NLG919 and sulconazole carrier-free self-assembled nano particle as well as preparation method and application thereof

Abstract

The invention provides an NLG919 and sulconazole carrier-free self-assembled nano particle as well as a preparation method and application thereof. According to the invention, carrier-free self-assembly is realized through intermolecular interaction of NLG919 and sulconazole, and the prepared nanoparticles are used for improving the drug utilization rate and the tumor inhibition effect. The two compounds both contain polycyclic aromatic structures and aza-aromatic rings and can generate pi-pi stacking and hydrophobic association; nitrogen-containing and oxygen-containing functional groups can be used as hydrogen bond acceptors / donors to form a multipoint hydrogen bond network; the halogenated aromatic ring of the thioconazole can also provide halogen bond / pi-halogen interaction. Under the condition that metal ions are not added, the system is mainly driven by hydrophobic-pi stacking-hydrogen bond synergistically to form a nano aggregate; when divalent metal ions (such as Cu < 2 + >) are optionally added, weak coordination crosslinking can further occur, and the stability of the nanoparticles is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a carrier-free self-assembled nanoparticle of NLG919 and thioconazole, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Breast cancer is one of the most common malignant tumors in women worldwide. While traditional surgery, chemotherapy, and radiotherapy can achieve short-term efficacy in some patients, their high drug resistance and recurrence rates result in persistently low long-term survival rates. In recent years, tumor immunotherapy has emerged as a novel treatment approach, opening new avenues for cancer treatment. The body's immune cells can recognize and eliminate incidentally occurring tumor cells as foreign substances. However, tumor cells can activate immune checkpoints, such as indoleamine 2,3-dioxygenase (IDO), through various pathways to evade immune clearance. Therefore, inhibiting IDO activity can effectively relieve the immunosuppressive state of T cells in the tumor microenvironment and restore their immune clearance capacity. Tumor immunotherapy exerts its anti-tumor effect by overcoming or mitigating tumor-induced immunosuppression. However, the low immunogenicity and immunosuppressive tumor microenvironment (TME) of breast cancer severely limit the durable effects of immunotherapy.

[0004] Tumor-associated fungal infections have become a challenge in clinical treatment, with invasive fungal infections (IFIs) showing a continuously rising incidence and mortality rate among cancer patients. Combination therapy with antitumor and antifungal drugs has demonstrated good therapeutic effects.

[0005] NLG919 is a small-molecule IDO1 inhibitor that can improve immunosuppressive tumor microenvironment (TME), but its application is limited due to poor water solubility, short half-life, and insufficient in vivo stability. Sulfonazole (SA), a commonly used antifungal drug in clinical practice, is also a hydrophobic small molecule with problems such as low solubility and poor bioavailability. The structural formulas of NLG919 and sulfonazole are shown below:

[0006] With the development of nanotechnology and the widespread application of nanomaterials in disease treatment, it has been found that using nanocarriers to load anti-tumor drugs can enhance efficacy and reduce toxicity in tumor treatment. Furthermore, nanomedicine delivery systems can improve the solubility of loaded drugs and increase drug permeability and retention.

[0007] Currently, various nanocarrier systems have been developed to improve drug delivery, such as liposomes and polymer micelles. However, these systems typically have complex preparation processes, limited drug loading capacity, and potential biosafety risks, making it difficult to meet the needs of clinical translation.

[0008] Therefore, it is of great significance to develop a novel nanosystem that does not require an external loading agent, has a simple preparation method, high drug loading capacity, and good stability. Summary of the Invention

[0009] To overcome the shortcomings of existing nanocarrier systems, this invention provides carrier-free self-assembled nanoparticles of NLG919 and thioconazole, along with their preparation method and applications. This invention enables the intermolecular self-assembly of two hydrophobic drugs into nanoparticles without relying on additional carrier materials, thereby significantly improving the water solubility and bioavailability of the drugs.

