A nanoparticle based on tumor microenvironment-activated golgi-targeting interfering agent

Abstract

The application provides a kind of nano-particle based on tumor microenvironment activated Golgi targeting interfering agent and a preparation method thereof.The nano-particle mainly comprises anthracycline antitumor antibiotic and tumor microenvironment activated Golgi targeting interfering agent, and other surface modification materials can also be added.The tumor microenvironment activated Golgi targeting interfering agent synthesized by the application can target to destroy the structure and function of Golgi, greatly improve the effect of Golgi interfering agent, and be suitable for various different drug delivery systems and disease models.The nano-particle prepared based on tumor microenvironment activated Golgi targeting interfering agent has simple preparation process, high drug loading capacity, can effectively treat tumor and metastasis, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to a nanoparticle based on a tumor microenvironment-activated Golgi apparatus-targeting interference agent, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] The Golgi apparatus is an organelle in cells responsible for protein modification, transport, and secretion. Its dysfunction is associated with various pathological processes, such as neurodegenerative diseases and cancer. Studies have shown that in rapidly proliferating tumor cells, the Golgi apparatus expands and its secretory function is highly active, participating in the secretion of various tumor and metastasis-related proteins. The expression levels of Golgi functional proteins are closely related to the prognosis of cancer metastasis. Therefore, disrupting the structure and function of the Golgi apparatus and blocking the modification and secretion of tumor and metastasis-related regulatory proteins can affect multiple signaling cascades in tumor progression, thereby inhibiting tumor growth and metastasis.

[0003] Currently, Golgi structure disruptors are mostly used for research on Golgi structure and function. However, they suffer from poor specificity, strong toxic side effects, and a lack of effective targeted delivery systems, thus limiting their use in disease treatment. Current research on Golgi targeting mainly focuses on the development of Golgi-specific fluorescent probes for disease diagnosis. For example, patents CN115161021A and CN104987862A disclose fluorescent carbon quantum dots with amino acids as targeting groups that can be used for Golgi imaging; patents CN114958363A, CN114032094A, CN112851556A, and CN112266351A disclose probes with benzenesulfonamide as targeting groups. Golgi-specific fluorescent probes or carbon dots; patents CN114621172A and CN114507204A disclose Golgi-targeted fluorescent probes with myristoyl as the targeting group; patents CN109280017A and CN108997363A disclose Golgi-targeting agents with sphingosine as the targeting group; patents CN111039866A and CN104529893A disclose Golgi-targeted fluorescent dyes with quinoline derivatives as the targeting group, etc. There are relatively few Golgi-targeted technologies used for disease treatment. For example, patent CN111317715A discloses a Golgi-targeted mucopolysaccharide nanomicelle, its preparation method, and its application; patent CN115177739A discloses a tissue-cell-organelle three-level targeted biomimetic agent, its preparation method, and its application, which utilizes Golgi-targeting peptides (amino acid sequence SXYQRL, where X represents any amino acid) to modify nanoparticles; patent CN114456152A discloses a photothermal reagent for Golgi-targeted covalently bound proteins, its preparation method, and its application, etc. However, these Golgi-targeted therapeutic technologies still suffer from drawbacks such as complex materials, difficulty in conversion, and limited application scope. Therefore, developing novel and effective Golgi-targeted therapeutic strategies is of great significance. Summary of the Invention

[0004] The purpose of this invention is to develop Golgi targeting interference agents and related nano-formulations that have the ability to target and disrupt the Golgi apparatus, respond to release in response to the tumor microenvironment, have simple preparation processes, and have a wider range of applications.

[0005] One objective of this invention is to provide a tumor microenvironment-activating Golgi-targeting disruptor, comprising a Golgi-targeting group indomethacin (IMC), an intermediate response group, and a Golgi-disrupting agent trans-retinoic acid (RA), with the following structural formula: in

[0006] One objective of this invention is to provide a method for synthesizing a tumor microenvironment-activated Golgi apparatus-targeting disruptor, characterized by the following steps:

[0007] (1) 2-Hydroxy-5-methylisophthalimide reacts with 4-nitrobenzyl bromide or 2,4-dinitrobenzenesulfonyl chloride or 4-bromomethylphenylboronic acid pinacol ester to generate intermediate compound 1, which is responsive to hypoxia, glutathione and reactive oxygen species, respectively. The structural formula of intermediate compound 1 is as follows: in

[0008] (2) Indomethacin reacts with intermediate compound 1 to generate intermediate compound 2. The structural formula of intermediate compound 2 is: in

[0009] (3) Trans-retinoic acid reacts with intermediate compound 2 to generate tumor hypoxia-activated Golgi target interference (INR), glutathione-activated Golgi target interference (IGR) and reactive oxygen species-activated Golgi target interference (IRR), respectively.

[0010] The intermediate response group described in this invention can be activated by nitroreductase overexpressed by tumor cells, high concentrations of glutathione, or high concentrations of reactive oxygen species, respectively, to further trigger a domino elimination reaction, thereby releasing the prototype drugs indomethacin and trans-retinoic acid.

