Self-synergistic enhanced ferroptosis nano assembly and preparation method and application thereof

Abstract

The invention relates to a nano assembly for self-synergistically enhancing ferroptosis as well as a preparation method and application of the nano assembly, and belongs to the technical field of pharmaceutical preparation combined treatment of new auxiliary materials and new dosage forms. The invention firstly provides a functional conjugate linker capable of automatically generating ROS, the functional conjugate linker comprises artesunate-ferrocene or dihydroartemisinin-ferrocene, and the structural formulas of the functional conjugate linker and the artesunate-ferrocene or dihydroartemisinin-ferrocene are respectively shown as a and b. According to the present invention, the ROS self-generating functional conjugate linker and the ferroptosis inducer are co-assembled to form the nanometer assembly, such that the ROS level in the tumor cells can be effectively and synergistically improved by the ROS self-generating functional conjugate linker and the ferroptosis inducer so as to cause the tumor cell lipid peroxidation storm, and the ferroptosis induction effect can be significantly improved. 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 requirements of efficient and diversified ferroptosis-based cancer treatment strategies on preparations in clinic. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a nanoassembly for self-synergistically enhancing ferroptosis, a preparation method and application thereof, and belongs to the technical field of new excipients and new dosage forms for combined treatment of pharmaceutical preparations. Background Art

[0002] Cancer is a disease that seriously threatens human health worldwide. Commonly used treatment strategies in clinical practice, such as surgery, chemotherapy, and radiotherapy, cannot achieve satisfactory therapeutic effects. There is an urgent need to develop more effective treatment methods based on the discovery of innovative treatment mechanisms and the development of cutting-edge drug delivery technologies. Ferroptosis is an emerging type of iron-dependent cell death that can be used as a promising target for cancer treatment. Intracellular iron and reactive oxygen species (ROS) dominate the occurrence of ferroptosis by promoting the accumulation of lipid peroxidation in tumor cells. In addition, the underlying mechanism of ferroptosis is closely related to the upregulation of endoplasmic reticulum stress and the activation of mitochondrial voltage-dependent anion channels. These processes play an important role in the ROS burst and subsequent lipid peroxidation storm in tumor cells. Strategies to induce ferroptosis include exogenous iron supplementation, depletion of glutathione (GSH), production of ROS through Fenton-like reactions, inhibition of Xc - system and inhibit glutathione peroxidase 4 (GPX4), etc.

[0003] Ferroptosis is an iron-dependent cell death pathway that involves the bioavailable ferrous iron (Fe 2+ ) is closely related to the existence of Fe 2+ The underlying mechanism of ferroptosis mediated by Fe 2+ It plays a vital role in the electrochemical Fenton reaction. 2+ Reaction with hydrogen peroxide (H2O2) to generate large amounts of highly lethal ROS has been identified as a key pathway for promoting ROS generation in iron-induced ferroptosis. Based on this chemical reaction, several iron nanomaterials, including hemoglobin, Fe3O4 nanoclusters, and iron-based organic frameworks, have been designed to promote tumor ferroptosis. However, these nanomaterials are often affected by the heterogeneity of redox and pH levels present in the tumor microenvironment and are highly dependent on cellular H2O2 levels and acidic conditions (pH 2-4). Therefore, there is an urgent need to develop cellular ROS generation modalities that are less dependent on the tumor microenvironment to promote tumor ferroptosis therapy.

[0004] Artesunate (ART) is a semisynthetic derivative of artemisinin that has been successfully used to treat malaria. In recent years, the potential value of ART in cancer treatment has gradually emerged. The endoperoxide bridge structure of ART can bind to Fe 2+ The reaction generates ROS and is not affected by pH conditions. Therefore, ART is widely considered to be a H2O2 substitute.2+ The triggered Fenton reaction induces tumor ferroptosis. 2+ The concentration of is crucial for ART-induced tumor ferroptosis. In addition, the redox balance mechanism in tumor cells triggers feedback upregulation of the antioxidant system to counteract ART-induced ferroptosis by preventing lipid peroxidation. It is worth noting that the cystine / glutamate antiporter (Xc - The Xc-GSH-GPX4 (glutathione peroxidase 4) axis plays a crucial role in the intracellular antioxidant system. In view of this pathway, many ferroptosis inducers have been developed by inhibiting Xc - Systems such as sorafenib (Sor) and erastin accelerate ferroptosis, which induce ferroptosis by inhibiting GPX4, such as FIN56 and RSL3. Therefore, the combination of ART and ferroptosis inducers is a promising therapeutic combination that can trigger a lipid peroxidation storm in tumor cells by upregulating cellular ROS while downregulating the antioxidant system in tumor cells. Although the simultaneous delivery of multiple drugs is of great significance in precise combination therapy, it remains challenging.

[0005] Biomedical nanotechnology offers a versatile co-delivery nanoplatform. However, conventional co-delivery nanodrugs are mostly prepared through non-covalent encapsulation using organic or inorganic nanocarriers. This suffers from low co-delivery efficiency, inconvenient dosage adjustment, and suboptimal synergistic effects. These limitations can be attributed to significant differences in the physicochemical properties of different drugs, leading to significant variations in the molecular affinity between the nanocarrier material and the drug. Therefore, there is an urgent need to develop a co-delivery nanoplatform that can simultaneously upregulate ROS and downregulate antioxidant systems. Summary of the Invention