[0010] Specifically, the present invention achieves the above-mentioned effects through the following technical solution: In a first aspect, the present invention provides a carrier-free self-assembled nanoparticle comprising NLG919 and thioconazole; the nanoparticle is a nano-aggregate spontaneously formed by NLG919 and thioconazole through non-covalent interactions, the core interactions including π-π stacking, hydrophobic interactions and hydrogen bonds.

[0011] Optionally, in the carrier-free self-assembled nanoparticles, the mass ratio of NLG919 to thioconazole is 1 to 3:1, preferably 1.7:1.

[0012] Both NLG919 and thioconazole contain polycyclic aromatic structures and nitrogen-containing heterocyclic aromatic rings, enabling π-π stacking and hydrophobic association. Their nitrogen- and oxygen-containing functional groups can act as hydrogen bond acceptors / donors to form multi-point hydrogen bond networks. The halogenated aromatic rings of thioconazole can also provide halogen bonds / π-halogen interactions. The inventors were pleasantly surprised to find that the system mainly forms nano-aggregates driven by hydrophobic-π-π stacking-hydrogen bonding, achieving a weak and transient aggregation effect for both.

[0013] Further research revealed that although the aforementioned intermolecular interactions exist in the system, their strength is insufficient to drive the formation of uniform and stable nanoparticles. In order to prepare more stable nanoparticles, this invention provides a technical solution of adding metal ions.

[0014] Optionally, the carrier-free self-assembled nanoparticles further contain divalent metal ions, such as Cu. 2+ Zn 2+ Fe 2+ Mn 2+ Co 2+ Ni 2+ The divalent metal ion selected in this invention is Cu. 2+;Optional addition of divalent metal ions (such as Cu) 2+ Under certain conditions, weak coordination interactions can further form to enhance structural stability. This connection is a reversible physical self-assembly and does not involve chemical bonding.

[0015] In a second aspect, the present invention provides a method for preparing carrier-free self-assembled nanoparticles, comprising the following steps: dissolving NLG919 and thioconazole in an organic solvent, dissolving a salt of divalent metal ions and albumin in water, mixing the solutions, adding water and vortexing to spontaneously form nanoparticles, and then centrifuging to remove residual solvent to obtain the nanoparticles.

[0016] Optionally, the organic solvent for dissolving NLG919 is selected from organic solvents such as DMSO, DMF, methanol, ethanol and acetonitrile, preferably ethanol, and the concentration of the active ingredient in the organic phase is preferably 1 to 25 mg / mL, preferably 8 to 14 mg / mL.

[0017] Optionally, the organic solvent for dissolving thioconazole is selected from organic solvents such as DMSO, DMF and acetonitrile, preferably DMSO, and the concentration of the active ingredient in the organic phase is preferably 1 to 125 mg / mL, preferably 4 to 8 mg / mL.

[0018] Optionally, the salt of the divalent metal ion is copper sulfate pentahydrate.

[0019] Optionally, the concentration of copper sulfate pentahydrate in water should preferably be 10–100 mM, preferably 40 mM. The concentration of albumin in water should preferably be 1–40 mg / mL, preferably 4–6 mg / mL.

[0020] Optionally, in the preparation method, the mass ratio of NLG919 to thioconazole is 3:1 to 1:1, preferably 1.7:1. The Cu² + The molar ratio of the drug to the total number of drug molecules is 1:1 to 1:4, preferably 1:1.4 to 1:3.6; the albumin mass fraction is 0.05% to 0.2%, preferably 0.05%.

[0021] In some embodiments of the present invention, the preparation method is as follows: NLG919 solution with a concentration of 10-13 mg / mL, thioconazole solution with a concentration of 5-8 mg / mL, and copper sulfate solution with a concentration of 8-20 mg / mL are mixed at a volume ratio of 1:1:0.3-0.4. Then, the mixture is added dropwise to an albumin aqueous solution with a concentration of 0.8-1.2 mg / mL under rapid stirring, and the volume ratio of the mixture to the albumin aqueous solution is 0.2-0.3:1. The mixture is then rapidly mixed at 300-1500 rpm for 10-60 s.

