A sensitizer for ferroptosis lipid nano-regulator and a preparation method and application thereof

Abstract

The application discloses a sensitized ferroptosis lipid nano regulator and a preparation method and application thereof, and belongs to the technical field of new adjuvants and new dosage forms of drug preparation combined treatment. The lipid nano regulator is co-assembled by a GPX4 inhibitor and a FASN inhibitor through intermolecular forces, and is modified with an oxidation-reduction sensitive PEG modifier, a molar ratio of the GPX4 inhibitor and the FASN inhibitor is 10:1-1:10, a mass ratio of the sum of the GPX4 inhibitor and the FASN inhibitor to the PEG modifier is 10:90-90:10, and the intermolecular forces include pi-pi stacking force, hydrophobic force and hydrogen bond. The co-assembled nano preparation provides a new strategy and more choices for the development of drug delivery, and meets the urgent needs of high-efficiency and diverse ferroptosis treatment strategies in preparations in the clinic.

Description

A lipid nanoregulator that enhances ferroptosis, its preparation method and application Technical Field

[0001] This invention belongs to the technical field of new excipients and dosage forms for combination therapy of pharmaceutical preparations, specifically relating to a lipid nanoregulator that sensitizes ferroptosis, its preparation method, and its application. Background Technology

[0002] Cancer remains one of the most serious threats to human health. Despite the application of various therapies in clinical settings, the treatment outcomes for most cancers remain unsatisfactory. In recent years, the discovery of ferroptosis has provided a new perspective on cancer treatment. Ferroptosis is an iron-dependent regulated form of cell death with unique characteristics, including alterations in iron homeostasis, reduced oxidative stress defense, and aberrant lipid peroxidation. Its pathogenesis stems from an imbalance between the production and degradation of intracellular lipid reactive oxygen species (ROS). Currently, various ferroptosis inducers have been reported to induce intracellular oxidative stress and trigger ferroptosis through different pathways. Among them, the System Xc-GSH-GPX4 axis is the central regulatory pathway for ferroptosis, with GPX4 being the most critical and classic regulatory target. Most LPO needs to accumulate on the cell membrane to induce membrane lipid peroxidation, ultimately leading to ferroptosis. Furthermore, ferroptosis signals can propagate between cells, depending on the release of oxidized lipids encapsulated within intact lipid membranes (e.g., extracellular vesicles). Therefore, lipid membrane rupture is generally considered a late event in ferroptosis. Successful induction of ferroptosis hinges on the effective triggering of lipid peroxidation in the cell membrane. Polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) in the cell membrane can make the cell membrane more flexible and promote changes in membrane curvature and morphology. However, excessive PUFA-PLs can trigger a series of reactions that produce lipid reactive oxygen species (ROS), promoting the induction of ferroptosis. Therefore, lipid metabolism can influence cellular susceptibility to ferroptosis by regulating the balance between monounsaturated fatty acid-containing phospholipids (MUFA-PLs) and PUFA-PLs. Lipid metabolism controls phospholipid composition through two main mechanisms: (i) regulating fatty acid (FA) supply through synthesis, uptake, storage, and β-oxidation, and (ii) regulating the expression and synthesis and remodeling of enzymes involved in phospholipids. Fatty acid synthase (FASN) is a key lipid regulatory enzyme that converts acetyl-CoA to palmitic acid. Upregulation of FASN can increase the synthesis of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs). High saturation levels of membrane lipids can protect cancer cells from ROS-induced damage, thereby preventing and repairing lipid peroxidation induced by ferroptosis inducers such as GPX4 inhibitors. Conversely, inhibition of FASN promotes the accumulation of polyunsaturated fatty acids (PUFAs) with multiple double bonds, thereby promoting the induction of lipid peroxidation and ferroptosis.

