Oxygen-enhanced fluorinated nano assembly for bidirectional amplification of tumor ferroptosis as well as preparation method and application of oxygen-enhanced fluorinated nano assembly

Abstract

The invention discloses an oxygen-enhanced fluorinated nano assembly for bidirectional amplification of tumor ferroptosis as well as a preparation method and application of the oxygen-enhanced fluorinated nano assembly, and belongs to the technical field of pharmaceutical preparation combined treatment of new auxiliary materials and new dosage forms. According to the invention, the oxygen-functionalized fluorinated adriamycin prodrug with oxygen carrying capacity is designed and synthesized for the first time, a hybrid nano-assembly composed of the disulfide-bridged fluorinated adriamycin prodrug and the ferroptosis inducer is constructed and can be used for tumor treatment, and a fluorinated functional side chain can efficiently carry oxygen to break tumor hypoxia trapping and relieve limitation of hypoxia on Sor, so that the drug can be applied to tumor treatment. DOX can activate NOXs and promote generation of ROS through supplemented oxygen as a catalytic substrate, redox balance is broken through in a two-way synergistic mode, excessive accumulation of lipid peroxide is caused, and therefore the ferroptosis treatment effect is enhanced, a new strategy and more choices are provided for drug delivery development, and the application prospect is wide. The urgent demand of the hypoxia solid tumor on efficient and diverse ferroptosis treatment strategies on preparations in clinic is met.

Description

Technical Field

[0001] The present invention relates to the technical field of new adjuvants and new dosage forms for combined treatment of pharmaceutical preparations, and more specifically, to an oxygen-enhanced fluorinated nanoassembly for bidirectionally amplifying tumor ferroptosis, and a preparation method and application thereof. Background Art

[0002] At present, cancer still poses a serious threat to human health. Although a variety of cancer treatment strategies have been applied clinically, their therapeutic effects are still unsatisfactory. Therefore, there is an urgent need to develop more effective treatment models. Ferroptosis, as an iron-dependent regulated cell death method, has attracted widespread attention in recent years. Studies have shown that the essence of ferroptosis lies in the regulation of the balance between oxidative damage and oxidative defense. Sorafenib (Sor), as a potent ferroptosis inducer, blocks the upstream regulatory factors of the cystine / glutamate antiporter system (system Xc-), thereby reducing the synthesis of glutathione in tumor cells, breaking oxidative defense and inducing tumor ferroptosis. However, a large number of studies have shown that tumor hypoxia seriously restricts the anti-tumor effect of sorafenib. In addition, it was found that hypoxia significantly inhibited the cytotoxicity of Sor to 4T1 breast cancer cells. Therefore, it is difficult to induce satisfactory ferroptosis by relying solely on Sor to consume intracellular GSH levels and effectively destroy the tumor redox balance.

[0003] In addition to strategies to inhibit antioxidant systems, various methods that simultaneously increase intracellular ROS levels have been used to synergistically promote the induction of ferroptosis. It has been reported that doxorubicin (DOX) can activate intracellular NADPH oxidases (NOXs) to catalyze the production of superoxide anions using oxygen as a substrate, thereby increasing intracellular ROS levels. In addition, it has been found that hypoxia significantly reduces the cytotoxicity of DOX to 4T1 tumor cells and the ability to induce ROS. Increasing evidence shows that the tumor hypoxic microenvironment has become a huge barrier to intracellular oxidative stress and the induction of ferroptosis, indicating that alleviating hypoxia is crucial.

[0004] Various advanced materials and drug delivery systems have been proposed to address the problem of tumor hypoxia. Among them, perfluorocarbons (PFCs) have excellent oxygen solubility due to their inherent weak intermolecular van der Waals interactions and minimal cohesion, making them promising candidates for alleviating tumor hypoxia. Despite the obvious advantages of the strategy of combining hypoxia alleviation and bidirectional synchronous disruption of redox balance to induce ferroptosis, it remains challenging to achieve simultaneous and efficient co-delivery of multiple drugs in vivo. Summary of the invention

[0005] In order to solve the problems of tumor hypoxic microenvironment and difficult-to-break redox balance, as well as poor water solubility, poor assembly ability, low drug loading due to encapsulation in polymers, drug leakage and excipient-related toxicity of fluorinated doxorubicin prodrug (FD) and sorafenib, the present invention provides an oxygen-enhanced fluorinated nanoassembly for bidirectionally amplifying tumor ferroptosis and its preparation method and application. The present invention designs and synthesizes an oxygen-functionalized fluorinated doxorubicin prodrug with oxygen-carrying capacity for the first time, and constructs a hybrid nanoassembly (FDS nanoparticle) composed of a disulfide-bridged fluorinated doxorubicin prodrug and a ferroptosis inducer through small molecule carrier-free hybrid nanoassembly technology, which can be used for tumor treatment. The fluorinated functional side chain can efficiently carry oxygen to break the tumor hypoxia dilemma and relieve the restriction of hypoxia on Sor, while DOX can activate NOXs to promote the generation of ROS by using supplemented oxygen as a catalytic substrate, bidirectionally synergistically breaking the redox balance, leading to excessive accumulation of lipid peroxides, thereby enhancing the ferroptosis efficacy.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A disulfide-bridged fluorinated doxorubicin prodrug having oxygen-carrying capacity, wherein the structural formula of the fluorinated doxorubicin prodrug is as follows:

[0008]

[0009] The present invention also discloses a method for synthesizing the above-mentioned disulfide-bridged fluorinated doxorubicin prodrug with oxygen-carrying capacity, which is characterized by comprising the following steps:

[0010] (1) dehydrating 3,3'-dithiodipropionic acid with acetic anhydride to obtain a dibasic acid anhydride;

[0011] (2) reacting 1H, 1H, 2H, 2H-perfluoro-1-octanol with the dibasic acid anhydride under the action of 4-dimethylaminopyridine to obtain an intermediate product;

[0012] (3) dissolving the intermediate product in DMF, adding HBTU and DIPEA dropwise thereto, activating the intermediate product in an ice bath, adding DOX to react, and purifying the reaction product by liquid phase separation to obtain the fluorinated doxorubicin prodrug;

[0013]