[0011] One objective of this invention is to provide nanoparticles based on a tumor microenvironment-activated Golgi apparatus-targeting interference agent, comprising 1 part anthracycline antitumor antibiotic and 1-10 parts a tumor microenvironment-activated Golgi apparatus-targeting interference agent; preferably, 1 part anthracycline antitumor antibiotic and 2-5 parts a tumor microenvironment-activated Golgi apparatus-targeting interference agent. These nanoparticles exhibit excellent pharmacological activity without the addition of any carrier.

[0012] The anthracycline antitumor antibiotics described in this invention are selected from one or more of doxorubicin, daunorubicin, epirubicin, pirarubicin, aclarubicin, amrubicin, zolrubicin, pentorubicin, idarubicin, or mitoxantrone.

[0013] The nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents described in this invention can also be modified with surfactants, amphiphilic polymers, or negatively charged substances that can be adsorbed onto the surface of the nanoparticles as surface modification materials.

[0014] One objective of this invention is to provide a lyophilized nanoparticle powder injection comprising anthracycline antitumor antibiotics, a tumor microenvironment-activating Golgi apparatus-targeting interferon, a surface modification material, and a lyophilization protectant. It may also contain other pharmaceutically acceptable excipients. Specifically, based on parts by weight, the anthracycline antitumor antibiotic comprises 1 part, the tumor microenvironment-activating Golgi apparatus-targeting interferon 1-10 parts, the surface modification material 0-100 parts, the lyophilization protectant 0-1000 parts, and other pharmaceutically acceptable excipients in appropriate amounts. Preferably, the anthracycline antitumor antibiotic comprises 1 part, the tumor microenvironment-activating Golgi apparatus-targeting interferon 2-5 parts, the surface modification material 0-10 parts, the lyophilization protectant 10-200 parts, and other pharmaceutically acceptable excipients in appropriate amounts.

[0015] The surface modification material can be selected from one or more of the surfactants Solutol HS15, Tween, Mize, Benzepine, and Poloxamer; one or more of the amphiphilic polymers PEGylated phospholipids, PEG-PLGA, PEG-PLA, and PEG-PCL; and one or more of the negatively charged substances that can be adsorbed onto the surface of nanoparticles, such as chondroitin sulfate, hyaluronic acid, chitosan, low molecular weight heparin, cyclodextrin, fucoidan, dextran, and alginate. The preferred surface modification material is mPEG. 2000 -One or more of DSPE, Solutol HS15, and chondroitin sulfate.

[0016] The freeze-drying protectant in the lyophilized nanoparticle powder injection of the present invention is selected from one or more of sucrose, glucose, maltose, lactose, mannitol, trehalose, glycine, and dextran; the preferred freeze-drying protectant is sucrose.

[0017] The other excipients described in this invention are selected from pharmaceutically acceptable excipients such as isotonic adjusters, antioxidants, preservatives, and pH adjusters.

[0018] One objective of this invention is to provide a method for preparing nanoparticles based on tumor microenvironment-activated Golgi targeting disruptors, characterized by the following steps:

[0019] (1) Take a pharmaceutically acceptable salt of anthracycline antitumor antibiotic, neutralize the corresponding acid with an alkaline substance, and then dissolve it in an organic solvent; or dissolve anthracycline antitumor antibiotic in an organic solvent.

[0020] (2) Dissolve the tumor microenvironment-activating Golgi-targeting interferon in an organic solution;

[0021] (3) Mix (1) and (2), add deionized water, and homogenize under high pressure or use a probe for ultrasonic emulsification;

[0022] (4) Remove the organic solvent by rotary evaporation to obtain the product.

[0023] In step (1), the alkaline substance is mainly used to neutralize the acid radicals in pharmaceutically acceptable salts of anthracycline antitumor antibiotics, obtaining a free base in the form of the drug molecule. The alkaline substance is selected from one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate, triethylamine, and ammonia water; sodium bicarbonate is preferred in step (1), and the amount of alkaline substance added can be determined according to the properties of the drug. The organic solvents in steps (1) and (2) are selected from chloroform, dichloromethane, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, methylpyrrolidone, or similar solvents; one or more of these solvents or mixtures can be selected; chloroform is preferred. The rotary evaporation temperature range in step (4) is 20–70°C, preferably 30–40°C.

[0024] The nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents also contain surface modification materials, which can be dissolved in an organic solvent in step (2) and / or dissolved in deionized water in step (3), and / or added directly after step (4).

[0025] As one of the specific implementation schemes, the nanoparticles obtained by the above method based on tumor microenvironment-activated Golgi targeting interference agents can be added with a freeze-drying protectant after step (4) and freeze-dried into powder by conventional methods in the art.

[0026] The nanoparticles have a particle size of 1–1000 nm, preferably 10–300 nm.

