Nano assembly for tumor hypoxia relief and ferroptosis / immunotherapy as well as preparation method and application of nano assembly

Abstract

The invention discloses a nano assembly for tumor hypoxia relief and ferroptosis / immunotherapy as well as a preparation method and application of the nano assembly, and belongs to the technical field of new auxiliary materials and new dosage forms for combined treatment of pharmaceutical preparations. Wherein the ferroptosis agonist and the fluorinated prodrug are co-assembled through intermolecular forces such as pi-pi accumulation, hydrophobic interaction, hydrogen bonds and the like, and the DSPE-PEG2K is modified after co-assembly. The molar ratio of the ferroptosis agonist to the fluorinated prodrug is (5: 1)-(1: 5), and the mass ratio of the drug to the PEG modifier is (100: 0)-(80: 20). The co-assembled nano preparation provided by the invention provides a new strategy and more choices for developing the delivery of drugs, and meets the urgent demand of the preparation for relieving tumor hypoxia and ferroptosis / immune combined treatment strategy in clinic.

Description

Technical Field

[0001] The present invention belongs to the technical field of new adjuvants and new dosage forms for combined treatment of pharmaceutical preparations, and specifically relates to the construction of a nanoassembly formed by the co-assembly of a ferroptosis agonist and a fluorinated prodrug, and the application of the nanoassembly in drug delivery. Background Art

[0002] Currently, a variety of therapies have been widely used in the clinical treatment of cancer, but the existing treatment methods mainly based on surgery, chemotherapy and radiotherapy still show unsatisfactory clinical efficacy. With the continuous deepening of research, ferroptosis has attracted widespread attention as a new type of cell death. In recent years, a variety of strategies to induce ferroptosis have been explored and developed, including increasing the iron content in tumor cells, reducing the synthesis of glutathione (GSH) in tumor cells or increasing the content of reactive oxygen species (ROS) and lipid peroxides, and providing unsaturated fatty acids to increase the level of lipid peroxides in tumor cells. It is worth noting that sorafenib (Sor), as an effective ferroptosis inducer, can inhibit the cystine / glutamate antiporter system (system Xc - ) to inhibit the uptake of cystine by tumor cells, thereby reducing the production of GSH in tumor cells, and further inhibiting the activity of downstream glutathione peroxidase 4 (GPX4), inducing ferroptosis of tumor cells. At the same time, the process of tumor ferroptosis is accompanied by strong oxidative stress, which stimulates tumor cells to release immunogenic antigens, induces the occurrence of immunogenic cell death (ICD), and finally activates the anti-tumor immune response. Among them, ICD is a special form of cell death. Dead tumor cells release cell damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), high mobility group protein 1 (HMGB1), and adenosine triphosphate (ATP), which significantly promotes the maturation of dendritic cells (DC) and the recruitment of cytotoxic T lymphocytes, thereby activating anti-tumor immune responses. However, the high expression of PD-L1 in tumor cells is one of the main mechanisms leading to tumor immune escape, which further leads to the occurrence of immune resistance, resulting in poor immune efficacy. It can be seen that the use of immune checkpoint inhibitors in tumor treatment can shield the "brake" function of the immune response, thereby promoting the immune system to recognize and kill tumor cells. Although ferroptosis combined with immunotherapy has potential for clinical application, there are still some problems and challenges that need to be addressed.

[0003] Typically, the oxygen tension of normal tissues is between 10 and 80 mmHg. However, most solid tumors present a hypoxic tumor microenvironment with an oxygen tension of less than 5 mmHg, which is significantly lower than the level of normal tissues due to their characteristics such as vascular abnormalities, blood flow fluctuations, and insufficient oxygen diffusion. This hypoxic state not only hinders the efficacy of ferroptosis and ICD, but also forms an immunosuppressive microenvironment, which seriously hinders the effectiveness of the anti-tumor immune response triggered by ICD. Specifically, under hypoxic conditions, tumor-derived cytokines can transform tumor-associated macrophages (TAMs) into immunosuppressive M2-like macrophages, promoting tumor growth and immune resistance. In addition, the hypoxic microenvironment activates hypoxia-inducible factor (HIF-1α), directly regulates the function and differentiation of myeloid-derived suppressor cells (MDSCs), and promotes immune tolerance. Therefore, improving the hypoxic microenvironment of tumor cells is the key to improving the efficacy of ferroptosis / immunotherapy. In recent years, perfluorocarbons (PFCs) have shown good clinical potential due to their special biocompatibility and high oxygen decomposition ability, and have even been used as artificial blood substitutes. In addition, oxygen exists in hemoglobin in a covalently bound manner, and the unique gas solubility of PFCs allows them to accelerate oxygen release when necessary. Therefore, PFCs show great application prospects in in vivo oxygenation and relief of tumor hypoxia. However, the simultaneous and effective delivery of oxygen and cytotoxic drugs in vivo remains a major challenge.

[0004] With the continuous advancement of bio-nanotechnology, many new nano drug delivery systems have attracted widespread attention from researchers. Among them, small molecule nano drug delivery strategies provide a promising path for the efficient administration of anti-tumor drugs. Unlike traditional nano drug delivery systems that rely on carrier materials, this carrier-free system avoids the challenges of drugability caused by carrier materials and exhibits multiple advantages, including easy preparation, high drug loading capacity, and no carrier-related toxicity. In addition, the carrier-free small molecule nano drug delivery system is universal and can not only deliver a single drug, but also co-deliver multiple cytotoxic drugs by adjusting the dose ratio. This flexibility makes multimodal combined treatment of cancer possible. The carrier-free small molecule nano drug delivery system has a high drug loading capacity and excellent tumor selectivity, which is expected to solve the problems of ferroptosis caused by tumor hypoxia and unsatisfactory immunotherapy effects. Summary of the invention

[0005] Based on the technical problems described in the background technology, the present invention further designs a kind of ferroptosis agonist and fluorinated prodrug co-assembled nanoparticles to solve the problems of poor ferroptosis and ICD induction effect and enhanced tumor immune escape caused by tumor hypoxia, so as to achieve good stability, high drug loading, and low toxic and side effects. The technical effects, thereby solving the problem of unsatisfactory ferroptosis and immunotherapy effects caused by tumor hypoxia, improve the effect of intracellular oxidative stress, and synergistically promote the improvement of tumor cell immunogenicity and the reduction of immune escape. The purpose of the present invention is to form co-assembled nanoparticles with ferroptosis agonists and fluorinated prodrugs, or nanoparticles assembled from ferroptosis agonists, fluorinated prodrugs and PEG modifiers.