[0014] Optionally, in step (1), 3,3'-dithiodipropionic acid is dissolved in acetic anhydride, and stirred at room temperature for 1.5h to 2.5h under nitrogen protection to react the dibasic acid into a dibasic acid anhydride. After the reaction is completed, toluene is added, and toluene and acetic anhydride are removed by vacuum evaporation at 30°C to 35°C; the molar ratio of the 3,3'-dithiodipropionic acid to the acetic anhydride is 1:(2 to 3); in step (2), 1H, 1H, 2H, 2H-perfluoro-1-octanol and 4-dimethylaminopyridine are taken and mixed with the dibasic acid anhydride. and dissolved in anhydrous dichloromethane, reacted for 10 h to 14 h at room temperature, and separated by chromatography to obtain an intermediate product; the molar ratio of the 4-dimethylaminopyridine, the 1H, 1H, 2H, 2H-perfluoro-1-octanol and the dibasic acid anhydride is 1:(0.2-0.4):(1-2); in step (3), the molar ratio of the DMF, the HBTU, the DIPEA, the DOX and the intermediate product is 1:(1-1.5):(1-1.5):(0.5-0.8).

[0015] The present invention also discloses an oxygen-enhanced fluorinated nanoassembly for bidirectionally amplifying tumor ferroptosis. The oxygen-enhanced fluorinated nanoassembly is a nanoparticle co-assembled with a fluorinated doxorubicin prodrug and a ferroptosis inducer, or a nanoparticle co-assembled with a fluorinated doxorubicin prodrug modified with a polyethylene glycol modifier and a ferroptosis inducer; the fluorinated doxorubicin prodrug is the fluorinated doxorubicin prodrug as described above, or the fluorinated doxorubicin prodrug obtained by the synthesis method as described above.

[0016] Optionally, the particle size of the oxygen-enhanced fluorinated nanoassembly is 90 nm to 100 nm; the fluorinated doxorubicin prodrug and the ferroptosis inducer are co-assembled through one or both of hydrophobic interaction and intermolecular hydrogen bonding.

[0017] Optionally, the ferroptosis inducer is one or more of sulfasalazine, laminophen, trigonelline and sorafenib; the polyvinyl alcohol modifier includes one or more of PLGA-PEG, DSPE-PEG, DSPE-SS-PEG, PCL-PEG, PE-PEG; the molecular weight of the polyvinyl alcohol modifier is 200 to 20,000; the molar ratio of the fluorinated doxorubicin prodrug to the ferroptosis inducer is 10:1 to 1:10; the mass ratio of the fluorinated doxorubicin prodrug to the polyvinyl alcohol modifier is 10:90 to 90:10.

[0018] The present invention also discloses a method for preparing the oxygen-enhanced fluorinated nanoassembly as described above, comprising the following steps:

[0019] The fluorinated doxorubicin prodrug and the ferroptosis inducer, or the fluorinated doxorubicin prodrug, the ferroptosis inducer and the polyethylene glycol modifier are dissolved in an organic solvent, and the mixed solution is slowly added dropwise to deionized water, and uniform co-assembled nanoparticles are spontaneously formed under stirring, and the organic solvent is removed to obtain the oxygen-enhanced fluorinated nanoassembly.

[0020] The present invention also discloses an application of the fluorinated doxorubicin prodrug or the oxygen-enhanced fluorinated nano-assembly in a drug delivery system.

[0021] The present invention also discloses a use of the fluorinated doxorubicin prodrug or the oxygen-enhanced fluorinated nano-assembly in the preparation of anti-tumor drugs.

[0022] The present invention also discloses an application of the fluorinated doxorubicin prodrug or the oxygen-enhanced fluorinated nano-assembly in an injection, oral administration or local administration system.

[0023] Optionally, the application specifically includes: charging the oxygen-enhanced fluorinated nanoassembly with pure oxygen at a constant flow rate of 5L / min to 8L / min for 20min to 30min to obtain oxygen-saturated FDSO nanoparticles, and adding the oxygen-saturated FDSO nanoparticles into deoxygenated water. Subsequently, external ultrasound triggers oxygen release, and a dissolved oxygen meter (WLDO-300, Shanghai Water Technology Co., Ltd.) is used to record changes in oxygen concentration.

[0024] Implementing the embodiments of the present invention will have the following beneficial effects:

[0025] 1. The present invention is the first to design and synthesize an oxygen-functionalized fluorinated doxorubicin prodrug with oxygen-carrying capacity, which can effectively carry oxygen to relieve tumor hypoxia and increase oxidative damage to tumor cells. Further, by co-assembling the fluorinated doxorubicin prodrug and the ferroptosis inducer into nanoparticles, technical effects such as high drug loading, good stability, and low toxic side effects are achieved, meeting the urgent clinical demand for high-efficiency and low-toxicity preparations, providing a new strategy for the assembly of homologous synergistic drug nanoparticles, and providing an effective nano-platform for the development of carrier-free prodrug hybrid nanoassemblies and combined therapy for alleviating hypoxia and enhancing ferroptosis.

[0026] 2. The present invention uses small molecule carrier-free hybrid nanoassembly technology to construct a hybrid nanoassembly (FDS nanoparticles) composed of disulfide-bridged fluorinated doxorubicin prodrugs and ferroptosis inducers. After carrying oxygen, FDS nanoparticles (FDSO nanoparticles) accumulate in the tumor. Due to the low oxygen partial pressure in the hypoxic tumor microenvironment, oxygen is quickly released to alleviate tumor hypoxia. At the same time, the disulfide bonds in the FDSO nanoparticles specifically respond to the tumor reduction microenvironment, quickly decompose and release DOX-SH and Sor. The oxygen carried not only effectively alleviates the limitation of hypoxia on Sor, but also promotes the process of DOX activating NOXs to catalyze the generation of ROS as a substrate. In addition, the NADPH consumed in the catalytic process also inhibits the conversion process of cysteine ​​to cystine, further helping sorafenib overcome the hypoxia dilemma.

[0027] In summary, the present invention proposes a novel fluorinated prodrug hybrid nanoassembly strategy to promote bidirectional synergistically driven ferroptosis by alleviating hypoxia in cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The structural formula of the fluorinated doxorubicin prodrug (FD) designed in Example 1 of the present invention is

[0029] Figure 2 This is the synthetic route of FD in Example 1 of the present invention.

[0030] Figure 3 This is the mass spectrum of FD in Example 1 of the present invention.

[0031] Figure 4 The FD of Example 1 of the present invention 1 H NMR spectrum.