[0006] In order to solve the problems of poor water solubility, poor assembly ability, low drug loading due to entrapment in polymers, drug leakage and excipient-related toxicity in the prior art, the present invention provides a self-synergistically enhanced ferroptosis nanoassembly and its preparation method and application. The present invention achieves the technical effects of high drug loading, good stability and low toxic side effects by co-assembling the prepared functional conjugate linker that self-generates ROS with the ferroptosis inducer, thereby solving the problem of unsatisfactory effect of single-drug therapy. In the present invention, the functional conjugate linker that self-generates ROS and the ferroptosis inducer synergistically enhance the ROS level in tumor cells, causing a lipid peroxidation storm in tumor cells, and significantly improving the induction of ferroptosis.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] One of the purposes of the present invention is to provide a functional conjugate linker that self-generates ROS, wherein the functional conjugate linker that self-generates ROS is artesunate-ferrocene (a) or dihydroartemisinin-ferrocene (b), and its structural formula is as follows:

[0009]

[0010] Preferably, the functional conjugate linker that self-generates ROS is artesunate-ferrocene.

[0011] A second object of the present invention is to provide a method for preparing a functional conjugate that self-generates ROS, comprising the following steps:

[0012]

[0013] When the functional conjugate linker that self-generates ROS is artesunate-ferrocene, the preparation process is as follows: artesunate, ferrocene methanol, and catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in an organic solvent, stirred for reaction, and purified to obtain artesunate-ferrocene;

[0014] When the functional conjugate linker that self-generates ROS is dihydroartemisinin-ferrocene, the preparation process is to dissolve dihydroartemisinin, ferrocenecarboxylic acid, and catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in an organic solvent, stir the reaction, and purify to obtain dihydroartemisinin-ferrocene.

[0015] Furthermore, the organic solvent is dichloromethane.

[0016] Furthermore, the molar ratio of artesunate or dihydroartemisinin, ferrocenylmethanol or ferrocenylcarbolic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:0.2-5:0.5-4:0.05-0.8.

[0017] Preferably, the molar ratio of artesunate or dihydroartemisinin, ferrocenylmethanol or ferrocenylcarbolic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:1:0.2.

[0018] Furthermore, the stirring reaction condition is to stir the reaction at 800 rpm at 15-45° C. for 24-144 hours.

[0019] Preferably, the stirring reaction condition is stirring the reaction at 800 rpm at 25° C. for 144 hours.

[0020] The third object of the present invention is to provide a nanoassembly that self-synergistically enhances ferroptosis, wherein the nanoassembly is a nanoparticle formed by co-assembling a functional conjugate linker that self-generates ROS and a ferroptosis inducer through intermolecular forces, or a nanoparticle formed by co-assembling a functional conjugate linker that self-generates ROS and a ferroptosis inducer modified by a PEG modifier through intermolecular forces.

[0021] Furthermore, the functional conjugate linker that self-generates ROS is the functional conjugate linker that self-generates ROS described above, or the functional conjugate linker that self-generates ROS obtained by the above preparation method.

[0022] Furthermore, the ferroptosis inducer is one or more of sorafenib, erastin, sulfasalazine, FIN56, RSL3 and simvastatin.

[0023] Preferably, the ferroptosis inducer is sorafenib.

[0024] Furthermore, the intermolecular force is one or both of hydrophobic interaction and hydrogen bonding.

[0025] In the above technical solution, the nanoassembly is uniformly spherical and has a particle size of 160 to 170 nm.

[0026] Furthermore, the PEG modifier is one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG or PE-PEG.

[0027] Furthermore, the molecular weight of the PEG modifier is 200 to 20,000.

[0028] Furthermore, the molar ratio of the functional conjugate linker that self-generates ROS to the ferroptosis inducer is 1:0.1-10.

[0029] Furthermore, the mass ratio of the sum of the functional conjugate linker that self-generates ROS and the ferroptosis inducer to the PEG modifier is 1:0.1-10.

[0030] The fourth object of the present invention is to provide a method for preparing a nanoassembly that self-synergistically enhances ferroptosis, comprising the following steps: dissolving a functional conjugate linker that self-generates ROS and a ferroptosis inducer, or dissolving a functional conjugate linker that self-generates ROS, a ferroptosis inducer and a PEG modifier in an organic solvent, stirring and mixing, slowly adding the mixed solution dropwise into water to spontaneously form a uniform nanoassembly, and removing the organic solvent to obtain the result.

[0031] Furthermore, the organic solvent is one of ethanol, tetrahydrofuran, and dimethyl sulfoxide, or a combination of any two thereof.

[0032] Preferably, the organic solvent for dissolving the functional conjugate linker that self-generates ROS and the ferroptosis inducer is a combination of tetrahydrofuran and anhydrous ethanol in a volume ratio of 1:1.

[0033] Preferably, the organic solvent for dissolving the PEG modifier is tetrahydrofuran.

[0034] Furthermore, the stirring rate of the stirring and mixing is 200 to 2000 rpm.

[0035] Furthermore, the method for removing the organic solvent may be a solvent evaporation method, an ultrafiltration method, and a membrane permeation method.

[0036] The present invention preferably has a technical solution:

[0037] A method for preparing a nanoassembly that self-synergistically enhances ferroptosis comprises the following steps:

[0038] (1) Synthesis of artesunate-ferrocene: Artesunate, ferrocene methanol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 4-dimethylaminopyridine (DMAP) were added to dichloromethane for reaction, and then purified by semi-preparative high performance liquid chromatography to obtain artesunate-ferrocene;

[0039] (2) Preparation of nanoassemblies: The artesunate-ferrocene and sorafenib obtained above were dissolved in a tetrahydrofuran-anhydrous ethanol mixed solution (v / v, 1:1), and DSPE-SS-PEG 2K The modifying agent is dissolved in tetrahydrofuran and mixed evenly. The solution is slowly added dropwise into deionized water under stirring to spontaneously form uniform nanoparticles. The organic solvent is removed by rotary evaporation to obtain the nanoparticles.