[0022] Optionally, the preparation method further includes centrifuging to remove residual solvent after reacting at room temperature for 8 to 16 hours, preferably 12 hours.

[0023] In a third aspect, the present invention provides a pharmaceutical composition or pharmaceutical formulation comprising the nanoparticles described in the first aspect.

[0024] In a fourth aspect, the present invention provides a drug carrier or drug delivery system, wherein the drug composition comprises the nanoparticles of the first aspect or the drug composition or drug formulation of the third aspect.

[0025] In a fifth aspect, the present invention provides the use of carrier-free self-assembled nanoparticles and / or pharmaceutical compositions or pharmaceutical formulations and / or drug delivery systems in the preparation of medicaments for the prevention and / or treatment of cancer and / or tumors.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention proposes to achieve carrier-free self-assembly of nanoparticles through intermolecular interactions between NLG919 and thioconazole, which can be used to improve drug utilization and antitumor effects. Both contain polycyclic aromatic structures and nitrogen-containing heterocyclic aromatic rings, enabling π-π stacking and hydrophobic association; their nitrogen- and oxygen-containing functional groups can act as hydrogen bond acceptors / donors to form a multi-point hydrogen bond network; the halogenated aromatic rings of thioconazole can also provide halogen bonds / π-halogen interactions. Without the addition of metal ions, the system mainly forms nanoaggregates driven by a synergistic effect of hydrophobic-π-π stacking-hydrogen bonding; with the optional addition of divalent metal ions (such as Cu), the nanoaggregates can be formed. 2+ When ), weak coordination crosslinking can further occur, which can improve the stability of nanoparticles.

[0027] 2. This invention is the first to construct carrier-free self-assembled nanoparticles composed of NLG919 and thioconazole. The preparation method is simple and the conditions are mild.

[0028] 3. The nanoparticles prepared by this invention have high drug loading capacity, uniform particle size, high stability, and good biocompatibility.

[0029] 4. In cell and animal experiments, the nanoparticles showed strong tumor-suppressing effects on breast cancer models, demonstrating promising application prospects. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 The above are transmission electron microscope images of SN nanoparticles in Example 1 of this invention, with scale bars of 500 nm. Figure 2This is a particle size distribution diagram of SN nanoparticles in Example 1 of the present invention; Figure 3 This is the ultraviolet absorption spectrum formed by SN nanoparticles in Example 1 of the present invention; Figure 4 This is a diagram showing the results of the in vitro cytotoxicity experiment of SN nanoparticles in Example 1 of the present invention; Figure 5 The Trp level in the cell supernatant after SN nanoparticle treatment in Example 1 of this invention; Figure 6 The ROS level in cells after treatment with SN nanoparticles in Example 1 of this invention; Figure 7 Figure A shows the in vivo anti-tumor experimental results of SN nanoparticles in Example 1 of the present invention; Figure B shows the change curve of tumor volume and the change curve of tumor weight. Figure 8 The images show the particle size distribution and transmission electron microscopy (TEM) image of the product obtained by the copper-free preparation method in Comparative Example 1 of this invention; A is the particle size distribution, and B is the TEM image. Figure 9 The particle size distribution and transmission electron microscopy (TEM) image of the product obtained by the preparation method without albumin in Comparative Example 1 of this invention are shown; A is the particle size distribution and B is the TEM image. Figure 10 This is a comparison chart of the particle size of SN nanoparticles in Comparative Example 1 of the present invention with the product obtained by the preparation method without copper and the product obtained by the preparation method without albumin. Figure 11 The solubility of NLG919, thioconazole technical and SN nanoparticles in water. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0033] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this disclosure. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0034] In some specific embodiments of the present invention, a nanoparticle is provided, which is a carrier-free self-assembled nanoparticle composed of two drugs, NLG919 and thioconazole.

[0035] Unless otherwise specified, the nanoparticles described in this invention may also be referred to as SN nanoparticles or abbreviated as SNNPs.

