total of four administrations. The mice were observed for 21 days, and tumor growth curves were plotted. The results showed that NLG919 alone had almost no effect on tumor growth in mice. NLG919@Epi-DP could limit tumor growth in mice to some extent; Epi.HCl, Epi-DBP, and NLG919 combined with Epi.HCl further inhibited tumor growth; after treatment with NLG919@Epi-DBP, the tumor volume in mice did not change significantly compared to before treatment, indicating that NLG919@Epi-DBP significantly inhibited tumor growth in mice. Figure 19 After 21 days of observation, the mice were euthanized, and their major organs and tumors were harvested. The tumors were weighed, and the tumor growth inhibition rate was calculated. The results showed that the tumor growth inhibition rates in the NLG919@Epi-DP group and the NLG919 combined with Epi.HCl group were 56.9% and 74.6%, respectively. The NLG919@Epi-DBP group had the highest tumor growth inhibition rate, reaching 85.9%. Figure 20More importantly, by the end of the experiment, mice in the NLG919 combined with Epi.HCl group had a 10% decrease in body weight, indicating that this treatment group had serious toxic side effects. Figure 21 ).

[0175] With a tumor volume exceeding 2000 mm 3 The experimental endpoint was defined as a decrease in mouse body weight exceeding 20%. All mice in the saline group died within 34 days; all mice in the NLG919 combined with Epi.HCl group and the NLG919@Epi-DP group died within 46 days; the survival rates of mice in the NLG919@Epi-DBP group were 62.5% and 37.5% at 50 and 60 days, respectively, indicating that the NLG919@Epi-DBP group significantly prolonged the survival of CT26 tumor-bearing mice. Figure 22 ).

[0176] Flow cytometry was used to analyze mature dendritic cells (DCs) in mouse lymph nodes, spleen, and tumors to explore the synergistic mechanism of IDO blockade combined with chemotherapy. First, the maturation status of DCs in mouse lymph nodes, spleen, and tumors was examined. Results showed that the number of mature DCs in mouse lymph nodes after NLG919@Epi-DBP treatment was significantly higher than in other groups (…). Figure 23 ); The number of mature DCs in the spleen of mice was significantly increased after treatment with Epi-DBP and NLG919@Epi-DBP. Figure 24 The proportion of mature DCs was very high in all mouse tumor groups, reaching 90%. After NLG919@Epi-DBP treatment, the proportion of mature DCs further increased, reaching 97%. Figure 25 ).

[0177] The secretion of cytokines, including interleukin-2 (IL-2), in mouse tumor tissue and blood was detected using ELISA. Figure 26 Interleukin-6 (IL-6) Figure 27 Interleukin-12 (IL-12) Figure 28 ), IFN-γ Figure 29 ) and TNF-α Figure 30 Consistent with the results of stimulating DC maturation in vivo, the NLG919@Epi-DBP treatment group induced the highest levels of cytokine secretion, with IL-6 and TNF-α levels being 1.84 times and 2.07 times higher than those in the saline group, respectively. Furthermore, serum indicators were significantly elevated after NLG919@Epi-DBP treatment compared to the saline group. These results indicate that, combined with NLG919's blockade of IDO, NLG919@Epi-DBP exerts a powerful chemotherapeutic effect, thereby inducing a strong ICD effect and evoking an immune response, achieving enhanced antitumor efficacy through combined chemotherapy and immunotherapy.

[0178] Flow cytometry was used to examine the types and numbers of immune cells infiltrating the tumor tissue. First, CD4+ cells were analyzed. + / CD8 + T cells were investigated, and NLG919@Epi-DBP treatment significantly increased CD4 count. + T cells ( Figure 31 ) and CD8 + T cells ( Figure 32 Infiltration of tumor tissue, particularly in the NLG919@Epi-DBP treatment group with CD4... + The levels of T cells were 6 times, 3.89 times, and 4.35 times higher in the saline group, the NLG919 combined with Epi.HCl group, and the NLG919@Epi-DP group, respectively. Natural killer (NK) cells, another potent anti-tumor immune cell, were present in the saline group (only 8%); NLG919 alone or Epi.HCl alone had almost no effect on NK cell levels; however, NLG919@Epi-DBP treatment significantly increased NK cell levels, reaching 2.2 times that of the saline group. Figure 33 ).

[0179] In addition to the presence of anti-tumor immune cells, tumor tissue also contains a large number of immunosuppressive cells. Among them, myeloid-derived suppressor cells (MDSCs) are one of the most important immunosuppressive cells, mainly involved in suppressing T cell-related anti-tumor immune responses, polarizing M1 macrophages into the immunosuppressive M2 type, and recruiting immunosuppressive regulatory T (Treg) cells. Results showed that after Epi-DBP treatment, the content of MDSCs decreased from 61.50±0.62% to 30.67±0.16%. After NLG919@Epi-DBP treatment, the content of MDSCs further decreased to 24.53±3.84%. Figure 34 M2 macrophages Figure 35 The number of Treg cells was also significantly reduced. Figure 36 This indicates that NLG919@Epi-DBP treatment remodels the tumor immune microenvironment and promotes positive regulation of the immune response.

[0180] In summary, this invention constructs Epi-DBP by bridging PEG and Epi dendritic macromolecules via borate ester bonds. Epi, used as a first-line chemotherapy drug and ICD inducer for tumors, is coupled to one end of the dendritic molecule via acid-sensitive hydrazone bonds, while PEG is coupled to the periphery of the dendritic molecule via borate ester bonds. Its amphiphilic structure endows Epi-DBP with self-assembly capability. The hydrophobic IDO1 inhibitor NLG919 can be encapsulated in the hydrophobic cavity of Epi-DBP, forming the assembly NLG919@Epi-DBP. The NLG919@Epi-DBP assembly possesses a stable nanostructure and can accumulate in tumor tissue. In the weakly acidic environment of tumor tissue, the weakening of the borate ester bonds exposes the PBA groups, which bind to the abundant sialic acid residues on the surface of tumor cells, enhancing the phagocytic efficiency of tumor cells. The intracellular acidic state triggers hydrazone bond breakage, leading to the release of Epi and NLG919. This invention integrates Epi and NLG919 into a tree-like delivery platform to reshape the tumor immune microenvironment by inducing ICD in tumor cells and blocking the IDO pathway, while simultaneously synergizing with chemotherapy to achieve enhanced chemoimmunotherapy effects.

Claims

1. A dendritic prodrug of epirubicin, characterized in that, Its structure is shown in Equation I: Formula I Where n is 10-30; The epirubicin dendritic prodrug contains 3-10 wt% epirubicin.

2. The epirubicin dendritic macromolecular prodrug according to claim 1, characterized in that, The epirubicin dendritic prodrug contains 6.3 wt% epirubicin.

3. A nano-assembly, characterized in that, It is formed by the self-assembly of epirubicin dendritic macromolecular prodrug as described in any one of claims 1-2 and an IDO inhibitor, wherein the IDO inhibitor is NLG919 and the mass ratio of epirubicin to IDO inhibitor is 1:(0.5-10).

4. The nano-assembly according to claim 3, characterized in that, In the nano-assembly, the mass ratio of epirubicin to IDO inhibitor is 1:(4-5).

5. A method for preparing the nano-assembly according to any one of claims 3-4, characterized in that, The method includes the following steps: mixing epirubicin dendritic macromolecular prodrug with an IDO inhibitor in an organic solvent, dropping the mixture into water, and self-assembling to obtain a nano-assembly.

6. Use of the nanoassembly according to any one of claims 3-4 in the preparation of an antitumor drug, wherein the tumor is colon cancer or breast cancer.

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