[0040] Preferably, in step (2), the molar ratio of artesunate-ferrocene to sorafenib is 1:0.1-6.

[0041] Further preferably, in step (2), the molar ratio of artesunate-ferrocene to sorafenib is 1:1.

[0042] The present invention forms a nanoassembly by combining artesunate-ferrocene, a functional conjugate linker that self-generates ROS, with the ferroptosis inducer sorafenib. Alternatively, the nanoassembly is modified with a PEG modifier. Studies have shown that artesunate-ferrocene and the ferroptosis inducer can synergistically elevate ROS levels in tumor cells, triggering a lipid peroxidation storm in tumor cells and significantly enhancing the induction of ferroptosis. Furthermore, the nanoassembly modified with the PEG modifier exhibits superior stability.

[0043] A fifth object of the present invention is to provide the use of the functional conjugate linker or the nanoassembly that self-generates ROS in the preparation of a drug delivery system.

[0044] A sixth object of the present invention is to provide the use of the above-mentioned functional conjugate linker or the above-mentioned nanoassembly that self-generates ROS in the preparation of anti-tumor drugs.

[0045] The seventh object of the present invention is to provide the use of the above-mentioned functional conjugate linker or the above-mentioned nanoassembly that self-generates ROS in the preparation of an injection, oral administration or local administration system.

[0046] Beneficial effects of the present invention:

[0047] 1. The present invention first prepares a functional conjugate that self-generates ROS (preferably artesunate-ferrocene), which is then co-assembled with a ferroptosis inducer (preferably sorafenib) to form a nanoassembly, which is then modified with a PEG modifier to obtain a nanoassembly that self-synergistically enhances ferroptosis, which can be used for tumor treatment. The functional conjugate that self-generates ROS prepared by the present invention can effectively increase ROS and iron levels in tumor cells, and the ferroptosis inducer can effectively inhibit the production of GSH in tumor cells, leading to excessive accumulation of lipid peroxides, thereby enhancing the efficacy of ferroptosis.

[0048] 2. The nanoassembly formed by co-assembling the functional conjugate linker that self-generates ROS and the ferroptosis inducer prepared by the present invention can achieve technical effects such as high drug loading, good stability, and low toxic side effects, meeting the urgent demand for high-efficiency and low-toxic preparations in clinical practice, providing a new strategy for the assembly of homologous synergistic drug nanoassemblies, and providing an effective nanoplatform for the development of carrier-free nanoassemblies and tumor ferroptosis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The mass spectrum and NMR spectrum of AF obtained in Example 1 of the present invention and the mass spectrum of DF obtained in Example 2 are shown.

[0050] Figure 2 This is a graph showing the degree of MB degradation by AF obtained in Example 1 of the present invention in ART solution, Fc solution, ART / Fc solution and AF solution.

[0051] Figure 3 AF obtained in Example 1 of the present invention is ART / Fe 2+ The degradation degree of MB in solution and AF solution.

[0052] Figure 4This is a qualitative graph of ROS after 4T1 cells were treated with AF obtained in Example 1 of the present invention in ART solution, Fc solution, ART / Fc solution and AF solution.

[0053] Figure 5 This is a quantitative graph of ROS after 4T1 cells were treated with AF obtained in Example 1 of the present invention in ART solution, Fc solution, ART / Fc solution and AF solution.

[0054] Figure 6 This is a graph showing the cytotoxicity of AF to 4T1 cells obtained in Example 1 of the present invention.

[0055] Figure 7 This is the synergistic index graph of AF obtained in Example 1 of the present invention and various ferroptosis inducers in 4T1 cells

[0056] Figure 8 This is the optimal synergistic ratio and synergistic index diagram of AF obtained in Example 1 of the present invention and various ferroptosis inducers in 4T1 cells.

[0057] Figure 9 The particle size distribution and electron microscopy image of the bare AFS nanoparticles obtained in Example 3 of the present invention are shown.

[0058] Figure 10 The particle size distribution and electron microscope image of the AFS nanoparticles obtained in Example 3 of the present invention are shown.

[0059] Figure 11 This is a stability diagram of naked AFS nanoparticles and AFS nanoparticles obtained in Example 3 of the present invention in PBS (pH 7.4).

[0060] Figure 12 This is a graph showing the stability of naked AFS nanoparticles and AFS nanoparticles obtained in Example 3 of the present invention in PBS (pH 7.4) containing 10% FBS.

[0061] Figure 13 This is the molecular docking diagram of AF and Sor in Example 4 of the present invention.

[0062] Figure 14 This is a diagram showing the force destruction of naked AFS nanoparticles in Example 4 of the present invention in PBS (pH 7.4) containing 10 mM sodium dodecyl sulfate and urea.

[0063] Figure 15 This is a graph showing the particle size changes of AFS nanoparticles in Example 5 of the present invention in PBS (pH 7.4) containing 0-10 mM DTT.

[0064] Figure 16These are confocal microscopy photos and three-dimensional intensity maps of the cellular uptake of Cy7 solution and Cy7-AFS nanoparticles in Example 6 of the present invention at 0.5 hours and 2 hours.

[0065] Figure 17 Flow cytometric quantitative graphs of cellular uptake of Cy7 solution and Cy7-AFS nanoparticles in Example 6 of the present invention at 0.5 hours and 2 hours.

[0066] Figure 18 This is a graph showing the 4T1 cytotoxicity of Example 7 of the present invention.