[0032] Figure 5 is the LogP of compound FD of Example 1 of the present invention

[0033] Figure 6 The Malvern particle size distribution diagram of the FDS nanoparticles and FDSO nanoparticles of Example 2 of the present invention and the transmission electron microscope image of the nanoparticles.

[0034] Figure 7 This is a quantitative analysis of the oxygen loading capacity of the FDSO nanoparticles of Example 3 of the present invention.

[0035] Figure 8 This is a stability diagram of the FDS naked nanoparticles, FDS nanoparticles and FDSO nanoparticles of Example 4 of the present invention incubated in PBS (pH 7.4).

[0036] Fig. 9The stability graph of the FDS naked nanoparticles, FDS nanoparticles and FDSO nanoparticles of Example 4 of the present invention incubated in PBS (pH 7.4) containing 10% FBS.

[0037] Fig.10 This is the molecular docking diagram of FD and Sor in Example 5 of the present invention.

[0038] Fig.11 The non-PEGylated nanoparticles of Example 5 of the present invention are destroyed by the action of forces in 10 mM sodium chloride, sodium dodecyl sulfate and urea.

[0039] Fig.12 The degradation degree of the prodrug under incubation with different concentrations of DTT in Example 6 of the present invention.

[0040] Fig.13 This is the liquid phase diagram of HPLC analysis of DTT-triggered FD degradation at different incubation times in Example 6 of the present invention (DTT=1 mM).

[0041] Fig.14 The particle size (10 mM) of FDSO nanoparticles in the presence of DTT in Example 6 of the present invention.

[0042] Fig.15 The transmission electron microscope of Example 6 of the present invention obtained the particle size distribution of FDS NAs and FDSO NAs under incubation with different concentrations of DTT

[0043] Fig.16 These are confocal microscope photographs of the cellular uptake of the Cy7 solution, Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles of Example 7 of the present invention at 0.5 hour and 2 hours.

[0044] Fig.17 Flow cytometry quantitative graph of cellular uptake of Cy7 solution, Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles of Example 7 of the present invention.

[0045] Fig.18 These are the cytotoxicity results of different preparations of FD solution, Sor solution, FDS mixed solution, FDS nanoparticles and FDSO nanoparticles on 4T1 cells under normoxic and hypoxic conditions in Example 8 of the present invention.

[0046] Fig.19 For the in vitro cellular hypoxia relief evaluation of Example 9 of the present invention, immunofluorescence images of HIF-1α protein in 4T1 cells containing PBS (pH 7.4), FD solution, Sor solution, FDS mixed solution, FDS nanoparticles and FDSO nanoparticles.

[0047] Fig. 20The changes in intracellular glutathione levels of PBS (pH 7.4), FD solution, Sor solution, FDS mixed solution, FDS nanoparticles and FDSO nanoparticles of Example 10 of the present invention.

[0048] Fig.21 It is the GPXs inhibition rate of PBS (pH 7.4), FD solution, Sor solution, FDS mixed solution, FDS nanoparticles and FDSO nanoparticles in Example 10 of the present invention.

[0049] Fig. 22 The intracellular NADP of PBS (pH 7.4), FD solution, Sor solution, FDS mixed solution, FDS nanoparticles and FDSO nanoparticles in Example 11 of the present invention is + Changes in NADPH ratio

[0050] Fig.23 The fluorescence images of ROS under normoxic and hypoxic conditions are shown in the inverted fluorescent 4T1 cells of Example 12 of the present invention after being treated with a specified formula.

[0051] Fig.24 The DCFH-DA of Example 12 of the present invention detects the intensity of ROS in 4T1 cells under normoxic and hypoxic conditions.

[0052] Fig.25 The intracellular content of LPOs in 4T1 cells under normoxic and hypoxic conditions after treatment with different formulations in Example 13 of the present invention is

[0053] Fig.26 This is a blood drug concentration-time curve of the Cy7 solution, Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles of Example 14 of the present invention.

[0054] Fig. 27 This is the in vivo imaging of mice administered Cy7 solution, Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles in Example 15 of the present invention.

[0055] Fig.28 These are fluorescence imaging images of isolated organs of mice administered Cy7 solution, Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles in Example 15 of the present invention.

[0056] Fig.29 This is a fluorescence quantitative graph of isolated organs of mice administered Cy7 solution, Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles in Example 15 of the present invention.

[0057] Fig.30 This is a photograph of the in vitro tumor in Example 16 of the present invention.

[0058] Fig.31This is a graph showing the mouse tumor growth curve in the in vivo anti-tumor experiment of Example 16 of the present invention.

[0059] Fig.32 This is a statistical graph of the tumor-bearing rate of mice in the in vivo anti-tumor experiment of Example 16 of the present invention.

[0060] Fig.33 H&E and HIF-1α of mouse tumors in vitro in the in vivo anti-tumor experiment of Example 16 of the present invention

[0061] Fig.34 This is a graph showing the changes in mouse body weight in the in vivo anti-tumor experiment of Example 16 of the present invention.

[0062] Fig.35 This is the mouse survival rate curve of the in vivo anti-tumor experiment of Example 16 of the present invention.

[0063] Fig.36 The liver and kidney function analysis results of Example 16 of the present invention.

[0064] Fig.37 This is a histopathological section diagram of Example 16 of the present invention. DETAILED DESCRIPTION

[0065] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0066] Example 1: Synthesis and characterization of FD compounds

[0067] This embodiment designs a fluorinated DOX prodrug (F 13 -SS-DOX, FD), which is expected to achieve tumor-specific release while alleviating tumor hypoxia.

[0068] The prodrug was synthesized according to the following route: 3,3′-dithiodipropionic acid (2mmol) and 6mL acetic anhydride were dissolved in a flask, and the rest was stirred at 25°C for 2h under nitrogen protection, and then 30ml toluene was added to the reaction solution and evaporated several times in vacuum. Subsequently, 25mL of anhydrous dichloromethane was immediately poured to dissolve the crude product, and then 0.2mmol of DMAP was dripped into the solution under stirring. Subsequently, 1H, 1H, 2H, 2H-perfluoro-1-octanol (2mmol) were added. The mixed solution was stirred at 25°C for 24h, and the product was separated by silica gel column chromatography (dichloromethane / methanol = 150:1, 0.1% glacial acetic acid) with a yield of 65%. Subsequently, the above-obtained product (1.5mmol) was dissolved in a flask containing DMF, and HBTU (3mmol) and DIPEA (3mmol) solutions were dripped into the system. The mixed solution was stirred for 2 h under nitrogen protection in an ice bath, and then 1 mmol of DOX was added and stirred at 25° C. for 48 h. The crude product was purified by preparative liquid chromatography using acetonitrile / water (75:25) as the mobile phase (yield 56%).