[0027] One objective of this invention is to provide an application of nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents in the treatment of tumors and their metastases.

[0028] Invention benefits

[0029] (1) The tumor microenvironment activating Golgi targeting interference agent of the present invention can improve Golgi targeting efficiency and improve the effect of Golgi targeting therapy;

[0030] (2) The tumor microenvironment-activating Golgi-targeting interferon of the present invention has a simple synthesis step, can be delivered by various different drug delivery systems, and can also be used to treat various different Golgi-related diseases, with a wide range of applications;

[0031] (3) The method for preparing nanoparticles of tumor microenvironment-activated Golgi targeting interference agents of the present invention is simple, the raw materials are readily available, the drug loading capacity is large, and it can play a role without any inactive drug delivery carrier.

[0032] (4) The nanoparticles of the present invention based on tumor microenvironment-activated Golgi targeting interference agent can significantly improve the anti-tumor and anti-tumor metastasis treatment effects and have good application prospects. Attached Figure Description

[0033] Figure 1 1H NMR spectrum of INR, a tumor hypoxia-activated Golgi body targeting interferon.

[0034] Figure 2 Transmission electron microscopy images of nanoparticles NP and PNP.

[0035] Figure 3 .4T1 cell uptake of free pirarubicin, NP, and PNP (n=4). ****p<0.0001.

[0036] Figure 4 The content of trans-retinoic acid in 4T1 cells after PNP incubation for different time periods (n=3). **p<0.01, ****p<0.0001. ns, no significant difference.

[0037] Figure 5 Results of in vitro cytotoxicity experiments (n=5).

[0038] Figure 6 Confocal micrographs of immunofluorescence-labeled Golgi apparatus structural proteins in 4T1 cells after treatment with free drugs or nanoparticles. Scale bar: 10 μm.

[0039] Figure 7 Scratch test results. Scale bar: 100 μm.

[0040] Figure 8 Transwell invasion test results. Scale bar: 100 μm.

[0041] Figure 9 In vivo antitumor effects. (A) Tumor growth curves for each group (n=6). **p<0.01, ****p<0.0001. (B) Tumor weight on day 22 (n=6). **p<0.01. (C) Tumor inhibition rate calculated based on tumor weight (n=6). **p<0.01. (D) Tumor tissue photographs taken on day 22. (E) Kaplan-Meier survival curves for different treatment groups (n=10).

[0042] Figure 10In vivo anti-metastatic effect. (A) Bioluminescence imaging of ex vivo lung tissue (top) and photographs of lung tissue fixed in Born's fixative (bottom, lung metastatic nodules are circled in black). (B) Semi-quantitative results of bioluminescence intensity of lung tissue and number of lung metastatic nodules (n=3). *p<0.05,***p<0.001 and****p<0.0001. ns, no significant difference. Detailed Implementation

[0043] The following embodiments are further illustrations of the present invention, but are by no means limitations on the scope of the invention. The present invention is further described in detail below with reference to the embodiments; however, those skilled in the art should understand that the present invention is not limited to these embodiments and the preparation methods used. Furthermore, those skilled in the art can make equivalent substitutions, combinations, improvements, or modifications to the present invention based on the description thereof, but all such substitutions and modifications will be included within the scope of the present invention.

[0044] Example 1

[0045] Taking tumor hypoxia-activated Golgi-targeting interferon (INR) as an example, the synthesis steps of INR are as follows:

[0046] (1) Dissolve 0.84 g of 2-hydroxy-5-methyl-isophthalic acid and 2.16 g of 4-nitrobenzyl bromide in 50 mL of acetone, add 3.45 g of K2CO3, reflux in an oil bath at 60 °C for 4 h, then cool to room temperature, remove acetone by rotary evaporation at 60 °C to obtain crude product, and purify by column chromatography to obtain yellow powder, which is intermediate compound 1;

[0047] (2) Dissolve 1.79 g of indomethacin in DMF, add 1.44 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.92 g of 4-dimethylaminopyridine, 1.94 g of N,N-diisopropylethylamine and 2.27 g of intermediate compound 1, and stir overnight at room temperature under nitrogen protection. DMF is then removed by pumping to obtain the crude product, which is purified by column chromatography to obtain a yellow solid, which is intermediate compound 2.

[0048] (3) Dissolve 1.2 g of trans-retinoic acid in dichloromethane, add 1.15 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.73 g of 4-dimethylaminopyridine, 1.55 g of N,N-diisopropylethylamine and 3.09 g of intermediate compound 2, and stir overnight at room temperature under nitrogen protection. Remove dichloromethane by rotary evaporation to obtain crude product, purify by column chromatography, and dry the product under vacuum to obtain a yellow solid powder, which is a tumor hypoxia-activated Golgi apparatus targeting interference agent (INR).

[0049] The synthesis steps for tumor glutathione-activated Golgi target interference (IGR) and tumor reactive oxygen species-activated Golgi target interference (IRR) are the same as above.