[0036] In the technical solution of this invention, NLG919 and thioconazole carrier-free self-assembled nanoparticles achieve synergistic use, solving the problems of poor water solubility and low bioavailability of hydrophobic drugs, such as... Figure 9 As shown, NLG919 and thioconazole, when used individually in water, easily aggregate due to hydrophobic interactions, resulting in visible precipitation. However, the nanoparticles self-assembled using the method of this invention are well dispersed in the aqueous phase, exhibiting a uniform and transparent appearance without visible aggregation or precipitation. This indicates that self-assembly significantly improves the drug's dispersibility and solubility in water. Furthermore, the inventors of this invention discovered that NLG919 in the nanoparticles provided by this invention can increase Trp and decrease Kyn, thereby modulating the immunosuppressive tumor microenvironment. Meanwhile, thioconazole can interfere with mitochondrial function and trigger various programmed cell deaths, thereby inducing pan-apoptosis and leading to immunogenic cell death, achieving a stronger immunotherapeutic effect against breast cancer.

[0037] In some specific embodiments of the present invention, divalent metal ions (such as Cu) are added. 2+ This further enhances structural stability by forming weak coordination interactions.

[0038] In some specific embodiments of the present invention, the mass ratio of NLG919 to thioconazole in the carrier-free self-assembled nanoparticles is 1:1, 2:1, 3:1 or any ratio between 1 and 3:1.

[0039] In some specific embodiments of the present invention, a method for preparing carrier-free self-assembled nanoparticles is provided, comprising the following steps: dissolving NLG919 and thioconazole in organic solvents respectively, dissolving copper sulfate pentahydrate and albumin in water, mixing them and adding water for vortexing to spontaneously form nanoparticles, and then removing the residual solvent by centrifugation to obtain the nanoparticles.

[0040] In some specific embodiments of the present invention, the organic solvent for dissolving NLG919 is selected from organic solvents such as DMSO, DMF, methanol, ethanol and acetonitrile, and the concentration of the active ingredient in the organic phase is preferably 1 to 25 mg / mL.

[0041] In some specific embodiments of the present invention, the organic solvent for dissolving thioconazole is selected from organic solvents such as DMSO, DMF and acetonitrile, and the concentration of the active ingredient in the organic phase is preferably 1 to 125 mg / mL.

[0042] In some specific embodiments of the present invention, the concentration of copper sulfate pentahydrate in water is preferably 10–100 mM. The concentration of albumin in water is preferably 1–40 mg / mL.

[0043] In some specific embodiments of the present invention, the above-mentioned organic phase is slowly added dropwise at an organic phase:water ratio of 1:5 to 1:20 and rapidly mixed by vortexing at a vortex speed of 300 to 1500 rpm for 10 to 60 s.

[0044] In some specific embodiments of the present invention, the preparation method further includes centrifugation to remove residual solvent after reacting at room temperature for 8 to 16 hours.

[0045] In some specific embodiments of the present invention, a pharmaceutical composition or pharmaceutical formulation is provided, wherein the pharmaceutical composition comprises the carrier-free self-assembled nanoparticles described above.

[0046] In some specific embodiments of the present invention, a drug carrier or drug delivery system is provided, wherein the drug composition comprises the carrier-free self-assembled nanoparticles described above.

[0047] In some specific embodiments of the present invention, the use of carrier-free self-assembled nanoparticles and / or pharmaceutical compositions or pharmaceutical formulations and / or drug delivery systems in the preparation of medicaments for the prevention and / or treatment of cancer and / or tumors is provided.

[0048] Example 1 Accurately weigh 11.3 mg NLG919 and dissolve it in 1 mL of anhydrous ethanol, 6.6 mg thioconazole and dissolve it in 1 mL of dimethyl sulfoxide (DMSO), and 3.5 mg copper sulfate pentahydrate and dissolve it in 350 μL of water. Mix 100 μL of NLG919, 100 μL of thioconazole and 35 μL of copper sulfate pentahydrate. Then, under rapid stirring, add the mixture dropwise to 1 mL of an aqueous solution containing 1 mg albumin. Vortex at 1000 rpm for 40 s to rapidly mix. Spontaneous nanoparticles are formed.