[0069] Using nuclear magnetic resonance spectroscopy ( 1 The structure of the prodrug was verified by H at 400MHz) and mass spectrometry. The purity of the prodrug was determined by high performance liquid chromatography.

[0070] FD NMR results are as follows Figure 3-4 As shown, 1H NMR (400MHz, DMSO-d6) δ7.979 (d, J=7.1Hz, Ar-H, 2H), 7.851 (d, J=7.2Hz, Ar-H, 2H),7.743(t,J=7.3Hz,Ar-H,1H),7.668-7.058(Ar-H,10H),6.292(s,10-CH,1 H),5.813(t,J=9.1Hz,13-CH,1H),5.525(t,J=8.7Hz,3'-CH,1H),4.892(dd,J= 12.1,7.3Hz,2'-CH,1H),4.584(s,5-CH,1H),4.112(d,J=7.0Hz,7-CH,1H),4.0 10(q,J=8.3Hz,20-CH2,2H),3.871-3.720(CH2SSCH2,4H),3.578(d,J=7.1Hz,3 -CH,1H),2.697-2.311(F8-CH2,4H),2.237(S,4-COCH3,3H),2.099(S,10-COCH 3,3H),1.782(s,18-CH3,3H),1.635(t,J=12.6Hz,6-CH2-αH,1H),1.496(s,19- CH3,3H),1.479(m,6-CH2-βH,1H),1.025(s,17-CH3,3H),0.998(s,16-CH3,3H).

[0071] In addition, if Figure 5 As shown, the FD (Log P=7.15) calculated by the software showed a relatively large hydrophobicity.

[0072] Example 2 Preparation of FDSO Nanoparticles

[0073] FD and Sor 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. FD and Sor spontaneously formed uniform nanoparticles, and then the organic solvent in the nanoparticles was removed by rotary evaporation at 30°C to obtain a nanoparticle without any organic solvent.

[0074] The particle size, particle size distribution and synergistic index of FD and Sor of the prepared nanoformulation were detected. The results are shown in Table 1.

[0075] Table 1. Particle size, particle size distribution and synergy index of FD and Sor of FDS nanoparticles

[0076]

[0077]

[0078] As shown in Table 1, FD and Sor were able to co-assemble into uniform nanoparticles at different molar ratios (FD / Sor) ranging from 1:5 to 5:1 without any excipients through a simple one-step nanoprecipitation method. The FD / Sor combination showed better synergistic effect at a molar ratio of 1:1, with a combination index (CI) of 0.50 and better co-assembly performance (80-90 nm, PDI < 0.2).

[0079] The molar ratio of FD to Sor was initially optimized to be 1:1, and nanoparticles were prepared with this molar ratio:

[0080] (1) Preparation of non-PEGylated FA nanoparticles (FDS naked nanoparticles): 0.70 mg FD and 0.30 mg (equimolar amount) sorafenib (Sor) were accurately weighed and dissolved in 200 μL tetrahydrofuran and anhydrous ethanol solution (v / v, 1:1). The solution was slowly added dropwise to 2 mL deionized water under stirring to spontaneously form uniform FDS naked nanoparticles. The organic solvent in the nanoformulation was then removed by rotary evaporation at 30°C to obtain a nanosolution free of any organic solvent.

[0081] (2) DSPE-PEG 2K Preparation method of modified FDS nanoparticles (FDS nanoparticles): accurately weigh 0.70 mg FD and 0.30 mg (equimolar amount) sorafenib (Sor), dissolve them in 200 μL tetrahydrofuran and anhydrous ethanol solution (v / v, 1:1); accurately weigh 0.25 mg DSPE-PEG 2K , dissolve in anhydrous ethanol to a 10 mg / mL stock solution. Mix 200 μL FDS solution with 25 μL DSPE-PEG 2K After mixing evenly, the solution was slowly added dropwise into 2 mL of deionized water under stirring to obtain uniform FDS nanoparticles. The organic solvent in the nanoparticle preparation was then removed by rotary evaporation at 30°C to obtain a nanoparticle solution free of any organic solvent.

[0082] (3) Preparation method of oxygen-carrying FDSO nanoparticles: Take the FDS nanoparticles prepared according to (2) of Example 2 above, fill them with pure oxygen (20 min) at a constant flow rate (5 L / min) to obtain oxygen-saturated FDSO nanoparticles.

[0083] The particle size and particle size distribution of the prepared FDS nanoparticles and the oxygenated FDSO nanoparticles were measured by dynamic light scattering. Figure 6 , indicating that the nanoparticles are uniform spherical and the particle size is about 90nm.

[0084] Example 3: Evaluation of the oxygen carrying capacity of nanoparticles

[0085] Pure oxygen was charged into water, FDS nanoparticles of Example 2, and FD at a constant flow rate (5L / min) for 20min to obtain oxygen-saturated pure water (H2OO), FDSO nanoparticles, and FDO nanoparticles. 5mL H2OO, FDSO nanoparticles, and FDO nanoparticles (equivalent to 1μM 1H, 1H, 2H, 2H-perfluoro-1-octanol) were added to 15mL of deoxygenated water. Subsequently, external ultrasound triggered oxygen release, and the oxygen concentration was recorded with a dissolved oxygen meter (WLDO-300, Shanghai Water Technology Co., Ltd.). The oxygen carrying capacity of 1mL nanoparticles (equivalent to 1μM / mL 1H, 1H, 2H, 2H-perfluoro-1-octanol) was calculated as follows.

[0086] Oxygen carrying capacity = increased oxygen concentration after adding oxygen-saturated nanoparticles × total volume of the final solution / volume of nanoparticles (n=3).

[0087] Oxygen carrying capacity Figure 7 As shown, FDSO nanoparticles and FDO nanoparticles exhibited very consistent absolute oxygen carrying capacity (70-80 mg / L), which indicates that the introduction of sorafenib will not affect the oxygen carrying capacity of fluorinated doxorubicin prodrug.