[0050] Example 2

[0051] Dissolve 3 mg of doxorubicin (THP) and 7.5 mg of INR in 0.3 mL of chloroform, add 6 mL of deionized water, sonicate with a probe for 8 min (power 200 W, sonication for 5 s, pause for 5 s), and then remove the chloroform by rotary evaporation at 37 °C to obtain nanoparticles NP.

[0052] Example 3

[0053] Take 3 mg THP, 7.5 mg INR and 2.1 mg mPEG. 2000 -DSPE was dissolved in 0.3 mL of chloroform, and 6 mL of deionized water was added. The mixture was sonicated for 8 min with a probe (power 200W, sonication for 5 s, pause for 5 s). Then, the chloroform was removed by rotary evaporation at 37 °C to obtain nanoparticle PNP.

[0054] Example 4

[0055] Dissolve 1.5 mg THP and 3 mg INR in 0.2 mL chloroform, add 3 mL deionized water, sonicate with a probe for 8 min (power 200 W, sonication for 5 s, pause for 5 s), then remove chloroform by rotary evaporation at 37 °C to obtain nanoparticles. Under stirring conditions, slowly add the obtained nanoparticles dropwise to 0.45 mL chondroitin sulfate solution (10 mg / mL), and continue stirring for 30 min to obtain the final product.

[0056] Example 5

[0057] Dissolve 5 mg of doxorubicin hydrochloride in 1 mL of water, add 20 μL of 50 mg / mL NaHCO3 solution, vortex for one minute, and centrifuge to obtain doxorubicin. Dissolve doxorubicin and 15 mg INR in 1 mL of chloroform, add 10 mL of deionized water, sonicate for 10 min (150 W power, 4 s sonication, 6 s pause), and then remove the chloroform by rotary evaporation at 37 °C to obtain the final product.

[0058] Example 6

[0059] Dissolve 2 mg THP, 5 mg INR and 2.1 mg Solutol HS15 in 0.25 mL chloroform, add 4 mL deionized water, sonicate for 10 min with a probe (power 200 W, sonication for 5 s, pause for 5 s), and then remove the chloroform by rotary evaporation at 40 °C.

[0060] Example 7

[0061] Dissolve 4 mg aclarubicin and 8 mg INR in 0.4 mL of chloroform, and dissolve 4.8 mg mPEG. 3400-DSPE was dissolved in 8 mL of deionized water, and the mixture was homogenized under high pressure for 5 min. Then, the chloroform was removed by rotary evaporation at 45 °C to obtain the final product.

[0062] Example 8

[0063] Take 3 mg THP, 7.5 mg INR and 2.1 mg mPEG. 2000 -DSPE was dissolved in 0.3 mL of chloroform, 6 mL of deionized water was added, and the mixture was sonicated for 8 min (power 200W, sonication for 5 s, pause for 5 s) with a probe. Then, the chloroform was removed by rotary evaporation at 37℃. After fully dissolving 300 mg of sucrose, the mixture was freeze-dried to obtain the lyophilized powder for injection.

[0064] Example 9

[0065] Dissolve 3 mg THP and 6 mg IGR in 0.3 mL of dichloromethane, add 6 mL of deionized water, sonicate for 10 min with a probe (150 W power, 5 s sonication, 5 s pause), and then remove the dichloromethane by rotary evaporation at 37 °C.

[0066] Example 10

[0067] Dissolve 5 mg of epirubicin hydrochloride in 1 mL of water, add 20 μL of 50 mg / mL NaHCO3 solution, vortex for one minute, and centrifuge to obtain epirubicin. Dissolve epirubicin and 12 mg IRR in 0.5 mL of chloroform, add 10 mL of deionized water, sonicate with a probe for 8 min (200 W power, 5 s sonication, 5 s pause), and then remove the chloroform by rotary evaporation at 37 °C to obtain the final product.

[0068] Experimental Example 1

[0069] The tumor hypoxia-activated Golgi apparatus-targeting disruptor INR synthesized in Example 1 was dissolved in deuterated chloroform and analyzed by proton nuclear magnetic resonance spectroscopy. The results are shown in [reference needed]. Figure 1 . Figure 1 This indicates that the tumor hypoxia-activated Golgi body-targeting interferon INR has been successfully synthesized.

[0070] Experimental Example 2

[0071] The nanoparticles obtained in Examples 2, 3, 4, 5, and 6 were diluted with deionized water to a suitable concentration, and then the particle size and potential of the nanoparticles were measured using a laser particle size analyzer.

[0072] The experimental results are shown in Table 1. Tumor microenvironment-activating Golgi targeting interference agents can form nanoparticles with anthracycline antitumor antibiotics. Without the addition of surface modification materials, the nanoparticles have a strong positive charge (Examples 2 and 5). The addition of surface modification materials can significantly change the potential of the nanoparticles (Examples 3, 4, 6, and 7).