[0049] After the reaction was completed, the nanoparticles were centrifuged (12000 g, 5 min) and washed three times with deionized water to obtain a light blue nanoparticle formulation.

[0050] Comparative Example 1 Under rapid stirring, a mixture of NLG919 and thioconazole was added dropwise to 1 mL of an aqueous solution containing 1 mg of albumin. After the reaction was completed under the same conditions, the solvent was obtained by centrifugation (12000 g, 5 min) and repeated washing three times with deionized water.

[0051] Comparative Example 2 Under rapid stirring, a mixture of NLG919, thioconazole, and copper sulfate pentahydrate was added dropwise to 1 mL of albumin-free aqueous solution. After the reaction was completed under the same conditions, the solvent was obtained by centrifugation (12000 g, 5 min) and repeated washing three times with deionized water.

[0052] Example 2 The nanoparticle formulation was freeze-dried and dissolved in 1 mL of methanol. The mixture was sonicated for 5 min to fully disintegrate the nanoparticle structure. The solution was then diluted appropriately with methanol. The NLG919 loading was quantified using high-performance liquid chromatography (HPLC) under the following conditions: mobile phase: water: methanol = 10:90, flow rate: 1 mL / min, wavelength: 268 nm, column temperature: 40 °C. Alternatively, the nanoparticle formulation was freeze-dried and dissolved in 1 mL of DMSO. The mixture was sonicated for 5 min to fully disintegrate the nanoparticle structure. The solution was then diluted appropriately with DMSO. The thioconazole loading was quantified using HPLC under the following conditions: mobile phase: water: acetonitrile = 15:85, flow rate: 1 mL / min, wavelength: 230 nm, column temperature: 25 °C. Calculations showed that the NLG919:thioconazole mass ratio in the SN nanoparticles was approximately 1:1, with an NLG919 encapsulation efficiency of approximately 45% and a thioconazole drug encapsulation efficiency of approximately 75%.

[0053] Example 3 Take 10 μL of the prepared SN nanoparticle solution, drop it onto a copper grid with a carbon support film, air dry at room temperature in the dark, and observe the morphology of the SN nanoparticles under a transmission electron microscope. The transmission electron microscope image is shown below. Figure 1 and Figure 2 As shown in the figure, the results indicate that the obtained nanoparticles are spherical, uniformly distributed, and have an average particle size of approximately 250 nm.

[0054] The particle sizes of the products of Comparative Example 1 and Comparative Example 2 were measured and compared with those obtained by electron microscopy. Figure 8 and Figure 9 As shown. The results indicate that nanoparticles were not synthesized under these conditions. The particle size comparisons after synthesis under different conditions are as follows: Figure 10 As shown in the figure. The results indicate that in the absence of metal ions and albumin, hydrophobicity, π-π stacking, and hydrogen bonding alone are insufficient to drive the formation of stable nanostructures; however, the introduction of copper ions and albumin into the system promotes the assembly and cross-linking of drug molecules, thereby significantly improving the formation efficiency and stability of nanoparticles.

[0055] Example 4 The absorption peaks of NLG919, thioconazole, and SN nanoparticles were determined using a UV-Vis spectrophotometer. Figure 3 As shown in the figure, the results indicate that the characteristic peaks of NLG919 and thioconazole appear simultaneously in the prepared SN nanoparticles, proving the successful preparation of the formulation.

[0056] Example 5 1. Cell culture The mouse breast cancer cell line 4T1 was selected as the research subject. Frozen cells were collected and treated with a solution containing 1% penicillin. High-glucose DMEM culture in streptomycin solution and 10% fetal bovine serum was conducted at 37°C and 5% CO2. When the cells reached high density, they were passaged, transferred to culture flasks proportionally for further culture, and cell counting was performed.