[0006] The present invention achieves the above object through the following technical solutions:

[0007] The present invention provides a nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy, wherein the nanoassembly is co-assembled by a ferroptosis agonist and a fluorinated prodrug through intermolecular forces, and modified with a PEG modifier; the parent drug of the fluorinated prodrug is a small molecule immune checkpoint inhibitor, the side chain of the fluorinated prodrug is a perfluorocarbon, and the parent drug and the side chain are bridged by a linker; the linker is a chemical bond corresponding to the tumor microenvironment; the molar ratio of the ferroptosis agonist to the fluorinated prodrug is 5:1-1:5, and the mass ratio of the sum of the mass of the ferroptosis agonist and the fluorinated prodrug to the PEG modifier is 100:0-70:30.

[0008] In the above technical scheme, further, the molar ratio of the ferroptosis agonist to the fluorinated prodrug is 1:1, and the mass ratio of the sum of the mass of the ferroptosis agonist and the fluorinated prodrug to the PEG modifier is 100:0 to 80:20.

[0009] In the above technical solution, further, the intermolecular forces include π-π stacking, hydrophobic interaction, electrostatic force and hydrogen bond.

[0010] In the above technical scheme, further, the ferroptosis agonist includes one or more of erastine, sorafenib, RSL-3 and sulfasalazine; the small molecule immune checkpoint inhibitor includes JQ1, PD1-PDL1 inhibitor 2 (BMS-202), BMS-1001, MAX-10181 or GS-4224; the linker is a monosulfide bond, a disulfide bond, or a diselenide bond; the side chain is perfluorohexanoic acid, perfluorooctanoic acid, perfluorodecanoic acid, perfluorododecanoic acid, hexanoic acid, octanoic acid, decanoic acid or dodecanoic acid.

[0011] In the above technical scheme, further, the PEG modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG and PE-PEG, and the molecular weight of PEG is 200-20000.

[0012] In the above technical solution, further, the PEG modifier is DSPE-PEG 2K .

[0013] In the above technical solution, further, the synthesis method of the prodrug includes:

[0014] The linker is subjected to an esterification reaction with perfluorohexanoic acid, perfluorooctanoic acid, perfluorodecanoic acid, perfluorododecanoic acid, hexanoic acid, octanoic acid, decanoic acid or dodecanoic acid using an esterification reaction catalyst, and the reaction product is purified and subjected to an esterification reaction with a small molecule immune checkpoint inhibitor to obtain a prodrug; the esterification reaction catalyst is EDCI and DMAP.

[0015] In the above technical solution, further, the aforementioned nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy, the preparation method comprises the following steps:

[0016] The ferroptosis agonist, the fluorinated prodrug and the PEG modifier are dissolved in an organic solvent respectively, mixed under vortex, and the mixed solution is slowly dropped into water, and uniform co-assembled nanoparticles are spontaneously formed under stirring; finally, the organic solvent is removed to obtain a nanoassembly;

[0017] Alternatively, the ferroptosis agonist, the fluorinated prodrug co-assembled nanoparticles and the organic solvent of the PEG modifier are prepared separately, and then the organic solvent of the PEG modifier is added dropwise to the co-assembled nanoparticles under vigorous stirring, and finally the organic solvent is removed to obtain the nanoassembly;

[0018] Or oxygen was charged into the nanoassemblies prepared by the above two methods (30 min) to obtain oxygen-saturated nanoassemblies;

[0019] The method for removing the organic solvent includes solvent evaporation, ultrafiltration and membrane permeation.

[0020] In the above technical solution, further, oxygen is charged at a flow rate of 5 L / min for 30 minutes.

[0021] In the above technical solution, further, the organic solvent includes one or a combination of any two of anhydrous ethanol, tetrahydrofuran, and dimethyl sulfoxide; preferably, tetrahydrofuran and anhydrous ethanol are mixed in equal volumes.

[0022] The present invention also provides the use of the aforementioned nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy in the preparation of a drug delivery system; or in the preparation of an injection, oral administration or local administration system.

[0023] The present invention also provides the use of the aforementioned nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy in the preparation of anti-tumor drugs.

[0024] The beneficial effects of the present invention compared with the prior art are as follows:

[0025] 1. The present invention prepares a co-assembled nanoparticle formed by a ferroptosis agonist and a fluorinated prodrug, which can be used for tumor treatment. The ferroptosis agonist can effectively inhibit the function of System Xc-, resulting in the inhibition of the synthesis of intracellular GSH, thereby destroying the redox balance, and promoting ICD of tumor cells through oxidative stress, thereby improving the immunogenicity of tumor cells. The fluorinated prodrug can effectively alleviate tumor hypoxia, inhibit the expression of tumor cell PD-L1, and enhance the recognition of tumors by cytotoxic T cells. The combined use of the two can achieve synergistically enhanced ferroptosis and immunotherapy efficacy by alleviating hypoxia.

[0026] 2. The ferroptosis agonist and fluorinated prodrug co-assembled nanoparticles of the present invention achieve technical effects such as high drug loading, good stability, and low toxic side effects, meeting the urgent clinical demand for high-efficiency and low-toxic preparations, providing a new strategy for the assembly of synergistic drug nanoparticles, and providing an efficient nano-platform for the development of carrier-free hybrid nanoassemblies, the alleviation of tumor hypoxia, and ferroptosis combined with immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0028] Figure 1 This is the synthetic route of Example 1 of the present invention.