[0088] Example 4: Stability test of nanoparticles

[0089] 1 mL of the FDS naked nanoparticles, FDS nanoparticles and FDSO nanoparticles (0.5 mg / mL) prepared in Example 2 was taken out and added to 10 mL of PBS (pH 7.4) and PBS (pH 7.4) containing 10% FBS, incubated in a shaker at 37°C for 8 hours, and the changes in particle size were measured by dynamic light scattering at predetermined time points (0, 1, 2, 4 and 8 hours).

[0090] The results are as follows Figure 8 As shown, compared with non-PEG-modified FDS bare nanoparticles, FDS nanoparticles and FDSO nanoparticles had better colloidal stability in PBS (pH 7.4), and the particle size did not change significantly within 8 hours.

[0091] The results are as follows Fig. 9 As shown, FDS nanoparticles and FDSO nanoparticles have better colloidal stability in PBS (pH 7.4) containing 10% FBS, and PEG-modified FDS nanoparticles and FDSO nanoparticles are preferred. At the same time, the results show that the process of oxygenation and oxygen carrying of the preparation will not affect the stability of the preparation.

[0092] Example 5: Analysis of the assembly mechanism of FD and Sor

[0093] The molecular docking simulation was performed using Autodock Vina software, and the intermolecular force analysis was performed with the optimal ratio of FD to Sor being 1:1. In addition, the presence of intermolecular forces in the nanoparticles was further verified using sodium chloride (NaCl), sodium dodecyl sulfate (SDS), and urea. FDS bare nanoparticles were dispersed in NaCl (10 mM), SDS (10 mM), and urea (10 mM) and incubated at 37 °C in a shaker. The particle size of these nanoparticles was measured by Zetasizer (Nano ZS, Malvern Co., UK) at predetermined time points.

[0094] The results are as follows Figure 10-11 As shown, there are multiple forces between FD and Sor molecules, such as hydrophobic forces and hydrogen bonds, which make a great contribution to the co-assembly of FD and Sor, and further explain that the fluorinated side chain gives the compound a stronger hydrophobic force that drives the assembly of FDS naked nanoparticles.

[0095] Example 6: In vitro nanoparticle disintegration and drug release

[0096] To investigate the drug release of fluorinated prodrug hybrid nanoparticles under the action of DTT, FDSO nanoparticles (0.035 mg / mL) were incubated with 30 mL of release medium (ethanol / PBS, V / V=3 / 7, pH 7.4, 1% Tween 80) with different concentrations of DTT in a shake flask (37°C). The degradation of FD in FDSO nanoparticles was monitored by HPLC at specified time intervals. The FD content was determined by chromatographic separation using a C18 column (4.6×150 mm, 5 μm). The mobile phase was acetonitrile and pure water in a ratio of 80:20 (V / V) at a flow rate of 1 mL / min, and the UV detection wavelength of FD was 254 nm. To further investigate the correlation between the disintegration of the formulation and the degradation of the prodrug during the incubation with DTT, the changes in the nanoparticle size and particle size of FDS nanoparticles and FDSO nanoparticles in the presence or absence of DTT were analyzed. Specifically, FDS nanoparticles and FDSO nanoparticles (1 mg / mL) were incubated in PBS (pH 7.4) containing 0 mM or 10 mM DTT in a shaker (37°C). Transmission electron microscopy (TEM) and ZetaSizer were used to observe the morphology and particle size changes of FDS nanoparticles and FDSO nanoparticles.

[0097] The release and nanoparticle disintegration results are shown in Figure 12-15As shown, the concentration of FD gradually decreased over time in a DTT concentration-dependent manner. Notably, to further verify that the destruction of the hybrid nanostructure had a synchronous temporal relationship with the decomposition of the prodrug FD, the particle size changes of FDSO nanoparticles were further studied in the presence of PBS (pH 7.4) and DTT. Under the action of 10 mM DTT, the particle size of FDSO nanoparticles changed significantly. The DTT-triggered cleavage of FD led to the disintegration of FDSO nanoparticles, which may be due to the weakening or disappearance of the intermolecular interactions and forces between FD and Sor after the degradation of the disulfide bond to DOX-SH. These results highlight the favorable decomposition properties of FDSO nanoparticles driven by the hydrophobic forces of the fluorinated side chains under redox stimulation, which is certainly beneficial to the on-demand release of drugs from FDSO nanoparticles and enhance their selectivity when exposed to overproduced glutathione in tumor cells.

[0098] Example 7: Cellular Uptake of Nanoparticles

[0099] By attaching Cy7 as a fluorescent probe to DSPE-PEG 2K The efficiency of cell uptake of nanoparticles was detected. 4T1 cells were plated at 5×10 4 Cells / well were seeded in 24-well plates and incubated for 12 h. The culture medium was then removed and replaced with fresh culture medium containing Cy7 solution, Cy7-labeled FDS nanoparticles and FDSO nanoparticles (Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles, equivalent to Cy7 = 2 μg / ml), and incubated for 0.5 h or 2 h, respectively. Afterwards, cells were washed three times with cold PBS (pH 7.4) to terminate cell uptake. 4% paraformaldehyde was added and fixed at room temperature for 15 min, Hoechst 33342 was added to stain the cell nucleus for 10 min, and the fluorescence image of cell uptake was observed using a laser scanning microscope (LSCM). In addition, for quantitative detection, 12-well plates (1×10 5 The other operations described above were performed on 100 cells / well), and cellular uptake was determined by flow cytometry.

[0100] like Figure 16-17 The results showed that the fluorescence intensity of Cy7 in 4T1 tumor cells treated with nanoparticles was significantly higher than that of Cy7 solution at 0.5 and 2 h. And the cellular internalization of nanoparticles showed time dependence. It is worth noting that the cellular fluorescence intensity of FDSO nanoparticles is very similar to that of FDS nanoparticles, which may be attributed to similar nanoparticle characteristics, including nanostructure, particle size and zeta potential. These results indicate that the effect of carried oxygen on the cellular internalization of nanoparticles is almost negligible.