[0057] 2. Cytotoxicity assay 4T1 cells were used to evaluate the cytotoxicity of different concentrations of NLG919, thioconazole, and SN nanoparticles. The target compounds NLG919, thioconazole, and SN nanoparticles were diluted in culture medium to 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL, respectively, according to the concentration of NLG919. The thioconazole concentration was diluted proportionally to the concentration in the SN nanoparticles. 4T1 cells in logarithmic growth phase were collected and cultured at a concentration of 1 × 10⁶ cells / mL. 4 Add cells / well to a 96-well plate at a concentration of [number] cells / well. Incubate overnight, then add 100 μL of different concentrations of the target compound solution and incubate for 24 hours. Discard the drug-containing medium and wash the plate. Add 10 μL of 5 mg / mL MTT solution to each well and continue incubation for 4 hours. Then discard the liquid in the wells, add 150 μL of DMSO to each well to dissolve the cells, and measure the absorbance at 490 nm using a microplate reader. Calculate the cell viability using the following formula: .

[0058] Where A-negative represents the absorbance of wells containing untreated cells, A-sample represents the absorbance of wells containing cells treated with various samples, and A-blank represents the absorbance of wells containing neither seeded cells nor treated with drugs. The absorbance is determined based on the concentration of the target compound and the corresponding viability, as follows: Figure 4 As shown, SN nanoparticles have an ideal inhibitory effect on the mouse breast cancer cell line 4T1, exhibiting an excellent cell inhibition rate of up to 90%.

[0059] 3. Extracellular Trp content detection experiment Collect 4T1 cells in logarithmic growth phase, and inoculate the cells at a rate of 5 × 10⁻⁶ cells / year. 4 Cells were cultured in 24-well plates at a concentration of [number] cells / well, supplemented with mouse recombinant IFN-γ (100 ng / mL) for 24 hours to stimulate IDO1 expression. Subsequently, cells were treated with NLG919, SA, or SN nanoparticles at an equivalent NLG919 concentration. The supernatant was collected and mixed with 30% trichloroacetic acid (100 μL). The mixture was stirred for 1 minute, allowed to stand for 5 minutes, and then centrifuged at 10,000 g to remove protein precipitates. Finally, the Trp content in the supernatant was measured by HPLC.

[0060] Depend on Figure 5 It can be seen that the SN nanoparticle group showed an increase in Trp compared to the blank group, but the difference was not significant compared to NLG919, indicating that the nanoparticles can increase Trp like NLG919, thereby regulating the immunosuppressive tumor microenvironment.

[0061] 4. Intracellular reactive oxygen species detection experiment Collect 4T1 cells in logarithmic growth phase, and inoculate the cells at a rate of 5 × 10⁻⁶ cells / year. 4 Cells were cultured in 24-well plates at a concentration of [number] cells / well. After 24 hours of culture, 4T1 cells were incubated with NLG919, SA, or SN nanoparticles at an equivalent SA concentration for 24 hours. Following treatment, cells were cultured at 37°C with 10 μM 2,7-dichlorodihydrofluoric acid diacetate (DCFH-DA) for 30 minutes. ROS levels were then analyzed by flow cytometry.

[0062] Depend on Figure 6 It can be seen that the ROS content of the SN nanoparticle group was significantly increased compared with other groups, indicating that the nanoparticles significantly improved the ability to damage mitochondrial function, thereby triggering oxidative stress, increasing the content of intracellular reactive oxygen species, and ultimately inducing panapoptosis and leading to immunogenic cell death.

[0063] Example 6 Animal model establishment 4T1 cells (2×10⁻⁶) in 100 μL PBS 6 The tumor model was established by subcutaneous injection into the flank of each mouse.