[0029] Figure 2 The mass spectrum and NMR spectrum of the FSSJ prodrug of Example 1 of the present invention.

[0030] Figure 3 This is the Malvern particle size distribution diagram of the FJS nanoparticles of Example 2 of the present invention.

[0031] Figure 4 This is the Malvern particle size distribution diagram of the FJSO nanoparticles of Example 2 of the present invention.

[0032] Figure 5 TEM images of FJS nanoparticles and FJSO nanoparticles according to Example 2 of the present invention.

[0033] Figure 6This is a stability diagram of the FJS nanoparticles and FJSO nanoparticles of Example 3 of the present invention.

[0034] Figure 7 This is the molecular docking diagram of Sor and FSSJ in Example 4 of the present invention.

[0035] Figure 8 This is the force destruction of the non-PEGylated nanoparticles of Example 4 of the present invention in water containing 10 mM sodium chloride, sodium dodecyl sulfate and urea.

[0036] Fig. 9 The oxygen carrying capacity of the FJS nanoparticles of Example 5 of the present invention.

[0037] Fig.10 The cumulative release of Sor from the FJS nanoparticles and FJSO nanoparticles of Example 6 of the present invention in PBS (pH 7.4) containing 0 mM and 10 mM GSH.

[0038] Fig.11 These are confocal microscope photographs of the cellular uptake of the Cy7 solution, Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles of Example 7 of the present invention at 0.5 hour and 2 hours.

[0039] Fig.12 This is a flow cytometric graph of the cellular uptake of the Cy7 solution, Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles of Example 7 of the present invention.

[0040] Fig.13 These are the cytotoxic results of JQ1 solution, Sor solution, SJ solution, FJS nanoparticles and FJSO nanoparticles in Example 8 of the present invention in 4T1 and 3T3 cells; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles.

[0041] Fig.14 These are the cytotoxic results of JQ1 solution, Sor solution, SJ solution, FJS nanoparticles and FJSO nanoparticles in 4T1 cells after adding different concentrations of ferroptosis inhibitor (Fer-1) in Example 8 of the present invention; I: JQ1 solution, II: Sor solution, III: SJ solution, IV: FJS nanoparticles, V: FJSO nanoparticles.

[0042] Fig.15 Schematic diagram of the changes in glutathione levels of PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles under normoxia and hypoxia in Example 9 of the present invention; II: Sor solution, IV: FJS nanoparticles, V: FJSO nanoparticles, VI: PBS (pH 7.4).

[0043] Fig.16 These are photographs of the lipid peroxidation levels of PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles under normoxia and hypoxia in Example 10 of the present invention, taken under a confocal microscope.

[0044] Fig.17 These are photographs of the active oxygen levels of PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles under normoxia and hypoxia according to Example 10 of the present invention, taken under a confocal microscope.

[0045] Fig.18 Flow cytometric graphs of the reactive oxygen levels of PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles under normoxia and hypoxia in Example 10 of the present invention; II: Sor solution, IV: FJS nanoparticles, V: FJSO nanoparticles, VI: PBS (pH 7.4).

[0046] Fig.19 These are photographs of the CRT levels of PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles under normoxia and hypoxia in Example 11 of the present invention, taken under a confocal microscope.

[0047] Fig. 20 These are photographs of the HMGB1 levels of PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles under normoxia and hypoxia in Example 11 of the present invention, taken under a confocal microscope.

[0048] Fig.21 PD-LI levels of PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles under normoxia and hypoxia in Example 12 of the present invention; Ⅰ: Sor solution, Ⅱ: FJS nanoparticles, Ⅲ: FJSO nanoparticles, Ⅳ: PBS (pH 7.4).

[0049] Fig. 22 The in vivo pharmacokinetic curves of Cy7 solution, Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles administered in Example 13 of the present invention.

[0050] Fig.23 These are in vivo fluorescence imaging images of mice administered Cy7 solution, Cy7-FJS nanoparticles, and Cy7-FJSO nanoparticles in Example 14 of the present invention.

[0051] Fig.24 These are fluorescence imaging images of isolated organs of mice administered Cy7 solution, Cy7-FJS nanoparticles, and Cy7-FJSO nanoparticles in Example 14 of the present invention.

[0052] Fig.25 This is a fluorescence quantitative graph of isolated organs of mice administered Cy7 solution, Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles in Example 14 of the present invention.

[0053] Fig.26 These are photos of mouse in vitro tumors from the in vivo anti-tumor experiment of Example 14 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0054] Fig. 27 This is a graph showing the tumor growth curve of mice in the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0055] Fig.28 These are the results of the mouse liver and kidney function analysis of the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0056] Fig.29 This is the H&E staining image of the mouse in vitro tumor in the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0057] Fig.30 This is a graph showing the changes in body weight of mice in the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0058] Fig.31 This is a graph of HIF-1α levels in mouse tumors in vitro in the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0059] Fig.32 This is a graph of GSH levels in mouse tumors in vitro in the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0060] Fig.33This is a graph of the PD-L1 level in mouse tumors in vitro in the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4).