[0003] However, most GPX4 inhibitors (such as RSL3 and Erastin) have poor water solubility, making efficient co-delivery with lipid-modulating compounds such as FASN inhibitors a significant challenge. With advancements in biotechnology and nanomedicine, there is growing interest in small-molecule nanoassembly strategies based on molecular engineering. Carrier-free small-molecule nanoassemblies (NAs) have attracted attention due to a series of significant advantages compared to traditional nanoformulations. First, they eliminate the need for traditional drug carriers, enhancing drug loading, reducing therapeutic doses, and mitigating potential toxicity and immune responses. Second, small-molecule NAs can integrate multiple functions through molecular engineering strategies, such as enabling visual monitoring, precise control of biocompatibility, and biodegradability. Furthermore, the unique design and precise control of this technology provide a flexible, efficient, and controllable solution for the co-delivery of small-molecule drugs, promising a major breakthrough in the field of drug delivery. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a lipid nanomodulator that enhances ferroptosis, its preparation method, and its application. This solves the technical problems of the GPX4 inhibitor RSL3, such as poor water solubility, poor assembly ability, low drug loading, drug leakage, and excipient-related toxicity due to polymer encapsulation. This invention designs a co-assembled nanoparticle of the FASN inhibitor Orlistat and the GPX4 inhibitor RSL3, thereby achieving high drug loading, good stability, and low toxicity, thus solving the problem of unsatisfactory therapeutic effects of single-drug therapy, improving lipid peroxidation capacity, and enhancing ferroptosis.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] In a first aspect, the present invention provides a lipid nanoregulator that sensitizes ferroptosis, wherein the lipid nanoregulator is co-assembled by a glutathione peroxidase 4 (GPX4) inhibitor and a fatty acid synthase (FASN) inhibitor through intermolecular forces and modified with a PEG modifier.

[0007] Furthermore, the molar ratio of the GPX4 inhibitor to the FASN inhibitor is 10:1 to 1:10; the mass ratio of the sum of the GPX4 inhibitor and the FASN inhibitor to the PEG modifier is 10:90 to 90:10.

[0008] Furthermore, the intermolecular forces include π-π stacking forces, hydrophobic forces, and hydrogen bonds.

[0009] Furthermore, the GPX4 inhibitor includes at least one of RSL3, FIN56, ML162, FINO2, DPI7, or Erastin, and the GPX4 inhibitor is a compound that has the function of inhibiting GPX4 expression.

[0010] Furthermore, the GPX4 inhibitor is preferably RSL3.

[0011] Furthermore, the FASN inhibitor includes at least one of Orlistat, FT113, Cerulenin, C75, or Fasnall, and the FASN inhibitor is a compound that has the function of inhibiting FASN expression.

[0012] Furthermore, the FASN inhibitor is preferably Orlistat.

[0013] Furthermore, the PEG modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, and PE-PEG, wherein the molecular weight of PEG in the PEG modifier is 200-20000.

[0014] Furthermore, the PEG modifier is preferably DSPE-SS-PEG. 2K .

[0015] Furthermore, the molar ratio of RSL3 to Orlistat is 1:5 to 1:10.

[0016] Secondly, this invention provides a method for preparing a lipid nanomodulator that sensitizes ferroptosis, comprising the following steps:

[0017] The GPX4 inhibitor and FASN inhibitor were dissolved in organic solvents respectively. The mixed solution was slowly added dropwise to deionized water, and co-assembled nanoparticles were spontaneously formed under stirring. The organic solvent containing the PEG modifier was added dropwise to the co-assembled nanoparticles under stirring. The organic solvent was then removed to obtain the final product.

[0018] Furthermore, the stirring speed is 200-2000 rpm.

[0019] Furthermore, the organic solvent is one or a combination of two or more of anhydrous ethanol, tetrahydrofuran, and dimethyl sulfoxide.

[0020] Furthermore, the organic solvent is preferably a mixture of tetrahydrofuran and anhydrous ethanol.

[0021] Furthermore, the method for removing organic solvents includes solvent evaporation, ultrafiltration, or membrane permeation.

[0022] Thirdly, this invention provides the application of lipid nanomodulators that sensitize ferroptosis in the preparation of antitumor drugs.