[0067] Figure 19 This is the L02 cytotoxicity graph of Example 7 of the present invention.

[0068] Figure 20 This is a cytotoxicity graph of the Sor solution, AF solution, AF solution and AFS nanoparticles according to Example 7 of the present invention and after the addition of ferroptosis inhibitors.

[0069] Figure 21 This is a graph showing the intracellular iron content in 4T1 cells after treatment with the Sor solution, AF solution, AFS solution, and AFS nanoparticles according to Example 8 of the present invention.

[0070] Figure 22 These are the qualitative graphs and three-dimensional intensity graphs of ROS after 4T1 cells were treated with the Sor solution, AF solution, AFS solution and AFS nanoparticles according to Example 9 of the present invention.

[0071] Figure 23 This is a quantitative graph of ROS after 4T1 cells were treated with the Sor solution, AF solution, AFS solution and AFS nanoparticles according to Example 9 of the present invention.

[0072] Figures 24-27 These are graphs showing GSH levels, GPX4 inhibition rates, LPOs contents, and MDA contents in 4T1 cells after treatment with Sor solution, AF solution, AFS solution, and AFS nanoparticles in Example 10 of the present invention.

[0073] Figure 28 The blood concentration-time curve of DiR solution and DiR / AFS nanoparticles in Example 11 of the present invention is shown in FIG.

[0074] Figure 29 This is an in vivo fluorescence imaging image of mice administered with DiR solution and DiR / AFS nanoparticles in Example 12 of the present invention.

[0075] Figure 30 This is a quantitative image of in vivo fluorescence imaging of mice administered DiR solution and DiR / AFS nanoparticles in Example 12 of the present invention.

[0076] Figure 31 These are fluorescence imaging images of isolated organs of mice administered DiR solution and DiR / AFS nanoparticles in Example 12 of the present invention.

[0077] Figure 32 This is a fluorescence quantitative graph of isolated organs of mice administered DiR solution and DiR / AFS nanoparticles in Example 12 of the present invention.

[0078] Figure 33 This is a graph showing the tumor growth curve of mice in the in vivo anti-tumor experiment in Example 13 of the present invention.

[0079] Figure 34 This is a photo of mouse in vitro tumors from the in vivo anti-tumor experiment in Example 13 of the present invention.

[0080] Figure 35 Statistical graph of the tumor-bearing rate of mice in the in vivo anti-tumor experiment in Example 13 of the present invention.

[0081] Figure 36 This is an H&E staining image of mouse in vitro tumors in the in vivo anti-tumor experiment in Example 13 of the present invention.

[0082] Figure 37 This is a graph showing changes in mouse body weight in the in vivo anti-tumor experiment in Example 13 of the present invention.

[0083] Figure 38 1 is a histopathological section diagram of the liver and kidney function analysis results in Example 13 of the present invention.

[0084] Figure 39 This is a histopathological section diagram in Example 13 of the present invention.

[0085] Figure 40 This is a diagram showing the results of the hemolysis experiment in Example 13 of the present invention. DETAILED DESCRIPTION

[0086] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0087] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0088] Example 1

[0089] A method for preparing a functional conjugate (artesunate-ferrocene) that self-generates ROS comprises the following steps:

[0090] Artesunate (ART, 2 mmol), ferrocene methanol (Fc, 2 mmol), DMAP (0.4 mmol), and EDCI (2 mmol) were added to 25 mL of dichloromethane and stirred at 800 rpm at 37°C for 144 hours. The mixture was then purified using a preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, pure acetonitrile as the mobile phase, and a flow rate of 10 mL / min) to obtain the product artesunate-ferrocene (AF), which was then characterized by HPLC, MS, and nuclear magnetic resonance spectroscopy.

[0091] Example 2

[0092] A method for preparing a functional conjugate (dihydroartemisinin-ferrocene) that self-generates ROS comprises the following steps:

[0093] Dihydroartemisinin (DHA, 2 mmol), ferrocenecarboxylic acid (Fc, 2 mmol), DMAP (0.4 mmol), and EDCI (2 mmol) were added to 25 mL of dichloromethane and stirred at 800 rpm at 37°C for 144 hours. The mixture was then purified using a preparative liquid chromatography (Shim-pack PREP-ODS 250×20 mm column, pure acetonitrile as the mobile phase, and a flow rate of 10 mL / min) to obtain the product dihydroartemisinin-ferrocene (DF), which was then characterized by HPLC and MS.

[0094] The characterization results of AF obtained in Example 1 and DF obtained in Example 2 are as follows: Figure 1 As shown, the molecular weight of AF was determined by MS and was 582.19, which was consistent with the actual molecular weight; the hydrogen of AF was analyzed by nuclear magnetic resonance hydrogen spectrum, confirming the successful synthesis of AF; the molecular weight of DF was determined by MS and was 496.15, confirming the successful synthesis of DF.

[0095] Determination of the ability of AF obtained in Example 1 to degrade methylene blue:

[0096] Methylene blue (MB) was used to detect the level of ROS generation in AF in vitro. ART solution, Fc solution, ART / Fc solution, and AF solution were incubated with PBS (pH 7.4) containing 20 μM MB, and the ultraviolet (UV) absorbance at 664 nm was measured using a multifunctional microplate reader (Thermo Scientific, USA) at designated time intervals (0, 10, 20, 60, 90, and 120 min). The results are shown in Table 1. Figure 2As shown in the figure, it can be seen that 20.4% of MB in the AF solution group were degraded, while no obvious degradation occurred in the ART solution, Fc solution and ART / Fc solution groups, indicating that the combination of ART and Fc can accelerate the production of ROS in ART. 2+ The AF solution and the AF solution were incubated with PBS (pH 7.4) containing 20 μM MB, and the ultraviolet (UV) absorbance at 664 nm was measured using a multifunctional microplate reader (Thermo Scientific, USA) after 24 h. Figure 3 As shown, it can be seen that the AF solution group showed a 2+ The higher MB degradation rate in the solution group indicated that the Fe supply was close to the 2+ and efficient intramolecular electron transfer with a higher ROS production rate.