[0061] Fig.34 This is a graph of CRT levels in mouse in vitro tumors of the in vivo anti-tumor experiment of Example 15 of the present invention; Ⅰ: JQ1 solution, Ⅱ: Sor solution, Ⅲ: SJ solution, Ⅳ: FJS nanoparticles, Ⅴ: FJSO nanoparticles, Ⅵ: PBS (pH 7.4). DETAILED DESCRIPTION

[0062] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0063] Example 1: Synthesis of fluorinated JQ1 prodrug (FSSJ)

[0064] In a 250 mL eggplant-shaped bottle, perfluorooctanoic acid (3 mmol) was dissolved in 15 mL of dichloromethane. EDCI (9 mmol) and HOBt (3 mmol) pre-dissolved in dichloromethane were then added to the eggplant-shaped bottle. The reaction was activated at 0°C for 2 h under nitrogen protection. After activation, the reaction solution was allowed to return to room temperature. 2,2'-dithiodiethanol (4 mmol) and DMAP (1.5 mmol) solution, which were also pre-dissolved in dichloromethane, were added dropwise with stirring. The reaction was then carried out at 25°C for 12 h under nitrogen environment. The intermediate product was obtained after rotary evaporation of the solvent and purified by silica gel column chromatography. In the subsequent step, JQ1 (1 mmol) was dissolved in 20 mL of DMF. EDCI (3 mmol) dissolved in dichloromethane was introduced and the reaction was started in an ice bath under nitrogen protection for 2 h. After activation, the intermediate (1 mmol) and DMAP (0.2 mmol) pre-dissolved in dichloromethane were added to the reaction mixture. Then the mixture was stirred at 30°C for 48 hours under nitrogen atmosphere. Finally, the target fluorinated technical (FSSJ) was purified by preparative liquid chromatography with an acetonitrile / water ratio of 85:15. 1 H NMR and mass spectrometry analysis confirmed the successful synthesis of the fluorinated JQ1 prodrug, the structure of which is shown below:

[0065]

[0066] Synthesis route such as Figure 1 The results of mass spectrometry and NMR are shown in Figure 2 shown.

[0067] Mass spectrum: [M+H]+=933.05;

[0068] NMR: 1 H NMR (400MHz, DMSO-d6) δ7.47(d,J=8.9Hz,2H,Ar-H),7.42(d,J=8.7Hz,2H,Ar-H),4.67(t,J=6.1Hz,2 H,-COOCH2-),4.51(dd,J=8.1,6.4Hz,1H,-OCOCH2-CH-),4.34(td,J=5.9,3.6Hz,2H,-COOCH2-),3.4 5(dd,J=7.3,5.0Hz,2H,-OCO-CH2-CH-),3.10(t,J=6.1Hz,2H,-CH2SSCH2-),3.01(t,J=6.2Hz,2H,-C H2SSCH2-),2.60(s,3H,triazole-CH3),2.40(s,3H,thiophene-CH3),1.62(s,3H,thiophene-CH3).

[0069] Example 2: Preparation of FJS nanoparticles

[0070] Sor and FSSJ in different molar ratios were dissolved in tetrahydrofuran and ethanol (v / v, 1:1) to obtain a 5 mg / mL drug-containing solution. Under stirring, 200 μL of the solution was slowly added dropwise to 2 mL of deionized water. Sor and FSSJ spontaneously formed uniform nanoparticles, and then the organic solvent in the nanoformulation was removed by rotary evaporation at 30°C to obtain a nanocolloid solution without any organic solvent.

[0071] The particle size and particle size distribution of the prepared nanoformulation were tested, and the results are shown in Table 1.

[0072] Table 1. Particle size and particle size distribution of FJS nanoparticles

[0073]

[0074]

[0075] As shown in Table 1, the particle size of the nanoparticles is between 70-120 nm. When Sor:FSSJ=1:1, the distribution of FJS nanoparticles is more uniform. The initial preferred ratio of Sor to FSSJ is 1:1.

[0076] (1) Preparation of non-PEGylated FJS nanoparticles: 0.46 mg of Sor and 0.993 mg of FSSJ (equimolar amounts) were accurately weighed and dissolved to 5 mg / mL with 200 μL of tetrahydrofuran and anhydrous ethanol solution (v / v, 1:1). The solution was slowly added dropwise to 2 mL of deionized water under stirring to spontaneously form uniform non-PEGylated FJS nanoparticles. The organic solvent in the nanoformulation was then removed by rotary evaporation at 30°C to obtain a nanocolloidal solution free of any organic solvent.

[0077] (2) DSPE-PEG 2K Preparation method of modified FJS nanoparticles: accurately weigh 0.46 mg of Sor and 0.993 mg of FSSJ (equimolar amounts), dissolve them to 5 mg / mL with 200 μL of tetrahydrofuran and anhydrous ethanol solution (v / v, 1:1) to obtain FJS solution; accurately weigh 0.25 mg of DSPE-PEG 2K DSPE-PEG was dissolved in tetrahydrofuran and anhydrous ethanol solution (v / v, 1:1) to form 10 mg / mL 2K Stock solution. Mix 200 μL FJS solution with 25 μL DSPE-PEG 2K The mother liquor was mixed evenly, and the solution was slowly added dropwise to 2 mL of deionized water under stirring to obtain uniform FJS nanoparticles. The organic solvent in the nanoformulation was then removed by rotary evaporation at 30°C to obtain a nanocolloid solution without any organic solvent. The particle size and particle size distribution of the prepared FJS nanoparticles were detected by dynamic light scattering ( Figure 3 ).

[0078] (3) Preparation of oxygen-carrying FJS nanoparticles: 0.46 mg of Sor and 0.993 mg of FSSJ (equimolar amounts) were accurately weighed and dissolved to 5 mg / mL with 200 μL of tetrahydrofuran and anhydrous ethanol solution (v / v, 1:1) to obtain FJS solution; 0.25 mg of DSPE-PEG was accurately weighed 2K DSPE-PEG was dissolved in tetrahydrofuran and anhydrous ethanol solution (v / v, 1:1) to form 10 mg / mL 2K Stock solution. Mix 200 μL FJS solution with 25 μL DSPE-PEG 2KThe mother liquor was mixed evenly, and the solution was slowly added dropwise to 2 mL of deionized water under stirring to obtain uniform FJS nanoparticles. Then, the organic solvent in the nanoformulation was removed by rotary evaporation at 30°C to obtain a nanocolloidal solution without any organic solvent. 5 mL of FJS nanoparticles were put into a capped vial, and the FJS nanoparticles were oxygenated steadily and evenly for 30 minutes through an oxygen pressure balance valve. Oxygen was charged at a flow rate of 5 L / min to obtain FJSO nanoparticles carrying oxygen. The particle size and particle size distribution of the prepared FJSO nanoparticles were detected by dynamic light scattering ( Figure 4 ).