[0073] Table 1 Physicochemical properties of nanoparticles

[0074]

[0075] Experimental Example 3

[0076] The nanoparticles (NP) prepared in Example 2 and the nanoparticles (PNP) prepared in Example 3 were diluted to a suitable concentration and dropped onto a copper grid. They were then negatively stained with 2% phosphotungstic acid solution at room temperature for 1 min. The excess dye solution was blotted off with filter paper, and the morphology of the nanoparticles was observed under a transmission electron microscope.

[0077] like Figure 2 As shown, NP and PNP are both spherical in shape. NP has a smaller particle size, while PNP has a relatively larger particle size. However, the size of NP is not as uniform as that of PNP.

[0078] Experiment Example 4

[0079] In vitro cell uptake assay. Healthy mouse 4T1 breast cancer cells were collected by trypsin digestion, centrifugation, and resuspending in complete culture medium. The cells were then seeded at an appropriate density into 12-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. After 24 hours, the culture medium was discarded, and the cells were washed with PBS. Then, 1 mL of free THP solution diluted with blood-free and antibiotic-free culture medium, nanoparticles (NP) prepared in Example 2, and nanoparticles (PNP) prepared in Example 3 were added to each well. The drug concentration was calculated as 5 μg / mL based on the THP dosage. Four replicates were set for each group. After incubation for 2 hours, the drug solution was discarded, and the cells were washed three times with PBS. After trypsin digestion, the cells were collected by centrifugation at 2000 rpm for 3 minutes. The supernatant was discarded, and the cells were washed three times with PBS. Finally, the cells were resuspended in 300 μL of PBS, and the cell uptake was detected by flow cytometry using THP fluorescence.

[0080] The experimental results are shown in Figure 3 The uptake of NP and PNP by 4T1 cells was 1.70 times and 1.96 times that of free drug, respectively, indicating that prodrug nanoparticles can significantly increase the uptake of drug by 4T1 cells.

[0081] Experimental Example 5

[0082] Hypoxia responsiveness verification of the prodrug. 4T1 cells in logarithmic growth phase were seeded in 6-well plates and incubated overnight. After discarding the culture medium, the cells were gently washed three times with sterile PBS. Nanoparticles (PNPs) prepared in Example 3 were added and incubated for different times at O2 concentrations of 0.1%, 6–12%, and 20% (normal culture conditions). The cells were then removed, washed three times with PBS, digested with trypsin, collected, washed, and then repeatedly freeze-thawed to lyse the cells. 100 μL of cell lysate was placed in a 2 mL centrifuge tube, 1 mL of ethyl acetate was added, vortexed for 10 min, centrifuged at 3000 rpm for 10 min, and the upper ethyl acetate layer was quantitatively aspirated. Nitrogen gas was used to evaporate the ethyl acetate, and 100 μL of methanol was added to the centrifuge tube to dissolve the residue. The tube was vortexed for 1 min, centrifuged at 10000 rpm for 5 min, and the supernatant was used to determine the content of the prototype drug RA by HPLC.

[0083] like Figure 4 As shown, at O2 concentrations of 6–12%, the release of the precursor drug RA showed little difference compared to normal O2 concentrations in the short term, but after 12 hours of incubation, the RA content significantly differed from that at normal O2 concentrations. However, at an O2 concentration of 0.1%, the release of the precursor drug RA increased significantly, exceeding that at all time points, indicating that a hypoxic environment can effectively promote the conversion of the prodrug to the precursor drug. Furthermore, RA could also be detected in cells at normal O2 concentrations, suggesting that 4T1 cells cultured at normal O2 concentrations also express a certain amount of nitroreductase, promoting the conversion of the prodrug to the precursor drug.

[0084] Experimental Example 6

[0085] Cytotoxicity assay. Healthy 4T1 cells in logarithmic growth phase were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. The supernatant was discarded, and a series of drug or formulation diluted with blood-free and antibiotic-free medium to a series of concentration gradients were added: free THP, free THP+IMC+RA, free THP+INR, nanoparticles (NP) prepared in Example 2, and nanoparticles (PNP) prepared in Example 3. Five replicates were set for each concentration, and a blank control group was added with blank medium. After drug addition, cells were incubated for 24 hours in an incubator with normal O2 concentration and in a closed culture box with O2 concentration of 0.1%, both at 37°C. After that, the drug solution was aspirated, and 100 μL of MTT solution (0.5 mg / mL) was added to each well. Cells were incubated at 37°C for another 4 hours. After that, the cells were removed, the supernatant in the wells was aspirated with a syringe, and DMSO (150 μL per well) was added. The cells were shaken for 10 minutes to fully dissolve the formazan crystals. The absorbance at 490 nm was then measured using an enzyme-linked immunosorbent assay (ELISA) reader, the results were recorded, and the cell viability was calculated.