[0101] Example 8: In vitro cytotoxicity and safety of nanoparticles

[0102] The cytotoxicity of nanoparticles was determined by MTT assay. 4T1 cells were seeded in 96-well plates at a density of 2×10 3 cells / well, cultured for 12h (37℃, O2 21%, CO2 5%), and then cultured for 12h under normoxia (O2 21%) or hypoxia (O2 5%). Then, the blank culture medium was replaced by 200μL culture medium containing different concentrations of FD solution, Sor solution, FDS solution, FDS nanoparticles and FDSO nanoparticles. After incubation for 24h in normoxia or hypoxia, all groups were placed for 24h. Then 20μL MTT (5mg / mL) was added to each well and cultured at 37℃ for 4h. The culture medium was discarded and DMSO (200μL) was replaced to dissolve the generated formazan. The UV absorbance at 490nm was measured by enzyme labeling to calculate the survival rate.

[0103] Cytotoxicity results such as Fig.18 As shown, the cytotoxicity of FDS solution to 4T1 cells was significantly stronger than that of FD solution and Sor solution, indicating that there is a significant synergistic mechanism between FD and Sor. At the same time, the cytotoxicity of FDS nanoparticles was significantly stronger than that of FDS solution, which may be due to the enhanced cellular uptake ability of FDS nanoparticles. It was also found that the cytotoxicity of 4T1 cells cultured in all formulations was inhibited under hypoxic conditions. In contrast, the cytotoxicity of FDSO nanoparticles was much less limited by hypoxia than that of FDS nanoparticles, indicating that the fluorinated oxygen-carrying strategy can break the hypoxia dilemma. It was confirmed that the combined use of FD and Sor can effectively kill tumor cells by synergistically inducing ferroptosis.

[0104] Example 9: In vitro hypoxia relief

[0105] Immunofluorescence staining was used to detect the expression of hypoxia-inducible factor (HIF-1α) in 4T1 cells with different dosage forms to explore their in vitro deoxygenation effects. Briefly, under normoxic conditions, 4T1 cells (5×10 4 Cells / well) were inoculated in 24-well plates for 12 h, then incubated under normoxic or hypoxic conditions for another 12 h, and then incubated with PBS (pH 7.4), FD solution, Sor solution, FDS solution, FDS nanoparticles, and FDSO nanoparticles (equivalent to 10 μM FD) for 4 h. Tumor cells were fixed with 4% paraformaldehyde, incubated with anti-HIF-1α antibody (1:400) in a 4 ° C refrigerator for 12 h, incubated with secondary antibody staining at room temperature for 2 h, and then incubated with Hoechst33342 for nuclear staining. Fluorescence signals were observed using CLSM (C2, Nikon, Japan).

[0106] The results are as follows Fig.19As shown, under normoxic conditions, the expression of HIF-1α in 4T1 cells treated with all formulations was very low. In contrast, under hypoxic conditions, obvious immunofluorescence indicating HIF-1α overexpression was observed in 4T1 cells treated with PBS (pH 7.4). It is worth noting that FDSO nanoparticles were able to significantly reduce the expression of HIF-1α in 4T1 cells. These results indicate that FDS nanoparticles can effectively attenuate the expression of HIF-1α under hypoxic conditions by loading oxygen, indicating the important role of hypoxia relief in promoting ferroptosis.

[0107] Example 10: GSH and GPXs detection:

[0108] 4T1 cells (2×10 6 Cells / well) were inoculated in a cell culture dish and incubated for 12h, then 4T1 cells were incubated under normoxic or hypoxic conditions for 12h, and then 4T1 cells were treated with PBS (pH 7.4), FD solution, Sor solution, FDS solution, FDS nanoparticles and FDSO nanoparticles (equal to 10μM FD) for 12h. After treatment, the cells were washed three times with ice-cold PBS (pH 7.4) and collected. 4T1 cells were suspended in PBS (pH 7.4, 0.3mL, 0.1M) and sonicated. The supernatant was added to an equal volume of precipitant and extracted at a speed of 3500rpm / min for 10 minutes. The supernatant was removed and quantified according to the instructions of the GSH detection kit and the instructions of the GPXs detection kit.

[0109] like Figure 20-21 As shown, the ability of FDS nanoparticles to inhibit GSH production and GPXs enzymes was limited by hypoxia, but FDSO nanoparticles showed lower GSH and GPXs levels, which can be attributed to the fact that FDS nanoparticles effectively alleviated the hypoxic limitation of Sor by loading oxygen.

[0110] Example 11: In vitro intracellular NADP + / NADPH detection

[0111] Detection of intracellular NADP + / NADPH detection, 4T1 cells (2×10 6 cells / ) were inoculated into the cell culture and incubated for 12 h. Subsequently, 4T1 cells were further incubated under normoxic or hypoxic conditions for 12 h. Then, the cells were treated with PBS (pH 7.4), FD solution, Sor solution, FDS mixed solution, FDS nanoparticles and FDSO nanoparticles (equivalent to 10 μM FD) for 4 h. After treatment, the cells were then lysed with 500 μL of cold NADPH extraction buffer. Centrifugation was performed at 12,000 rpm for 10 min at 4 °C. Commercial NADP +The NADP+ / NADPH ratio was analyzed by the NADP+ / NADPH assay kit (n=3).

[0112] like Fig. 22 As shown, NADP + The change in the ratio of NADPH to cysteine ​​indicated that FD consumed NADPH in the process of catalyzing NOXs to generate ROS using oxygen as substrate in cells. In addition, the decrease in NADPH level after oxygen supply also inhibited the conversion of cysteine ​​to GSH synthesis in cells.

[0113] Example 12: Cellular Reactive Oxygen Species (ROS) Detection

[0114] DCFH-DA probe was used to detect ROS generation in 4T1 cells treated with different preparations. 4 Cells / well) were seeded on 24-well plates under normoxic conditions for 12 h, and then incubated under normoxic or hypoxic conditions for another 12 h. Then 4T1 cells were incubated with PBS (pH 7.4), FD solution, Sor solution, FDS solution, FDS nanoparticles and FDSO nanoparticles (equivalent to 10 μM FD) and 10 μM DCFH-DA for 30 min. Finally, the green fluorescence intensity of DCF was detected using an Eclipse Ti-U inverted microscope.

[0115] like Figure 23-24 As shown, we found that under hypoxic conditions, the ability of FDS nanoparticles to induce ROS was significantly inhibited, while FDSO nanoparticles as substrates significantly removed this barrier and generated higher ROS by supplementing oxygen during the NOX catalysis process.