[0097] Determination of ROS production ability of AF obtained in Example 1:

[0098] The production of intracellular ROS was detected by FACS calibur flow cytometer (FCM, BD Corp., USA) and confocal laser scanning microscope (CLSM, Nikon Corp., Tokyo, Japan) using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, a fluorescent probe for detecting ROS). 4T1 cells were cultured at 5×10 4 The cells were seeded into 35 mm glass-bottomed cell culture dishes at a density of 100 cells / dish and cultured for 12 hours. Then, 4T1 cells were treated with ART solution, Fc solution, ART / Fc solution and AF solution for 4 hours, with the ART concentration of 200 nM in all groups. After this incubation, the cells were washed three times with PBS (pH 7.4) and then incubated with 10 μM DCFH-DA for 30 minutes. The green fluorescence intensity of DCF was observed by CLSM. In addition, 4T1 cells were cultured at a density of 2×10 5 Cells were seeded into 12-well plates at a density of 100 cells / well and cultured for 12 hours. The cells were then treated and collected using the same method as above. The cells were suspended in PBS (pH 7.4) and the intracellular fluorescence of ROS was quantified using FCM. Figure 4 and Figure 5 As shown in the figure, it can be seen that AF has a stronger ROS production ability than ART / Fc, which indicates that the supply of Fe at close range 2+ and efficient intramolecular electron transfer with a higher ROS production rate.

[0099] Cytotoxicity determination of AF obtained in Example 1:

[0100] The cytotoxicity of ART, Fc, ART / Fc and AF was compared by MTT assay. 4T1 cells were cultured at 2×10 3 The cells were seeded in a 96-well plate at a density of 100 μL / well for 12 hours. Then, 4T1 cells were treated with ART solution, Fc solution, ART / Fc solution and AF solution for 48 hours, respectively. Afterwards, the cells were incubated with 5 mg / mL MTT (20 μL / well) at 37°C for 4 hours. Then, formazan was dissolved in dimethyl sulfoxide (200 μL / well). Finally, the UV absorbance at 490 nm was measured using a multifunctional microplate reader (Thermo Scientific, USA). The results are shown in Figure 2. Figure 6 As shown in the results, it can be seen that AF has stronger cytotoxicity than ART / Fc, which further indicates that AF has stronger ROS production ability.

[0101] Screening of the best synergistic index between AF obtained in Example 1 and various ferroptosis inducers:

[0102] The MTT assay was used to evaluate the synergistic effect of AF with various ferroptosis inducers (sorafenib (Sor), erastin (Era), sulfasalazine (Sul), FIN56, RSL3, and simvastatin (SIM)). 4T1 cells were cultured at 2×10 3 The cells were seeded at a density of 100 cells / well in a 96-well plate for 12 hours, and then the 4T1 cells were treated with different concentrations of AF solution, ferroptosis inducer solution or mixed solutions of AF and ferroptosis inducer at different molar ratios. After 48 hours, the cells were incubated with 5 mg / mL MTT (25 μL / well) at 37°C for 4 hours, and then the formazan generated by the cells was dissolved with DMSO (200 μL / well). Finally, the ultraviolet absorbance at 490 nm was measured with a microplate reader. The cell viability and synergy index (CI) were calculated to evaluate the synergistic effect of AF and various ferroptosis inducers. The results are shown in Figure 2. Figure 7 and Figure 8 As shown, it can be seen that AF and Sor have the best synergistic effect when the molar ratio is 1:1, so the ferroptosis inducer is preferably Sor, and the molar ratio is preferably 1:1.

[0103] Example 3

[0104] A method for preparing non-PEGylated AFS nanoparticles (hereinafter referred to as naked AFS nanoparticles) comprises the following steps:

[0105] 0.56 mg of AF obtained in Example 1 and 0.44 mg (equimolar amount) of Sor were accurately weighed and dissolved in 200 μL of a mixed solution of tetrahydrofuran and anhydrous ethanol (v / v, 1:1) to obtain an AFS solution. The solution was slowly added dropwise to 2 mL of deionized water under stirring to spontaneously form uniform bare AFS nanoparticles. The organic solvent was then removed by rotary evaporation at 30°C to obtain a nanocolloidal solution free of any organic solvent.

[0106] A DSPE-SS-PEG 2K The preparation method of modified AFS nanoparticles (hereinafter referred to as AFS nanoparticles) comprises the following steps:

[0107] Accurately weigh 0.56 mg of AF obtained in Example 1 and 0.44 mg (equimolar amount) of Sor, dissolve them in 200 μL of a mixed solution of tetrahydrofuran and anhydrous ethanol (v / v, 1:1) to obtain an AFS solution; accurately weigh 0.25 mg of DSPE-SS-PEG 2K , dissolved in tetrahydrofuran to a 10 mg / mL stock solution; 200 μL AFS solution was mixed with 25 μL DSPE-SS-PEG 2K The mixture was mixed evenly and the solution was slowly added dropwise to 2 mL of deionized water under stirring to obtain uniform AFS nanoparticles. The organic solvent was then removed by rotary evaporation at 30°C to obtain a nanocolloidal solution free of any organic solvent. The particle size, particle size distribution, and Zeta potential of the prepared bare AFS nanoparticles and AFS nanoparticles were determined by dynamic light scattering. The results are shown in Table 1. The particle size and morphology were determined by transmission electron microscopy. Figure 9 and Figure 10 As shown, it can be seen that both bare AFS nanoparticles and AFS nanoparticles are uniformly spherical, with a particle size of about 160 nm.