[0086] according to Figure 5As shown, the cytotoxicity of the combination therapy group was significantly higher than that of the single-drug-treated free THP group under both normoxic and hypoxic conditions. Furthermore, PNP exhibited the strongest cytotoxic effect on 4T1 cells under both conditions, attributed to its good stability and high cellular uptake. For the free THP, free THP+RA+IMC, and NP groups, cell survival rates were significantly higher under hypoxic conditions than under normoxic conditions, suggesting that cells under hypoxic conditions may possess stronger survivability, thus reducing the effectiveness of the drugs. There was no significant difference in cytotoxicity between hypoxic and normoxic conditions in the PNP group. This is because under hypoxic conditions, the prodrug in PNP is more readily converted to the parent drug, meaning that even with enhanced cell viability, PNP can still effectively exert its cytotoxic effect.

[0087] Experimental Example 7

[0088] Golgi apparatus structural characterization. Well-grown 4T1 cells were seeded in glass-bottomed culture dishes and cultured overnight at 37°C in a 5% CO2 cell culture incubator. Cells were then washed three times with PBS. PBS, free THP+IMC, free THP+IMC+RA, free THP+INR, nanoparticles (NP) prepared in Example 2, and nanoparticles (PNP) prepared in Example 3 were then added, with a drug concentration of 100 ng / mL based on THP. After incubation for 12 h, the drug solution was discarded, and cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 10 min, washed three times with PBS, and then 0.1% Triton was added. Incubate with X-100 at room temperature for 5 min, then wash 3 times with PBS, add 5% BSA and incubate at 37°C for 1 h, wash 3 times with PBS, add GM130 antibody and incubate overnight at 4°C, then wash 3 times with PBS, add CoraLite594 labeled secondary antibody and incubate at room temperature for 1 h, wash 3 times with PBS, finally stain cell nuclei with DAPI at room temperature for 10 min, wash 3 times with PBS, and observe fluorescence distribution under a laser confocal microscope.

[0089] like Figure 6 As shown, THP and IMC had virtually no effect on the structure of the Golgi apparatus, while the addition of RA changed the Golgi apparatus from a perinuclear aggregated state to a scattered distribution throughout the cell. The number of Golgi fragments in the free THP+INR group was significantly greater than that in the free THP+RA+IMC group, indicating that the prodrug had a stronger Golgi apparatus disrupting effect. Because the prodrug can target the Golgi apparatus, RA can reach the Golgi apparatus more extensively to exert its effect. In addition, PNP has good stability and can be taken up by cells in greater quantities, resulting in the strongest Golgi apparatus disrupting effect.

[0090] Experimental Example 8

[0091] Cell migration assay. Healthy 4T1 cells were seeded into 12-well cell culture plates and cultured for 24 hours until the cell density reached 80%. The culture medium was then discarded. A 10 μL sterile pipette tip was used to make a slit along the diameter in the center of each well. The cells were washed three times with sterile PBS to remove excess cell debris. Then, 1 mL of blood-free and antibiotic-free medium diluted with free THP, free THP+IMC+RA, free THP+INR, nanoparticles (NP) prepared in Example 2, and nanoparticles (PNP) prepared in Example 3 were added, respectively. The dosage was 100 ng / mL based on THP. Blood-free and antibiotic-free medium served as the control group. Each group had three replicates. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, the solution was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde. Images were acquired at 0 h and 24 h using an inverted fluorescence microscope.

[0092] Tumor cell migration is an important step in the process of tumor metastasis, such as... Figure 7 As shown, THP has virtually no effect on cell migration, while RA and IMC have significant effects on inhibiting cell migration. The free and nanoparticle groups of the prodrug both showed considerable anti-migration ability, especially the PNP group, which had the best effect.

[0093] Experimental Example 9

[0094] Transwell invasion assay. Matrigel was diluted 1:8 with serum-free medium pre-chilled at 4°C. 100 μL of the diluted Matrigel was added to each Transwell chamber under ice bath conditions, and incubated at 37°C for 4 h to allow the Matrigel to solidify. The collected 4T1 cells were resuspended in blood-free and antibiotic-free medium and cultured at a rate of 1 × 10⁻⁶ cells / mL. 6Inoculate 100 μL of a solution at a concentration of / mL into a Transwell chamber, then add 100 μL of blank blood-free and antibiotic-free medium, free THP, free THP+IMC+RA, or free THP / mL medium, respectively. THP+INR, NP prepared in Example 2, and PNP prepared in Example 3 (all diluted with blood-free and antibiotic-free medium) were administered at a dose of 200 ng / mL based on THP. Additionally, 600 μL of DMEM high-glucose complete medium containing 10% fetal bovine serum was added to the lower chamber. After incubation at 37°C and 5% CO2 for 24 h, the liquid in the Transwell chamber was aspirated. Cells that had not passed through the matrix gel and the sieve were wiped off with a cotton swab. After washing with PBS, cells that had passed through the matrix gel and remained on the other side of the sieve were fixed with 4% paraformaldehyde at room temperature for 10 min. The cells were washed three times with PBS, and then the chamber was permeabilized in methanol at room temperature for 20 min. After washing three times with PBS, the chamber was stained in 0.1% crystal violet solution at room temperature for 30 min. Finally, the chamber was gently washed with PBS, and observed and photographed under an inverted fluorescence microscope.