[0023] Fourthly, this invention provides the application of lipid nanomodulators that sensitize ferroptosis in the preparation of drug delivery systems.

[0024] Fifthly, the present invention provides the application of lipid nanomodulators that sensitize ferroptosis in the preparation of injectable, oral, or topical drug delivery systems.

[0025] The advantages of this invention over the prior art are as follows:

[0026] 1. This invention prepares a co-assembled nanoparticle formed by a FASN inhibitor and a GPX4 inhibitor, which can be used for tumor treatment. RSL3 can effectively inhibit the expression of GPX4 in tumors, and Orlistat can effectively inhibit the expression of FASN, thereby enhancing the efficacy of ferroptosis.

[0027] 2. The FASN inhibitor and GPX4 inhibitor co-assembled nanoparticles of the present invention achieve high drug loading, good stability and low toxicity, meeting the urgent clinical need for highly effective and low-toxicity formulations. This provides a new strategy for the assembly of isomorphic synergistic drug nanoparticles and an effective nanoplatform for the development of carrier-free hybrid nanoassemblies and ferroptosis combination therapy. Attached Figure Description

[0028] Figure 1 shows the particle size distribution and transmission electron microscopy image of the nanoparticles prepared in Example 1 of the present invention; wherein, A is RO nanoparticles; B is sp-RO nanoparticles.

[0029] Figure 2 shows the PBS stability of RO nanoparticles and sp-RO nanoparticles in Example 2 of the present invention.

[0030] Figure 3 shows the stability of sp-RO nanoparticles in PBS (10% FBS) in Example 2 of this invention.

[0031] Figure 4 is a molecular docking diagram of RSL3 and Orlistat in Example 3 of the present invention.

[0032] Figure 5 shows the disruption of the interaction forces of the non-PEGylated nanoparticles in PBS containing sodium chloride, sodium dodecyl sulfate, and urea in Example 3 of the present invention.

[0033] Figure 6 shows confocal microscopy images of the Cy7 solution and Cy7-sp RO nanoparticles after 0.5 hours and 2 hours of cell uptake in Example 4 of this invention.

[0034] Figure 7 shows the flow cytometry results of cellular uptake of Cy7 solution and Cy7-sp RO nanoparticles in Example 4 of the present invention.

[0035] Figure 8 shows the cytotoxicity results of Example 5 of the present invention; where A is the cytotoxicity result of Orlistat solution; and B is the cytotoxicity result of RSL3 solution, RO solution and sp-RO nanoparticles.

[0036] Figure 9 shows the cytotoxicity results of RSL3 solution, RO solution and sp-RO nanoparticles after adding different concentrations of ferroptosis inhibitor (Fer-1) in Example 6 of the present invention.

[0037] Figure 10 shows the changes in glutathione levels in Orlistat solution, RSL3 solution, RO solution, and sp-RO nanoparticles in Example 7 of the present invention.

[0038] Figure 11 shows the malondialdehyde level changes of Orlistat solution, RSL3 solution, RO solution and sp-RO nanoparticles in Example 8 of the present invention.

[0039] Figure 12 is a confocal image of the reactive oxygen species levels of Orlistat solution, RSL3 solution, RO solution and sp-RO nanoparticles in Example 9 of the present invention.

[0040] Figure 13 shows the flow cytometry results of the reactive oxygen species levels of Orlistat solution, RSL3 solution, RO solution and sp-RO nanoparticles in Example 9 of the present invention.

[0041] Figure 14 shows the cellular protein imprinting results of Orlistat solution, RSL3 solution, RO solution and sp-RO nanoparticles in Example 10 of the present invention.

[0042] Figure 15 is a blood drug concentration-time curve of Cy7 solution and Cy7-sp RO nanoparticles in Example 11 of the present invention.

[0043] Figure 16 shows in vivo imaging of mice given Cy7 solution and Cy7-sp RO nanoparticles in Example 12 of this invention.