[0108] Table 1. Nanoparticle size, size distribution, and zeta potential

[0109]

[0110] Colloidal stability test of bare AFS nanoparticles and AFS nanoparticles:

[0111] 1 mL of the bare AFS nanoparticles and AFS nanoparticles (0.5 mg / mL) prepared in Example 3 was taken out and added to 10 mL of PBS (pH 7.4) and PBS containing 10% FBS (pH 7.4), and incubated in a shaker at 37°C for 12 hours. The changes in particle size 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 11 and Figure 12As shown, it can be seen that: compared with naked AFS nanoparticles, AFS nanoparticles have better colloidal stability in PBS, and the particle size does not change significantly within 12 hours; naked AFS nanoparticles and AFS nanoparticles have better colloidal stability in PBS containing 10% FBS, so PEG-modified AFS nanoparticles are preferred.

[0112] Example 4

[0113] Analysis of AF and Sor assembly mechanism:

[0114] Through computer simulation, the assembly mechanism of AF and Sor was explored, and molecular docking calculations were performed using the Vina program of the Yinfu cloud computing platform. Compounds AF and Sor were energy minimized under the MMFF94 force field to obtain a 3D structure, forming a stable nanoassembly. Semi-flexible docking was performed using the AutoDockVina program, and sodium chloride, sodium dodecyl sulfate, and urea were used for force destruction. The results are as follows Figure 13 and Figure 14 As shown, it can be seen that there are multiple forces between AF and Sor molecules, such as hydrophobic force and hydrogen bond, which make great contributions to the co-assembly of AF and Sor.

[0115] Example 5

[0116] Research on reduction-triggered disintegration of nanoassemblies:

[0117] Good colloidal stability is crucial for the in vivo delivery of nanoassemblies, but rapid release of drugs in tumors is also a prerequisite for exerting antitumor activity. 2K Modified on the surface of the nanoassembly, reduction-responsive nanoparticles (AFS nanoparticles) were obtained. In order to study the disintegration behavior of the nanoparticles triggered by reduction, the stability of the nanoparticles (AFS nanoparticles) under reduction stimulation was first evaluated using PBS (pH 7.4) containing DTT (10mM). AFS nanoparticles (0.5mg / mL) were incubated at 37°C in PBS (pH 7.4) containing 0mM and 10mM DTT, respectively. The particle size of the nanoparticles at different time points was measured using a Malvern particle size analyzer. The results are shown in Figure 2. Figure 15 As shown in the figure, it can be seen that under the action of 10mM DTT, the particle size of AFS nanoparticles increased significantly, while the particle size of AFS nanoparticles in PBS without DTT remained almost unchanged. The results showed that after DSPE-SS-PEG 2K The modified AFS NAs have the property of dissociating from reducing substances.

[0118] Example 6

[0119] Cellular uptake of AFS nanoparticles:

[0120] Confocal microscopy and flow cytometry were used to qualitatively and quantitatively determine the uptake of the AFS nanoparticles prepared in Example 3 in 4T1 cells (mouse breast cancer cells). 4 Cells were seeded into 24-well plates at a density of 100 cells / well and incubated in an incubator for 12 hours to allow them to adhere. After the cells adhered, Cy7 solution and Cy7-AFS nanoparticles were added. The concentration of Cy7 was 2 μM. After incubation at 37°C for 0.5 hours and 2 hours, the cells were washed and fixed. Finally, confocal microscopy was used to analyze the cellular uptake of various agents. The quantitative operation was to seed 4T1 cells at a density of 2×10 5 Cells were seeded onto 12-well plates at a density of 100 cells / well and incubated in an incubator for 12 hours to allow them to adhere. After the cells adhered, Cy7 solution and Cy7-AFS nanoparticles were added. The concentration of Cy7 was 2 μM. After incubation at 37°C for 0.5 hours and 2 hours, the cells were washed and digested and collected. Finally, the cellular uptake of various preparations was analyzed by flow cytometry. The experimental results are shown in the figure. Figure 16 and Figure 17 As shown in the figure, it can be seen that the cellular uptake showed a time-dependent uptake, and the cells treated with Cy7-AFS nanoparticles had a higher intracellular fluorescence intensity than the cells treated with Cy7 solution. Therefore, the prepared Cy7-AFS nanoparticles have a higher cellular uptake efficiency than the Cy7 solution.

[0121] Example 7

[0122] Cytotoxicity of AFS nanoparticles:

[0123] The cytotoxicity of Sor solution, AF solution, AF and Sor mixed solution (AFS solution) and AFS nanoparticles on mouse breast cancer (4T1) cells was investigated by MTT assay. 4 After the cells are adhered to the wall, they are cultured with a medium containing Sor solution, AF solution, AFS solution and AFS nanoparticles, 200 μL per well. The control group is cultured with a medium without drug solution. After 48 hours, the 96-well plate is removed and 5 mg mL of the suspension is added to each well. -125 μL of MTT solution was added to the plate and incubated in an incubator for 4 hours. The plate was then shaken and the 96-well plate was inverted on filter paper to fully absorb the residual liquid. 200 μL of DMSO was added to each well and shaken on a shaker for 10 minutes to dissolve the blue-purple crystals. The absorbance value of each well after zeroing was measured at 490 nm using a microplate reader. The changes in cytotoxicity after the addition of Fer-1 (10 μM) were also examined. Using the same method, a cytotoxicity experiment was performed to compare the cytotoxicity of Sor solution, AF solution, AFS solution, and AFS nanoparticles on L02 cells (normal human liver cells).