[0095] Tumor cell invasion is also a key step in the process of tumor metastasis. Figure 8 Experimental results showed that PNP had the strongest inhibitory effect on cell invasion.

[0096] Experimental Example 10

[0097] In vivo antitumor pharmacodynamics study. 4T1 cells in good growth condition and in logarithmic growth phase were collected, resuspended in PBS, and the cell concentration was adjusted to 1×10⁻⁶. 7 0.1 mL of cell suspension was injected into the right axillary fat pad of mice to establish a 4T1-bearing breast cancer mouse model. On day 6 after 4T1 cell inoculation, the successfully modeled tumor-bearing mice were randomly divided into 6 groups of 16 mice each. Each group received a tail vein injection of saline, free THP, free THP+IMC+RA, free THP+INR, NP prepared in Example 2, or PNP prepared in Example 3, respectively. The dosage was THP 4 mg / kg and INR 10 mg / kg, administered every 3 days for 4 consecutive days. From the first administration, the long and short diameters of the mouse tumors were measured with calipers every two days, and the tumor volume was calculated. On day 22 after tumor inoculation, 6 mice from each group were euthanized by cervical dislocation, and the tumor tissue was dissected, washed with saline, blotted dry with filter paper, photographed, and weighed to calculate the tumor inhibition rate. The remaining mice were used for survival recording.

[0098] like Figure 9As shown, after inoculation with 4T1 tumor cells, the tumor volume of mice in the saline group continued to increase. Free THP solution inhibited tumor growth to some extent, but the tumor growth rate of mice in the combined drug administration group was significantly reduced, especially the tumor volume growth of mice in the PNP group, which showed the most significant inhibition. After weighing the tumor tissue, the tumor inhibition rates of the free THP, free THP+RA+IMC, free THP+INR, NP, and PNP groups were calculated to be 37.39±10.65%, 76.59±7.33%, 75.80±6.44%, 73.38±9.48%, and 89.19±4.88%, respectively, indicating that PNP exerted the strongest tumor inhibitory effect. Furthermore, the median survival times for each group were 35 (Saline), 35.5 (free THP), 43 (free THP+RA+IMC), 44.5 (free THP+INR), 40.5 (NP), and 49 (PNP) days, respectively. PNP significantly prolonged the survival time of mice, and 20% of the mice had complete tumor disappearance without recurrence and were still alive 3 months later, demonstrating the good therapeutic effect of PNP.

[0099] Experimental Example 11

[0100] Efficacy study of nanoparticles in treating lung metastases of in situ breast cancer. 4T1-Luc cells in good growth condition and logarithmic growth phase were collected, resuspended in PBS, and the cell concentration was adjusted to 1×10⁻⁶. 7 Cells were injected at a rate of 0.1 mL / mL into the right axillary mammary fat pad of mice using a syringe. On day 6 after 4T1-Luc cell inoculation, successfully modeled tumor-bearing mice were randomly divided into 6 groups of 3 mice each. Each group received a tail vein injection of saline, free THP, free THP+IMC+RA, free THP+INR, NP, or PNP, respectively. The dosage was THP 4 mg / kg and INR 10 mg / kg, administered every 3 days for 4 consecutive days. On day 36 after tumor inoculation, mice were intraperitoneally injected with D-luciferin potassium salt (15 mg / mL, 10 μL / g), a substrate for luciferase. Ten minutes after injection, the mice were euthanized by cervical dissection, and the lung tissue was dissected, cleaned with saline, and immediately subjected to bioluminescence imaging using a live imaging system to record fluorescence intensity. The lung tissue was then fixed with Bouin's fixative for 24 hours, and the number of metastatic tumor nodules on the lung tissue was observed and recorded.

[0101] Experimental results are as follows Figure 10As shown, large areas of bioluminescence were observed in the lungs of mice in the untreated group, indicating numerous tumor metastases. Free THP solution showed poor inhibitory effects on lung metastasis, while the combined treatment group exhibited a strong anti-metastasis effect. In particular, no bioluminescence was detected in the lungs of the PNP group. After fixing lung tissue with Born's fixative, normal lung tissue appeared yellowish-brown, while metastatic nodules appeared bright yellow, easily distinguishable from normal tissue and facilitating nodule counting. The average number of metastatic nodules in the saline group reached 36, while the free THP, free THP+IMC+RA, free THP+INR, and NP groups had 27, 12, 10, and 9 nodules, respectively. No metastatic nodules were observed in the lung tissue of the PNP group. These results indicate that PNP can effectively inhibit lung metastasis of in situ breast cancer.

Claims

1. A tumor microenvironment-activating Golgi apparatus-targeting disruptor, characterized in that, It contains the Golgi targeting group indomethacin, an intermediate responsive group, and the Golgi interfering agent trans-retinoic acid, with the following structural formula: in .