[0116] Example 13: Lipid Peroxides (LPOs) Detection

[0117] 4T1 cells (2×10 6 Cells / well) were inoculated in a cell culture dish, incubated in an incubator at 37°C, O2 21%, CO2 5% for 12 h, then incubated under normoxic or hypoxic conditions for 12 h, then treated with blank culture medium, FD solution, Sor solution, FDS solution, FDS nanoparticles, and FDSO nanoparticles (equal to 10 μM FD) for 12 h, washed three times with cold PBS (pH 7.4), and the cells were collected. The experiment was performed according to the relevant operations of the LPOs content determination kit.

[0118] like Fig.25As shown, it is noteworthy that the LPOs accumulation level of the combined treatment group (FDS solution and FDS nanoparticles) was significantly higher than that of the other groups, indicating a synergistic effect of Sor and FD. Surprisingly, under hypoxic conditions, the LPOs level was the highest in the FDSO nanoparticle-treated group, while the LPOs levels of the other formulation-treated groups were suppressed. As expected, FDSO nanoparticles, with their ability to improve cellular uptake efficiency and relieve hypoxia, induced the strongest LPOs accumulation in cells, which is consistent with the observed cytotoxic results. These findings clearly confirm that the combined use of Sor and FD induces a bidirectional enhancement of tumor ferroptosis after relieving tumor hypoxia.

[0119] Example 14: Pharmacokinetic study of nanoparticles

[0120] Take SD rats weighing between 200-250g, randomly group them, fast for 12h before administration, and drink water freely. Cy7 solution and Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles (all in terms of Cy7 2mg / kg) prepared according to Example 7 were injected intravenously respectively, and about 500μL of blood samples were collected from the rat ophthalmic vein at predetermined time points of 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours and 12 hours after intravenous injection, and then centrifuged (8000 rpm, 3 minutes) to obtain plasma samples. Finally, Cy7 was extracted from the plasma sample by protein precipitation, and the concentration of Cy7 was detected by an ELISA instrument with an excitation wavelength of 748nm and an emission wavelength of 780nm.

[0121] The experimental results are as follows Fig.26 As shown in the figure, the fluorescence intensity emitted by the Cy7 solution decays rapidly, indicating that the Cy7 solution is quickly cleared from the bloodstream after intravenous injection. In contrast, the blood circulation time of the nanoparticles (Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles) was prolonged, and the t1 / 2 and AUC values ​​increased by 2.6 times and 5.3 times, respectively. It is worth noting that there was no significant difference in the pharmacokinetic characteristics of FDS nanoparticles and FDSO nanoparticles, indicating that oxygen loading had no significant effect on the in vivo delivery fate of FDS nanoparticles. These results indicate that good stability and PEGylated nanoparticles significantly prolong the drug circulation in vivo, which will certainly enhance the tumor-specific accumulation of nanomedicines. Due to its short half-life, the Cy7 solution is quickly metabolized and cleared. Compared with the solution, the circulation time of Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles was significantly prolonged, and the AUC was significantly increased, which provided a good foundation for the accumulation of drugs in tumors in vivo.

[0122] Example 15: Tissue distribution experiment of nanoparticles

[0123] The biodistribution of the nanoparticles in vivo was evaluated in 4T1 breast tumor-bearing mice. 4T1 cells were digested, collected, and redispersed in PBS (pH 7.4) at a concentration of 5 × 10 7 cells / ml. 100 μL of the above cell suspension was injected subcutaneously into the right back of the mouse to establish a 4T1 tumor-bearing mouse model. When the tumor volume reached about 500 mm 3 At 1, 2, 4, 8, and 12 h after injection, the fluorescence intensity of the mice was analyzed using an IVIS imaging system (PerkinElmer) at 748 nm excitation and 780 nm emission. Fig. 27 shown.

[0124] In order to quantify whether oxygen is responsible for the distribution of the nanoparticles in major organs and tumors by fluorescence intensity, the time point with the highest in vivo accumulation in each group was selected for fluorescence intensity analysis of ex vivo tissues and organs. Figure 28-29 shown.

[0125] The above results show that if Figure 27-29 As shown, Cy7-solution was rapidly cleared from the body after intravenous administration, while the nanoparticles showed higher tumor accumulation fluorescence at the tumor site within 12 h than Cy7 solution. Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles accumulated tumors more effectively at 4 h after injection than Cy7 solution at 1 h after injection. It is worth noting that the difference in tumor fluorescence intensity between Cy7-FDS nanoparticles and Cy7-FDSO nanoparticles was negligible through in vivo and in vitro biodistribution, which is consistent with the pharmacokinetic results. In summary, FDS nanoparticles loaded with oxygen can not only significantly prolong the systemic circulation time, but also show tumor-specific drug accumulation, which will certainly be beneficial for alleviating hypoxia and anti-tumor therapy.

[0126] Example 16: In vivo anti-tumor experiment of nanoparticles

[0127] 4T1 cell suspension (10 7 cells / 100 μL) were inoculated subcutaneously on the ventral side of female mice. 3The mice were randomly divided into groups of 5, each group was given PBS, Sor solution, FD solution, FA solution, FDS nanoparticles prepared in Example 1 and FDSO nanoparticles, respectively. The drug was administered once every other day for 5 consecutive times, and the dosage was equivalent to 10 mg / kg of doxorubicin according to FD or 8 mg / kg according to Sor. 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, lungs, and kidneys) were collected and fixed with 4% tissue fixative for H&E staining and HIF-1α staining. CLSM analysis confirmed that the fluorinated prodrug hybrid nanoparticles can effectively relieve hypoxia in the body and enhance its anti-tumor effect.