[0124] Cytotoxicity results such as Figures 18-20 As shown, in 4T1 cells, AFS solution and AFS nanoparticles exhibited a synergistic effect, resulting in a more significant inhibitory effect on cell growth compared to the free drug alone. AFS nanoparticles exhibited the most potent antitumor activity in vitro, attributed to their efficient cellular uptake and tumor-sensitive intracellular drug release. Co-treatment of 4T1 cells treated with Sor solution, AF solution, AFS solution, and AFS nanoparticles with Fer-1 significantly increased cell survival, confirming that AF and Sor effectively kill tumor cells by synergistically inducing ferroptosis. No significant cell death was observed in L02 cells treated with AFS nanoparticles, indicating a favorable safety profile.

[0125] Example 8

[0126] Effect of AFS nanoparticles on iron content:

[0127] 4T1 cells in good condition were digested, diluted with culture medium and plated at 2×10 6 The cells were inoculated into the culture dish at a density of 10 cells / dish and incubated in the incubator for 12 hours to allow them to adhere. After the cells adhered, they were cultured with a culture medium containing Sor solution, AF solution, AFS solution and AFS nanoparticles, 200 μL per well, and the concentration of AF was 200 nM or the concentration of Sor was 200 nM. After culturing for 4 hours, the cells were washed 3 times with ice PBS, and the cells were collected. The 4T1 cells were suspended in 300 μL PBS and further destroyed by ultrasound. The cell iron content was detected according to the instructions of the cell iron detection kit. The results are as follows Figure 21 As shown, it can be seen that the intracellular iron content in all AF-treated groups was significantly increased, indicating that AF has the function of iron supply and induces cellular iron overload.

[0128] Example 9

[0129] Effects of AFS nanoparticles on cellular reactive oxygen species (ROS) content:

[0130] The production of intracellular ROS was detected by FACS calibur flow cytometer (FCM, BD Corp., USA) and confocal laser scanning microscope (CLSM, Nikon Corp., Tokyo, Japan) using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, a fluorescent probe for detecting ROS). 4 The cells were seeded into 35 mm glass-bottomed cell culture dishes at a density of 100 cells / dish and cultured for 12 hours. 4T1 cells were then treated with Sor solution, AF solution, AFS solution, and AFS nanoparticles for 4 hours. The AF concentration in each solution was equal, all at 200 nM. After this incubation, the cells were washed three times with PBS (pH 7.4) and then incubated with 10 μM DCFH-DA for 30 minutes. The green fluorescence intensity of DCF was observed using CLSM. In addition, 4T1 cells were cultured at a density of 2×10 5 Cells were seeded into 12-well plates at a density of 100 cells / well and cultured for 12 hours. Then, the cells were treated and collected using the same method. The cells were suspended in PBS (pH 7.4) and the intracellular fluorescence of ROS was quantified using FCM. Figure 22 and Figure 23 As shown, it can be seen that the combined treatment group AFS solution and AFS nanoparticles significantly caused an increase in intracellular ROS levels and both were higher than the AF or Sor solution alone treatment group, indicating that AF and Sor can synergistically increase ROS levels, among which AFS nanoparticles caused the most significant increase in ROS levels.

[0131] Example 10

[0132] Effects of AFS nanoparticles on GSH levels, GPX4 inhibition rate, LPOs content and MDA content:

[0133] 4T1 cells in good condition were digested, diluted with culture medium and plated at 2×10 6 The cells were inoculated into the culture dish at a density of 10 cells / dish, and then placed in an incubator and incubated for 12 hours to allow them to adhere. After the cells adhered, they were cultured with a culture medium containing Sor solution, AF solution, AFS solution and AFS nanoparticles, 200 μL per well, and the concentration of AF was 200 nM or the concentration of Sor was 200 nM. After culturing for 4 hours, the cells were washed 3 times with ice PBS, and the cells were collected. The 4T1 cells were suspended in 300 μL PBS and further destroyed by ultrasound. The GSH content detection kit, GPX activity detection kit, LPO content detection kit, and MDA content detection kit were used for detection. The results are shown in the figure. Figures 24-27As shown in the figure, it can be seen that AFS nanoparticles can significantly reduce the intracellular GSH content, inhibit GPX activity, and increase the intracellular LPOs and MDA content, which is attributed to the good synergistic ferroptosis-inducing effect of AF and Sor and the efficient cellular uptake of nanoparticles.

[0134] Example 11

[0135] Pharmacokinetic study of AFS nanoparticles:

[0136] SD rats weighing 200-250 g were randomly divided into groups and fasted for 12 hours before administration, with free access to water. DiR solution and AFS nanoparticles prepared in Example 3 (2 mg / kg of DiR each) were injected intravenously. Blood was collected from the eye sockets at the specified time points and plasma was separated. DiR was then extracted by ultrasonication, centrifugation, and protein precipitation. Finally, the pharmacokinetic behavior of each preparation was detected using a microplate reader (excitation wavelength of 748 nm, emission wavelength of 780 nm). The results are shown in Figure 2. Figure 28 As shown, it can be seen that due to its short half-life, the DiR solution is quickly metabolized and cleared. Compared with the solution, the circulation time of DiR / AFS nanoparticles is significantly prolonged, which significantly improves the AUC of DiR and provides a good foundation for the accumulation of the drug in tumors in the body.