[0064] 2. Animal in vivo anti-tumor experiments When the tumor reaches ~80mm 3 Mice were weighed and randomly divided into 5 groups: a control group receiving saline, an experimental group receiving a direct mixture of NLG919 (8.6 mg / kg), thioconazole (5 mg / kg), NLG919 (8.6 mg / kg) and thioconazole (5 mg / kg), and an experimental group receiving SN nanoparticles (equivalent to 5 mg / kg thioconazole). In situ treatment was administered every two days for a total of three treatments. Tumor volume was measured every two days, and the tumor volume (V) was calculated using the following equation: After treatment, the mice were euthanized and the tumors were removed and weighed.

[0065] Depend on Figure 7 The changes in tumor volume and weight in groups A and B show that the SN nanoparticle group exhibits significantly slower tumor growth compared to other groups, indicating that the nanoparticles greatly enhance the anti-tumor effect. This is mainly due to the immunogenic cell death effect caused by thioconazole-induced pan-apoptosis, which enhances the immunotherapy effect. In addition, NLG919's ability to reverse the tumor immune microenvironment also contributes to the improved therapeutic effect.

[0066] This invention is the first to prepare stable nanoparticles containing two drugs, the hydrophobic drug NLG919 and thioconazole. These nanoparticles not only overcome the shortcomings of poor water solubility and low bioavailability of hydrophobic drugs, but also overcome the low immunogenicity of tumors and the resistance of the tumor immunosuppressive microenvironment in immunotherapy, thereby improving the efficacy of immunotherapy.

[0067] Matters not covered in this invention are common knowledge.

[0068] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A carrier-free self-assembled nanoparticle, characterized in that, It contains NLG919 and thioconazole; the nanoparticles are nano-aggregates spontaneously formed by NLG919 and thioconazole through non-covalent interactions, and the core interactions include π-π stacking, hydrophobic interactions and hydrogen bonds.

2. The carrier-free self-assembled nanoparticles according to claim 1, characterized in that, In the carrier-free self-assembled nanoparticles, the mass ratio of NLG919 to thioconazole is 1:1 to 3.

3. The carrier-free self-assembled nanoparticles according to claim 1, characterized in that, The carrier-free self-assembled nanoparticles also contain divalent metal ions, which are selected from Cu. 2+ Zn 2+ Fe 2+ Mn 2+ Co 2+ and Ni 2+ Cu is preferred 2 + The divalent metal ions are added in the form of salts or hydrates.

4. The method for preparing carrier-free self-assembled nanoparticles according to claim 1, characterized in that, The process includes the following steps: dissolving NLG919 and thioconazole in organic solvents, dissolving copper sulfate and albumin in water, mixing them under vortex conditions to spontaneously form nanoparticles, and then removing the residual solvent by centrifugation to obtain the final product.

5. The method for preparing carrier-free self-assembled nanoparticles according to claim 4, characterized in that, The organic solvents for dissolving NLG919 are selected from DMSO, DMF, methanol, ethanol and acetonitrile, and the concentration of NLG919 in the organic phase is 1 to 25 mg / mL.

6. The method for preparing carrier-free self-assembled nanoparticles according to claim 4, characterized in that, The organic solvents for dissolving thioconazole are selected from DMSO, DMF and acetonitrile, and the concentration of thioconazole in the organic phase is 1 to 125 mg / mL.

7. The method for preparing carrier-free self-assembled nanoparticles according to claim 4, characterized in that, The concentration of copper sulfate in water is 10–100 mM; the concentration of albumin in water is 1–40 mg / mL.

8. A pharmaceutical composition or pharmaceutical preparation, characterized in that, The pharmaceutical composition comprises the carrier-free self-assembled nanoparticles of claim 1.

9. A drug carrier or drug delivery system, characterized in that, It includes the carrier-free self-assembled nanoparticles described in claim 1.

10. The use of the carrier-free self-assembled nanoparticles of claim 1 and / or the pharmaceutical composition or pharmaceutical formulation of claim 8 and / or the drug carrier or drug delivery system of claim 9 in the preparation of medicaments for the prevention and / or treatment of cancer and / or tumors.

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