2. The method for synthesizing a tumor microenvironment-activating Golgi apparatus-targeting disruptor according to claim 1, characterized in that, Includes the following steps: (1) 2-Hydroxy-5-methyl-isophthalic acid reacts with 4-nitrobenzyl bromide to generate intermediate compound 1, which is responsive to hypoxia, glutathione, and reactive oxygen species. The structural formula of intermediate compound 1 is as follows: in ; (2) Indomethacin reacts with intermediate compound 1 to generate intermediate compound 2. The structural formula of intermediate compound 2 is: in (3) Trans-retinoic acid reacts with intermediate compound 2 to generate tumor microenvironment-activated Golgi apparatus-targeting interference agent.

3. A nanoparticle based on a tumor microenvironment-activated Golgi apparatus-targeting disruptor, characterized in that, Based on weight, it contains 1 part of anthracycline antitumor antibiotic and 1 to 10 parts of the tumor microenvironment activating Golgi targeting interferon as described in claim 1.

4. The nanoparticles based on tumor microenvironment-activated Golgi apparatus-targeting interference agents according to claim 3, characterized in that, Based on parts by weight, 1 part is an anthracycline antitumor antibiotic and 2-5 parts are the tumor microenvironment-activating Golgi targeting interferon.

5. The nanoparticles based on tumor microenvironment-activated Golgi apparatus-targeting interference agents according to claim 3, characterized in that, The anthracycline antitumor antibiotics are selected from one or more of doxorubicin, daunorubicin, epirubicin, pirarubicin, aclarubicin, amrubicin, zolrubicin, pentorubicin, idarubicin, or mitoxantrone.

6. The nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents according to claim 3, characterized in that, The nanoparticles are modified with a surfactant, an amphiphilic polymer, or a negatively charged substance that can be adsorbed onto the surface of the nanoparticles. The amount of the surface modification material is 0 to 100 parts by weight. The surfactant is selected from one or more of Solutol HS15, Tween, Mize, benzyl ether, and poloxamer. The amphiphilic polymer is selected from one or more of PEGylated phospholipids, PEG-PLGA, PEG-PLA, and PEG-PCL. The negatively charged substance that can be adsorbed onto the surface of the nanoparticles is selected from one or more of chondroitin sulfate, hyaluronic acid, chitosan, low molecular weight heparin, cyclodextrin, fucoidan, dextran, and alginate.

7. The nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents according to claim 6, characterized in that, The amount of surface finishing material used is 0 to 10 parts.

8. The nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents according to claim 6, characterized in that, The surface modification material is one or more of mPEG2000-DSPE, Solutol HS15, and chondroitin sulfate.

9. The nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents according to claim 3, characterized in that, The nanoparticles also contain a lyophilization protectant to form a lyophilized powder injection. The lyophilization protectant is selected from one or more of sucrose, lactose, glucose, trehalose, maltose, mannitol, glycine, and dextran, and the amount of lyophilization protectant used is 0 to 1000 parts by weight.

10. The nanoparticles based on tumor microenvironment-activated Golgi apparatus-targeting interference agents according to claim 9, characterized in that, The freeze-drying protectant is sucrose.

11. The nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents according to claim 9, characterized in that, The amount of the freeze-drying protectant used is 10 to 200 parts.

12. The nanoparticles based on tumor microenvironment-activated Golgi apparatus-targeting interference agents according to claim 3, characterized in that, The nanoparticles also contain other excipients to form a pharmaceutically acceptable formulation, wherein the other excipients are selected from one or more of isotonicity modifiers, antioxidants, preservatives, and pH modifiers.

13. A method for preparing nanoparticles based on tumor microenvironment-activated Golgi targeting interference agents as described in any one of claims 3-12, characterized in that... Includes the following steps: (1) Dissolve anthracycline antitumor antibiotics in an organic solvent; (2) Dissolve the tumor microenvironment-activating Golgi-targeting interferon in an organic solution; (3) Mix the solutions obtained in (1) and (2), add deionized water, and homogenize under high pressure or use a probe for ultrasonic emulsification; (4) Remove the organic solvent by rotary evaporation to obtain the product.

14. The preparation method according to claim 13, characterized in that, The organic solvent is selected from one or more of chloroform, dichloromethane, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone; the rotary evaporation temperature range is 20–70°C, and the surface modification material is dissolved in the organic solvent in step (2) and / or dissolved in deionized water in step (3).

15. The preparation method according to claim 13, characterized in that, The organic solvent is chloroform; the rotary evaporation temperature range is 30-40℃.

16. The use of the tumor microenvironment-activating Golgi-targeting interferon of claim 1, the nanoparticles of any one of claims 3-12, the interferon obtained by the synthesis method of claim 2, or the nanoparticles prepared by the preparation method of any one of claims 13-15 in the preparation of drugs for treating tumors or tumor metastases.

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