[0079] The particle size and morphology of the PEG-modified FJS nanoparticles and FJSO nanoparticles prepared in Example 1 were measured by transmission electron microscopy. Figure 5 ,The transmission electron microscopy image shows that the nanoparticles are uniform spherical and the particle size is about 70nm.

[0080] Example 3: Stability test of nanoparticles

[0081] 1 mL of the PEG-modified FJS nanoparticles and FJSO nanoparticles (0.5 mg / mL) prepared in Example 1 was taken out and added to 10 mL of PBS (pH 7.4), incubated in a shaker at 37°C for 12 hours, and the particle size changes were measured by dynamic light scattering at predetermined time points (0, 1, 2, 4, 8 and 12 hours). The results are shown in FIG. Figure 6 As shown, compared with non-PEG-modified FJS nanoparticles, FJS nanoparticles and FJSO nanoparticles had better stability in PBS, and the particle size did not change significantly within 12 hours.

[0082] Example 4: Analysis of the assembly mechanism of Sor and FSSJ

[0083] Through computer simulation, the mechanism of Sor and FSSJ assembly was explored, and the molecular docking calculation was completed using the Vina program of Yinfu cloud computing platform. The energy of compounds Sor and FSSJ was minimized under the MMFF94 force field to obtain a 3D structure and form a stable nanoassembly. The AutoDock Vina program was used for semi-flexible docking, and sodium chloride, sodium dodecyl sulfate and urea were used for force destruction. The results are as follows Figure 7-8 As shown, there are multiple forces between Sor and FSSJ molecules, such as π-π stacking, hydrophobic force, electrostatic force and hydrogen bonding, which make great contributions to the co-assembly of Sor and FSSJ molecules.

[0084] Example 5: Oxygen carrying capacity of nanoparticles

[0085] The oxygen carrying capacity of the preparation was determined using a dissolved oxygen meter. The specific method is as follows: 5 mL of fluorinated prodrug (5 mL of PBS, pH 7.4 as a blank control) was placed in a 50 mL centrifuge bucket for oxygenation for 30 minutes; 15 mL of deionized water that had been boiled several times in advance was placed in a small beaker, and the small beaker was placed in an ultrasound machine (not turned on), and the probe of the dissolved oxygen meter was placed below the liquid level in the beaker. After the reading stabilized, the oxygenated preparation was quickly poured into 15 mL of deionized water, and the ultrasound device was turned on at the same time. During this process, the probe of the dissolved oxygen meter was always below the liquid level, and the reading of this process was recorded. The results are as follows: Fig. 9 As shown, the fluorinated preparation phase has excellent oxygen carrying capacity, and the co-assembly with Sor does not affect the oxygen carrying capacity of the fluorinated preparation.

[0086] Example 6: In vitro drug release

[0087] The in vitro drug release behavior of the PEG-modified FJS nanoparticles and FJSO nanoparticles prepared in Example 1 was evaluated by an in vitro dialysis method. PBS (pH 7.4) containing 30% anhydrous ethanol and 1% Tween 80 was used as the release medium. 0 mM and 10 mM DTT were added thereto, 1 mL of FJS nanoparticles and FJSO nanoparticles (0.5 mg / mL) were added to the dialysis membrane, and 30 mL of the release medium was placed in a conical flask. The flask was placed in a shaker at 37°C. At preset time points (0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours), 200 μL of the release medium was taken out, and the same volume of the medium was added to the conical flask. The cumulative release of Sor was measured using a high performance liquid chromatograph. The results are shown in Figure 2. Fig.10 As shown, there was basically no obvious Sor release from FJS nanoparticles and FJSO nanoparticles in the blank release medium (0 mM DTT). In the presence of 10 mM DTT, about 70% of Sor was released from FJS nanoparticles and FJSO nanoparticles within 24 hours.

[0088] Example 7: Cellular Uptake of Nanoparticles

[0089] The DSPE-PEG in Example 2 2K Replaced with Cy7-labeled Cy7-DSPE-PEG 2K Fluorescently labeled Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles were prepared, and the uptake of Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles in 4T1 cells was measured using confocal microscopy. 4The cells were inoculated into 24-well plates at a density of 10 cells / well and incubated in an incubator for 24 hours to allow them to adhere to the wall. After the cells adhered to the wall, Cy7 solution, Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles were added. The concentration of Cy7 was 24 μg / mL. After incubation at 37°C for 0.5 hours and 2 hours, the cells were washed and fixed. Finally, the cell uptake of various preparations was analyzed by confocal microscopy and the fluorescence was quantified by flow cytometry. The experimental results are shown in Figure 2. Figure 11-12 shown.

[0090] The above experimental results show that the cell uptake shows concentration-dependent uptake, and the cells treated with Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles have higher intracellular fluorescence intensity than the cells treated with Cy7 solution. Therefore, the prepared Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles have higher cell uptake efficiency than Cy7 solution.

[0091] Example 8: Cytotoxicity of Nanoparticles

[0092] The MTT method was used to investigate the cytotoxicity of JQ1 solution, sorafenib solution (Sor solution), Sor and JQ1 mixed solution (SJ solution), PEG-modified FJS nanoparticles prepared in Example 1, and FJSO nanoparticles on mouse breast cancer (4T1) cells. The cells in good condition were digested and diluted to 1×10 4 The cell density was 1 cell / ml, and 200 μL of cell suspension was added to each well of the 96-well plate after blowing evenly, and the cells were incubated in an incubator for 12 hours to adhere to the wall. After the cells adhered to the wall, they were cultured in a hypoxic incubator for 12 hours, and then the cells were cultured with a medium containing JQ1 solution, Sor solution, SJ solution, FJS nanoparticles and FJSO nanoparticles, 200 μL per well. The control group was cultured with a medium without drug solution. After 48 hours, the 96-well plate was taken out, 20 μL of 5 mg / mL MTT solution was added to each well, and the plate was shaken after incubation in an incubator for 4 hours. After the 96-well plate was turned upside down on the filter paper to fully absorb the residual liquid, 200 μL DMSO was added to each well and oscillated on an oscillator for 10 minutes to dissolve the blue-purple crystals. The absorbance value of each well after zeroing was measured at 490 nm using an enzyme reader. The changes in cytotoxicity after the addition of Fer-1 (0, 5 and 10 μM) were also investigated. The cytotoxicity of JQ1 solution, Sor solution, SJ solution, FJS nanoparticles and FJSO nanoparticles in normal 3T3 cells was examined in the same manner.