[0137] Example 12

[0138] Tissue distribution experiment of AFS nanoparticles:

[0139] 4T1 cell suspension was inoculated subcutaneously on the ventral side of BALB / c mice. When the tumor volume reached 300 mm 3 At 4h, 8h, 12h, 24h, and 36h after administration, mice were anesthetized and subjected to in vivo imaging analysis. Figure 29 and Figure 30 As shown, the time point with the highest accumulation in each group was selected for fluorescence intensity analysis of isolated tissues and organs. The results are shown in Figure 31 and Figure 32 As shown, it can be seen that the fluorescence intensity of the DiR / AFS group in tumor tissue increased significantly compared with the DiR solution and reached the maximum accumulation at 24 hours.

[0140] Example 13

[0141] In vivo anti-tumor experiments of AFS nanoparticles:

[0142] 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 normal saline, Sor solution, AF solution, AFS solution, and AFS nanoparticles prepared in Example 3, respectively. The drug was administered once every other day for 5 consecutive times, with a dose of 10 mg / kg according to AF 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, kidneys) were collected and fixed with 4% tissue fixative for H&E staining.

[0143] Tumor growth curve, tumor growth photos and tumor-bearing rate results are shown in Figure 2. Figure 33 、 34 As shown in Figures 35 and 40, it can be seen that the antitumor effects of the Sor solution and the AF solution alone were limited, while the AFS solution showed a stronger antitumor effect than the individual solutions, indicating that there is a synergistic antitumor effect between AF and Sor. In addition, the AFS nanoparticles with tumor-specific drug release characteristics showed stronger antitumor activity, and tumor growth was negligible during the entire treatment process. Figures 36-40 As shown, it can be seen that the H&E staining shows that AFS nanoparticles have a strong tumor killing effect ( Figure 36 ); AFS nanoparticles had little effect on the body weight of tumor-bearing mice ( Figure 37 ); After treatment, no obvious abnormalities were found in H&E staining sections of major organs (heart, liver, spleen, lung, and kidney) and in the blood indicators of liver and kidney ( Figure 38 and Figure 39 ); In addition, AFS nanoparticles do not cause hemolysis ( Figure 40 In summary, AFS nanoparticles can be used as an efficient and safe nanoassembly for tumor ferroptosis therapy.

Claims

1. A functional conjugate linker that self-generates ROS, characterized in that: The functional conjugate linker for self-generating ROS is artesunate-ferrocene or dihydroartemisinin-ferrocene, and its structural formula is shown below:

2. A method for preparing a functional conjugate that self-generates ROS, characterized by: Artesunate, ferrocene methanol, catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, or dihydroartemisinin, ferrocenecarboxylic acid, catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in an organic solvent, stirred for reaction, and purified to obtain a functional conjugate linker artesunate-ferrocene or dihydroartemisinin-ferrocene that self-generates ROS.

3. The preparation method according to claim 2, wherein: The organic solvent is dichloromethane; The molar ratio of artesunate or dihydroartemisinin, ferrocenylmethanol or ferrocenylcarbolic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:0.2-5:0.5-4:0.05-0.8; The reaction conditions are stirring at 800 rpm at 15-45° C. for 24-144 hours.

4. A nanoassembly that self-synergistically enhances ferroptosis, characterized by: The nanoassembly is a nanoparticle formed by co-assembling a functional conjugate linker that generates ROS and a ferroptosis inducer through intermolecular forces, or a nanoparticle formed by co-assembling a functional conjugate linker that generates ROS and a ferroptosis inducer that is modified by a PEG modifier through intermolecular forces, wherein: The functional conjugate linker that self-generates ROS is the functional conjugate linker that self-generates ROS according to claim 1, or the functional conjugate linker that self-generates ROS obtained by the preparation method according to claim 2 or 3; The ferroptosis inducing agent is one or more of sorafenib, erastin, sulfasalazine, FIN56, RSL3 or simvastatin; The intermolecular force is one or both of hydrophobic interaction and hydrogen bonding.

5. The nanoassembly according to claim 4, characterized in that: The nano-assembly is uniformly spherical and has a particle size of 160-170 nm.

6. The nanoassembly according to claim 4, characterized in that: The PEG modifier is one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG or PE-PEG; The molecular weight of the PEG modifier is 200 to 20,000; The molar ratio of the functional conjugate linker that self-generates ROS to the ferroptosis inducer is 1:0.1-10; The mass ratio of the sum of the functional conjugate linker that self-generates ROS and the ferroptosis inducer to the PEG modifier is 1:0.1-10.

7. A method for preparing a nanoassembly that self-synergistically enhances ferroptosis, characterized by: The functional conjugate linker that self-generates ROS and the ferroptosis inducer, or the functional conjugate linker that self-generates ROS, the ferroptosis inducer and the PEG modifier are dissolved in an organic solvent respectively, stirred and mixed, and the mixed solution is slowly added dropwise to water to spontaneously form a uniform nanoassembly, and the organic solvent is removed to obtain the nanoassembly.

8. Use of the functional conjugate linker capable of self-generating ROS according to claim 1 or the nanoassembly according to any one of claims 4 to 6 in the preparation of a drug delivery system.

9. Use of the functional conjugate linker capable of self-generating ROS according to claim 1 or the nanoassembly according to any one of claims 4 to 6 in the preparation of anti-tumor drugs.

10. Use of the functional conjugate linker for self-generating ROS according to claim 1 or the nanoassembly according to any one of claims 4 to 6 in the preparation of an injection, oral administration or topical administration system.

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