[0044] Figure 17 is a fluorescence imaging image of an isolated mouse organ given Cy7 solution and Cy7-sp RO nanoparticles in Example 12 of the present invention.

[0045] Figure 18 shows the quantitative fluorescence spectroscopy of isolated mouse organs treated with Cy7 solution and Cy7-spRO nanoparticles in Example 12 of this invention.

[0046] Figure 19 is a photograph of an ex vivo tumor in Example 13 of the present invention.

[0047] Figure 20 is a mouse tumor growth curve in the in vivo anti-tumor experiment of Example 13 of the present invention.

[0048] Figure 21 is a statistical chart of the tumor bearing rate in mice in the in vivo anti-tumor experiment of Example 13 of the present invention.

[0049] Figure 22 shows the H&E staining of mouse tumors in an in vivo antitumor experiment in Example 13 of this invention.

[0050] Figure 23 shows the changes in mouse body weight in the in vivo anti-tumor experiment in Example 13 of the present invention.

[0051] Figure 24 is a histopathological section of tissue in Example 13 of the present invention.

[0052] Figure 25 shows the results of liver and kidney function analysis in Example 13 of the present invention.

[0053] Figure 26 is a Western blot analysis of mouse tumor proteins in the in vivo anti-tumor experiment of Example 14 of the present invention. Detailed Implementation

[0054] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. The embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0055] Example 1: Screening the sensitizing dose ratio of RSL3 and Orlistat

[0056] Different molar ratios of RSL3 (a GSH peroxidase 4 inhibitor) and orlistat were dissolved in a 1:1 volume ratio of tetrahydrofuran and anhydrous ethanol. Under magnetic stirring, 400 μL of this solution was slowly added dropwise to 2 mL of deionized water. RSL3 and orlistat spontaneously formed uniform nanoparticles. The organic solvent in the nanoparticles was then removed by rotary evaporation at 30 °C to obtain solvent-free co-assembled nanoparticles. An organic solvent containing PEG modifier (tetrahydrofuran and anhydrous ethanol, 1:1 volume ratio) was then added dropwise to the co-assembled nanoparticles under magnetic stirring. After removing the organic solvent, sp-RO nanoparticles were obtained.

[0057] The particle size, particle size distribution, and IC50 of the prepared sp-RO nanoparticles were measured. 50 The values ​​are shown in Table 1.

[0058] Table 1. Particle size, particle size distribution, and IC50 of sp-RO nanoparticles 50 value

[0059]

[0060] As shown in Table 1, the particle size of sp-RO nanoparticles ranges from 160 to 210 nm. When RSL3:Orlistat = 1:5, the distribution of sp-RO nanoparticles is more uniform, and the IC50 concentration is higher. 50 The value is the smallest. Therefore, the initial preferred ratio of RSL3 to Orlistat is 1:5.

[0061] (1) Preparation method of non-PEGylated RO nanoparticles: Accurately weigh 0.5 mg RSL3 and 2.811 mg Orlistat (mol:mol = 1:5), dissolve them in 400 μL tetrahydrofuran and anhydrous ethanol (volume ratio 1:1), and slowly add the solution dropwise to 2 mL deionized water under magnetic stirring to spontaneously form uniform nanoparticles. Then, under 30 °C, remove the organic solvent in the nano-formulation by rotary evaporation to obtain a nano-formulation without any organic solvent, which is denoted as RO nanoparticles.

[0062] (2)DSPE-SS-PEG 2K Preparation method of modified sp-RO nanoparticles: 0.5 mg RSL3 and 2.811 mg Orlistat (mol:mol = 1:5) were dissolved in 400 μL tetrahydrofuran and anhydrous ethanol (volume ratio 1:1) to obtain an RO solution; 1.00 mg DSPE-SS-PEG was accurately weighed. 2K Dissolved in tetrahydrofuran to form 20 mg / mL DSPE-SS-PEG 2K Mother liquor. Mix 400 μL of DF solution with 29.2 μL of DSPE-SS-PEG. 2K The mother liquor was slowly added dropwise to 2 mL of deionized water under stirring to obtain uniform nanoparticles. Then, at 30 °C, the organic solvent in the nanoparticle formulation was removed by rotary evaporation to obtain a nanoparticle formulation free of any organic solvent, denoted as sp-RO nanoparticles. The particle size, particle size distribution, and zeta potential of the prepared RO nanoparticles and sp-RO nanoparticles were determined by dynamic light scattering.