[0093] Cytotoxicity results such as Figure 13-14As shown in the figure, JQ1 solution, Sor solution, and SJ solution have weak cytotoxicity under normoxic conditions, while the antitumor effect is significantly inhibited under hypoxic conditions. In addition, compared with the solution, the inhibitory effect of hybrid nanoparticles on tumor cells under both normoxic and hypoxic conditions is significantly enhanced. More importantly, the cytotoxicity of FJS nanoparticles and FJSO nanoparticles under normoxic conditions is negligible, while under hypoxic conditions, the cytotoxicity of FJSO nanoparticles is better than that of FJS nanoparticles, indicating that FJSO NAs have the ability to overcome the deficiency of poor antitumor effect caused by hypoxia. In addition, compared with JQ1 solution, Sor solution, and SJ solution, the cytotoxicity of FJS nanoparticles and FJSO nanoparticles in 3T3 cells is significantly weakened, which may be attributed to the disulfide bonds in the original drug structure of the nanoparticles, which can achieve specific drug release at the tumor site.

[0094] Example 9: Glutathione detection

[0095] The cells in good condition were digested, diluted with culture medium and plated at 2×10 6 The cell density of 1 cell / dish was inoculated into the culture dish, and the culture dish was incubated in an incubator for 12 hours to make it adhere to the wall. After the cells adhered to the wall, they were cultured in a hypoxic incubator for 12 hours, and then the cells were cultured with a culture medium containing PBS (pH 7.4), Sor solution, PEG-modified FJS nanoparticles prepared in Example 1, and FJSO nanoparticles, 200 μL per well, and the concentration of equal amounts of Sor was 5 μM. After 4 hours of cultivation, the cells were washed 3 times with ice PBS, and the cells were collected, and the 4T1 cells were suspended in 300 μL PBS, and further destroyed by ultrasound. 100 μL of broken cells were mixed with 100 μL GSH probe precipitant, centrifuged at 3500 rpm / min for 10 minutes, and the supernatant was detected according to the instructions of the GSH detection kit. The ultraviolet absorbance at 405 nm was measured with a multifunctional microplate reader.

[0096] like Fig.15 As shown in the figure, under normoxic conditions, compared with the control group (PBS (pH 7.4)), the Sor solution can reduce the level of GSH, and this inhibitory effect is further amplified by the action of nanoparticles; under hypoxic conditions, the cells' ability to consume GSH is significantly reduced after administration of the control group, Sor solution and FJS nanoparticles, indicating that ferroptosis is inhibited under hypoxic conditions. It is worth noting that the ability of FJSO nanoparticles to consume GSH under hypoxic conditions is basically the same as that under normoxic conditions, indicating that it can effectively alleviate the ferroptosis-inducing niche caused by tumor hypoxia by carrying oxygen.

[0097] Example 10: Lipid peroxide and reactive oxygen species detection

[0098] C11 BODIPY581 / 591 fluorescent probe and DCFH-DA fluorescent probe were used to detect the level of intracellular lipid peroxides. The cells in good condition were digested, diluted with culture medium and then concentrated at 5×10 4 The cells were inoculated into a 24-well plate at a cell density of 1 cell / well and incubated in an incubator for 12 hours to adhere to the wall. After the cells adhered to the wall, they were cultured in a hypoxic incubator for 12 hours, and then the cells were cultured with a medium containing PBS (pH 7.4), Sor solution, PEG-modified FJS nanoparticles prepared in Example 1, and FJSO nanoparticles, 200 μL per well, and the concentration of Sor was 5 μM. After incubation for 4 hours, the cells were incubated with C11 BODIPY581 / 591 fluorescent probe and DCFH-DA fluorescent probe for 1 hour, the cells were washed, and the cells were fixed. Finally, intracellular lipid peroxides and reactive oxygen species were analyzed by confocal microscopy, and the reactive oxygen fluorescence generated was quantified by flow cytometry.

[0099] like Figure 16-18 As shown in the results, under normoxic conditions, Sor solution can enhance the expression of reactive oxygen species and lipid peroxides to some extent, while FJS nanoparticles and FJSO nanoparticles show stronger induction levels. However, hypoxic environment significantly inhibited the expression of reactive oxygen species and lipid peroxides after treatment with Sor solution and FJS nanoparticles. FJSO nanoparticles break the ferroptosis inhibitory niche under hypoxia by alleviating tumor hypoxia and show potent expression of reactive oxygen species and lipid peroxides under hypoxia.