[0128] Tumor growth curve Figure 30-Figure 37 As shown, the single-drug treatment groups such as FD solution and Sor solution had poor tumor growth inhibition due to rapid elimination and less tumor accumulation in the body. Compared with FD solution and Sor solution, FDS solution showed stronger anticancer activity, indicating that the two drugs have a synergistic effect. As expected, FDSO nanoparticles effectively inhibited tumor growth throughout the treatment regimen. In contrast, FDS nanoparticles only showed a moderate antitumor response, indicating the extreme importance of relieving hypoxia in the body. And its therapeutic safety was preliminarily explored. The survival rate of mice treated with doxorubicin solution (equivalent DOX dose of 10 mg / ml) was significantly reduced, while no significant weight loss or death was found in other treatment groups. After multiple FDSO nanoparticle treatments, there were no obvious abnormalities in liver and kidney function parameters, body weight, and H&E staining of organ sections. These findings indicate that the cumulative toxicity associated with drug combinations can be significantly reduced by formulating drugs as fluorinated prodrugs. Compared with saline, the FD groups (FD solution, FDS solution, FDS nanoparticles) showed relatively low hypoxic zones, which may be due to the fact that the fluorinated side chains can capture endogenous oxygen at a position higher than the tumor oxygen partial pressure. However, obvious tumor hypoxic zones still appeared after treatment with FDSO nanoparticles. As expected, the tumor hypoxic zones in the FDSO nanoparticle-treated group were very small, which was highly consistent with the pharmacokinetics, distribution, and in vitro hypoxia relief of 4T1 cells. These results verify that FDSO nanoparticles can effectively achieve tumor accumulation and hypoxia relief, which will effectively explain their superior antitumor efficiency. These results further confirm that the combined induction strategy of GSH inhibition and ROS generation based on oxygen carrying bidirectionally drives ferroptosis in tumor cells through redox balance disruption.

[0129] In summary, the FDS nanoparticles of the present invention can be used as a highly efficient and safe lipid peroxidation nano-amplifier for ferroptosis-driven tumor treatment by loading oxygen.

[0130] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A disulfide-bridged fluorinated doxorubicin prodrug having oxygen-carrying capacity, characterized in that: The structural formula of the fluorinated doxorubicin prodrug is as follows:

2. A method for synthesizing the disulfide-bridged fluorinated doxorubicin prodrug with oxygen-carrying capacity as claimed in claim 1, characterized in that: The following steps are involved: (1) dehydrating 3,3'-dithiodipropionic acid with acetic anhydride to obtain a dibasic acid anhydride; (2) reacting 1H, 1H, 2H, 2H-perfluoro-1-octanol with the dibasic acid anhydride under the action of 4-dimethylaminopyridine to obtain an intermediate product; (3) dissolving the intermediate product in DMF, adding HBTU and DIPEA dropwise thereto, activating the intermediate product in an ice bath, adding DOX to react, and purifying the reaction product by liquid phase separation to obtain the fluorinated doxorubicin prodrug; 3. The method for synthesizing the disulfide-bridged fluorinated doxorubicin prodrug with oxygen-carrying capacity according to claim 2, characterized in that: In step (1), 3,3'-dithiodipropionic acid is dissolved in acetic anhydride, and stirred at room temperature for 1.5 h to 2.5 h under nitrogen protection to react the dibasic acid into dibasic anhydride. After the reaction is completed, toluene is added, and toluene and acetic anhydride are removed by vacuum evaporation at 30° C. to 35° C.; the molar ratio of the 3,3'-dithiodipropionic acid to the acetic anhydride is 1:(2 to 3); In step (2), 1H, 1H, 2H, 2H-perfluoro-1-octanol and 4-dimethylaminopyridine are dissolved in anhydrous dichloromethane together with the dibasic acid anhydride, reacted at room temperature for 10 h to 14 h, and separated by chromatography to obtain an intermediate product; the molar ratio of the 4-dimethylaminopyridine, the 1H, 1H, 2H, 2H-perfluoro-1-octanol and the dibasic acid anhydride is 1:(0.2-0.4):(1-2); In step (3), the molar ratio of the DMF, the HBTU, the DIPEA, the DOX and the intermediate product is 1:(1-1.5):(1-1.5):(0.5-0.8).

4. An oxygen-enhanced fluorinated nanoassembly for bidirectionally amplifying tumor ferroptosis, characterized in that: The oxygen-enhanced fluorinated nanoassembly is a nanoparticle co-assembled with a fluorinated doxorubicin prodrug and a ferroptosis inducer, or a nanoparticle co-assembled with a fluorinated doxorubicin prodrug modified with a polyethylene glycol modifier and a ferroptosis inducer; The fluorinated doxorubicin prodrug is the fluorinated doxorubicin prodrug according to claim 1, or the fluorinated doxorubicin prodrug obtained by the synthesis method according to any one of claims 2 to 3.

5. The oxygen-enhanced fluorinated nanoassembly for bidirectionally amplifying tumor ferroptosis according to claim 4, characterized in that: The oxygen-enhanced fluorinated nanoassembly has an oxygen carrying capacity of 70 mg / L to 80 mg / L; The particle size of the oxygen-enhanced fluorinated nanoassembly is 90nm to 100nm; The fluorinated doxorubicin prodrug is co-assembled with the ferroptosis inducer through one or both of hydrophobic interaction and intermolecular hydrogen bonding.

6. The oxygen-enhanced fluorinated nanoassembly for bidirectionally amplifying tumor ferroptosis according to claim 4, characterized in that: The ferroptosis inducing agent is one or more of sulfasalazine, lampirizone, trigonelline and sorafenib; The polyvinyl alcohol modifier includes one or more of PLGA-PEG, DSPE-PEG, DSPE-SS-PEG, PCL-PEG, and PE-PEG; The molecular weight of the polyvinyl alcohol modifier is 200 to 20000; The molar ratio of the fluorinated doxorubicin prodrug to the ferroptosis inducing agent is 10:1 to 1:10; The mass ratio of the fluorinated doxorubicin prodrug to the polyvinyl alcohol modifier is 10:90 to 90:

10.

7. A method for preparing the oxygen-enhanced fluorinated nanoassembly according to any one of claims 4 to 6, characterized in that: The following steps are involved: The fluorinated doxorubicin prodrug and the ferroptosis inducer, or the fluorinated doxorubicin prodrug, the ferroptosis inducer and the polyethylene glycol modifier are dissolved in an organic solvent, and the mixed solution is slowly added dropwise to deionized water, and uniform co-assembled nanoparticles are spontaneously formed under stirring, and the organic solvent is removed to obtain the oxygen-enhanced fluorinated nanoassembly.

8. Use of the fluorinated doxorubicin prodrug of claim 1 or the oxygen-enhanced fluorinated nanoassembly of any one of claims 4 to 6 in a drug delivery system.

9. Use of the fluorinated doxorubicin prodrug according to claim 1 or the oxygen-enhanced fluorinated nanoassembly according to any one of claims 4 to 6 in the preparation of anti-tumor drugs.

10. Use of the fluorinated doxorubicin prodrug of claim 1 or the oxygen-enhanced fluorinated nanoassembly of any one of claims 4 to 6 in an injection, oral administration or topical administration system.

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