[0100] Example 11: CRT and HMGB1 detection

[0101] 4T1 cells were cultured at 5×10 4The density of cells / well was inoculated into a 24-well plate with a pre-placed crawling sheet, 1 mL per well, and the 24-well plate was placed in a 37°C constant temperature incubator for adherent culture for 12 hours. Next, the cells were further cultured in the above-mentioned normoxic and hypoxic (oxygen concentration constant 5%) incubators for 12 hours to obtain a tumor cell model under normoxic conditions and a tumor cell model under hypoxic conditions. The old culture medium was discarded and replaced with 5 mL of empty culture medium (control group (PBS (pH 7.4))) and fresh culture medium containing drugs (Sor solution, PEG-modified FJS nanoparticles and FJSO nanoparticles prepared in Example 1, Sor equivalent concentration of 5 μM). Incubate in a 37°C constant temperature incubator for 5 hours, discard the drug-containing culture medium, terminate the uptake with cold PBS (pH 7.4), and wash three times (replace the pipette tip each time). Then, fix with 4% paraformaldehyde for 15 minutes (500 μL per well) and wash three times with cold PBS (pH 7.4). The primary antibody (CRT and HMGB1, respectively) was diluted 200 times with 5% milk powder solution, 200 μL was added to each well and incubated for 12 hours, washed three times with cold PBS (pH 7.4), then the secondary antibody was added and incubated for 4 hours, washed three times with cold PBS (pH 7.4), then stained with Hoechst33342 staining solution for 10 minutes, and washed three times with cold PBS (pH 7.4). The slides were fixed on a slide with an anti-fluorescence attenuation mounting medium, and the images were observed and photographed under a laser confocal microscope.

[0102] like Figure 19-20 , compared with Sor solution, 4T1 cells treated with FJS nanoparticles and FJSO nanoparticles showed stronger HMGB1 release and CRT exposure, while 4T1 cells without any treatment obviously did not secrete the above DAMPs. In addition, hypoxia reduced the levels of HMGB1 and CRT in 4T1 cells treated with Sor solution and FJS nanoparticles, but had no effect on FJSO nanoparticles, because it can achieve hypoxia relief by carrying oxygen and further promote effective oxidative stress, thereby ensuring sufficient secretion of DAMPs and inducing the occurrence of ICD.

[0103] Example 12: Detection of PD-L1 expression level

[0104] Dilute the digested and centrifuged cell suspension to a concentration of 4 × 10 4Cells / mL of cell suspension, and the cell suspension was transferred to a 6-well plate, 5 mL per well, and the 6-well plate was placed in the above-mentioned normoxic cell culture incubator for adherent culture for 12 hours. Next, the cells were further cultured in the above-mentioned normoxic or hypoxic incubator for 12 hours to obtain a tumor cell model under normoxic conditions and a tumor cell model under hypoxic conditions. Then, the old cell culture medium in the 6-well plate was discarded, and the culture medium containing PBS (pH 7.4), Sor solution, FJS nanoparticles and FJSO nanoparticles was added to culture the cells, 5 mL per well, 3 parallel wells for each concentration, and the concentration of Sor was 5 μM. 12 hours after administration, pour out the original drug-containing culture medium in the well, wash three times with cold PBS (pH 7.4), aspirate the cold PBS (pH 7.4), scrape off the cells of each group, add 500 μL cold PBS (pH 7.4), blow evenly, and transfer to a 1.5 mL centrifuge tube. The remaining cells are moistened with 500 μL cold PBS (pH 7.4), and all are transferred to a centrifuge tube. Centrifuge at 4°C for 10 min (1000 rpm), then pour out the supernatant, add 200 μL of the prepared lysis solution (PMSF concentration is 1 mM), blow 3 times with a 200 μL pipette, and lyse for 15 min (operate on ice). After the lysis is completed, centrifuge at 4°C for 15 min (15000 rpm) to obtain the sample for use. Then operate according to the instructions of the kit.

[0105] like Fig.21 As shown in the figure, when cells were co-incubated with JQ1-containing formulations (FJS nanoparticles and FJSO nanoparticles), PD-L1 expression was significantly downregulated. Although hypoxia can lead to a moderate increase in PD-L1 expression, the oxygenation strategy of FJSO nanoparticles can effectively alleviate hypoxia and exert excellent PD-L1 inhibition even under hypoxic conditions, thereby effectively reducing the occurrence of tumor immune escape.

[0106] Example 13: Pharmacokinetic study of nanoparticles

[0107] SD rats weighing between 200-250g were randomly divided into groups, fasted for 12h before administration, and allowed free access to water. Cy7 solution and Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles prepared in Example 1 (2mg / kg of Cy7 each) were injected intravenously, blood was collected from the eye sockets at the specified time points, and plasma was separated. Cy7 was then extracted by ultrasonic fragmentation, centrifugation, and protein precipitation, and the pharmacokinetic behavior of each group of preparations was finally detected using an ELISA instrument (excitation wavelength and emission wavelength were 748nm and 776nm, respectively). The experimental results are shown in the figure below. Fig. 22 As shown in the figure, due to its short half-life, Cy7 solution is quickly metabolized and cleared. Compared with Cy7 solution, the circulation time of Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles is significantly prolonged, which significantly improves the AUC of Cy7, laying a good foundation for the accumulation of drugs in tumors in vivo.

[0108] Example 14: Tissue distribution experiment of nanoparticles

[0109] BALB / c mice were inoculated with 4T1 cell suspension and the tumor volume reached 300 mm. 3 At 2, 4, 6, 12, 24, and 36 hours after administration, mice were anesthetized and subjected to in vivo imaging analysis. Fig.21 As shown in the figure, the fluorescence intensity of Cy7 solution in the tumor is weak, and the fluorescence signal is the strongest at 2 hours. In contrast, the fluorescence signal of Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles in the tumor within 36 hours is significantly higher than that of Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles, and the fluorescence accumulation signal is the largest at 4 hours.

[0110] In addition, ex vivo tissue fluorescence imaging of mice treated with Cy7 solution, Cy7-FJS nanoparticles, and Cy7-FJSO nanoparticles was performed at the peak time points of fluorescence signals in tumors and major organs (heart, liver, spleen, lung, and kidney). Figure 23-24 As shown, the fluorescence signals of Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles in tumors are stronger than those of Cy7 solution. In addition, there is no significant difference in the fluorescence intensity between Cy7-FJS nanoparticles and Cy7-FJSO nanoparticles. These results indicate that PEGylated nanoparticles greatly improve pharmacokinetics and biodistribution, thus laying the foundation for improving the anti-tumor efficiency in vivo.

[0111] Example 15: In vivo anti-tumor experiment of nanoparticles

[0112] 4T1 cell suspension (10 7 cells / 100 μL) were inoculated subcutaneously on the ventral side of female mice. 3At the same time, the mice were randomly divided into groups of 5, each group was given PBS (pH 7.4), JQ1 solution, Sor solution, SJ solution, FJS nanoparticles prepared in Example 1, and FJSO nanoparticles. The drug was administered once every other day for 5 consecutive times, and the Sor dose was 15 mg / kg. The survival status of the mice was observed every day, the body weight was weighed, and the tumor volume was measured. After the last administration, the mice were killed after an interval of 1 day, and the organs and tumors were obtained for further analysis and evaluation. The main organs (heart, liver, spleen, lung, kidney) were collected and fixed with 4% tissue fixative for H&E staining. The tumor tissue was sectioned and the expression of HIF-1α, PD-L1 and CRT was analyzed. The tumor tissue was ground into a homogenate, and the GSH content in the tumor tissue was detected according to the GSH kit.

[0113] like Figure 25-27 As shown, the inhibitory effects of Sor solution, JQ1 solution and SJ solution on tumor growth were limited, which may be due to their rapid metabolism in the body and low accumulation in tumors. As expected, FJSO nanoparticles showed significant therapeutic advantages compared with other preparations, effectively and completely inhibiting tumor progression in the 4T1 breast cancer model. In contrast, the anti-tumor effect of FJS nanoparticles was not strong, indicating that tumor hypoxia must be alleviated to enhance the effect of tumor elimination. In addition, a preliminary evaluation of the safety of the treatment was also conducted.

[0114] like Figure 28-30 As shown, multiple treatments did not lead to significant abnormalities in liver and kidney function parameters, organ sections observed by H&E staining, or body weight. These results indicate that FJSO nanoparticles have potent antitumor activity and show good safety throughout the treatment process.

[0115] like Figure 31-34 As shown in the figure, the expression of HIF-1α, GSH and PD-L1 in mouse tumors treated with FJSO nanoparticles was significantly inhibited, while the expression of CRT was significantly enhanced. In summary, these results show that FJSO nanoparticles can be used as an efficient and safe nanodrug delivery system to effectively alleviate tumor hypoxia, significantly enhance tumor ferroptosis and immune response under oxidative stress, effectively inhibit immune escape, and achieve a strong tumor inhibition effect under the combined application of ferroptosis and immunotherapy.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy, characterized in that: The nanoassembly is co-assembled by a ferroptosis agonist and a fluorinated prodrug through intermolecular forces and modified with a PEG modifier; the parent drug of the fluorinated prodrug is a small molecule immune checkpoint inhibitor, the side chain of the fluorinated prodrug is a perfluorocarbon, and the parent drug and the side chain are bridged by a linker; the linker is a chemical bond corresponding to the tumor microenvironment; the molar ratio of the ferroptosis agonist to the fluorinated prodrug is 5:1 to 1:5, and the mass ratio of the sum of the mass of the ferroptosis agonist and the fluorinated prodrug to the PEG modifier is 100:0 to 80:

20.

2. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy according to claim 1, characterized in that: The intermolecular forces include π-π stacking, hydrophobic interaction, electrostatic force and hydrogen bonding.

3. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy as claimed in claim 1, characterized in that: The ferroptosis agonist includes one or more of erastin, sorafenib, RSL-3 and sulfasalazine; the small molecule immune checkpoint inhibitor includes JQ1, PD1-PDL1 inhibitor 2 (BMS-202), BMS-1001, MAX-10181 or GS-4224; the linker is a monosulfide bond, a disulfide bond, or a diselenide bond; the side chain is perfluorohexanoic acid, perfluorooctanoic acid, perfluorodecanoic acid, perfluorododecanoic acid, hexanoic acid, octanoic acid, decanoic acid or dodecanoic acid.

4. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy as claimed in claim 1, characterized in that: The PEG modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG and PE-PEG, and the molecular weight of PEG is 200-20000.

5. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy as claimed in claim 4, characterized in that: The PEG modifier is DSPE-PEG 2K .

6. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy according to claim 1, characterized in that: The synthesis method of the prodrug comprises: The linker is subjected to an esterification reaction with perfluorohexanoic acid, perfluorooctanoic acid, perfluorodecanoic acid, perfluorododecanoic acid, hexanoic acid, octanoic acid, decanoic acid or dodecanoic acid using an esterification reaction catalyst, and the reaction product is purified and subjected to an esterification reaction with a small molecule immune checkpoint inhibitor to obtain a prodrug; the esterification reaction catalyst is EDCI and DMAP.

7. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: The ferroptosis agonist, the fluorinated prodrug and the PEG modifier are dissolved in an organic solvent respectively, mixed under vortex, and the mixed solution is slowly dropped into water, and uniform co-assembled nanoparticles are spontaneously formed under stirring; finally, the organic solvent is removed to obtain a nanoassembly; Alternatively, the ferroptosis agonist, the fluorinated prodrug co-assembled nanoparticles and the organic solvent of the PEG modifier are prepared separately, and then the organic solvent of the PEG modifier is added dropwise to the co-assembled nanoparticles under vigorous stirring, and finally the organic solvent is removed to obtain the nanoassembly; Or oxygen was charged into the nanoassemblies prepared by the above two methods (30 min) to obtain oxygen-saturated nanoassemblies; The method for removing the organic solvent includes solvent evaporation, ultrafiltration and membrane permeation.

8. A nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy as claimed in claim 7, characterized in that: The organic solvent includes one of ethanol, tetrahydrofuran and dimethyl sulfoxide or a combination of any two thereof.

9. Use of the nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy according to any one of claims 1 to 8 in the preparation of a drug delivery system; or in the preparation of an injection, oral administration or local administration system.

10. Use of the nanoassembly for tumor hypoxia relief and ferroptosis / immunotherapy according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

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