[0063] (3)DSPE-SS-PEG 2K Preparation method of Cy7-modified sp-RO nanoparticles: 0.5 mg RSL3 and 2.811 mg Orlistat (mol:mol = 1:5) were dissolved in 400 μL tetrahydrofuran and anhydrous ethanol (volume ratio 1:1) to obtain an RO solution; 1.00 mg DSPE-SS-PEG was accurately weighed. 2K -Cy7, dissolved in tetrahydrofuran to form 20 mg / mL DSPE-SS-PEG 2K -Cy7 stock solution. Add 400 μL of RO solution to 36.5 μL of SPE-SS-PEG.2K Cy7 mother liquor was slowly added dropwise to 2 mL of deionized water under stirring to obtain uniform nanoparticles. Then, at 30 °C, the organic solvent in the nanoparticle formulation was removed by rotary evaporation to obtain a nanoparticle formulation free of any organic solvent, denoted as Cy7-sp RO nanoparticles.

[0064] The particle size and morphology of the RO nanoparticles and sp-RO nanoparticles prepared above were determined by transmission electron microscopy. The results are shown in Figure 1. The transmission electron microscopy images show that both RO nanoparticles and sp-RO nanoparticles are uniform spherical with a particle size of about 170 nm.

[0065] Example 2: Stability test of nanoparticles

[0066] One mL of the RO nanoparticles and sp-RO nanoparticles prepared in Example 1 were added to 9 mL of PBS (pH 7.4) and incubated in a shaker at 37°C. The particle size change was measured at predetermined time points using dynamic light scattering (LPLS). The stability of the nanoparticles was evaluated by measuring the particle size change using LPLS. The results are shown in Figure 2. The non-PEG-modified RO nanoparticles were unstable in PBS. In contrast, the sp-RO nanoparticles exhibited better stability in PBS.

[0067] One mL of the sp-RO nanoparticles prepared in Example 1 was added to 9 mL of PBS (pH 7.4) containing 10% FBS and incubated in a shaker at 37°C. The particle size change was measured at predetermined time points using dynamic light scattering. The stability of the nanoparticles was evaluated by measuring the particle size change using dynamic light scattering. The results are shown in Figure 3, indicating that the sp-RO nanoparticles also showed good stability in PBS containing 10% FBS.

[0068] Example 3: Assembly Mechanism Analysis of RSL3 and Orlistat

[0069] Computer simulations were used to explore the assembly mechanism of RSL3 and Orlistat in sp-RO nanoparticles. Molecular docking calculations were performed using the Vina scheme of the Infineon Cloud Computing Platform. Compounds RSL3 and Orlistat underwent energy minimization under an MMFF94 force field to obtain 3D structures, forming stable nanoassemblies. Semi-flexible docking was performed using the AutoDock Vina program, and force disruption was achieved using sodium chloride, sodium dodecyl sulfate, and urea. The results are shown in Figures 4 and 5. Multiple forces exist between RSL3 and Orlistat molecules, such as π-π stacking forces, hydrophobic interactions, and hydrogen bonding. These forces make a significant contribution to the co-assembly of RSL3 and Orlistat.

[0070] Example 4: Cellular uptake of nanoparticles

[0071] Confocal microscopy and flow cytometry were used to qualitatively and quantitatively determine the uptake of Cy7-sp RO nanoparticles in 4T1 cells (mouse breast cancer cells) using Cy7 solution. The qualitative procedure involved injecting 4T1 cells at 5 × 10⁻⁶ ppm. 4 Cells were seeded at a density of 10 cells / well in 24-well plates and incubated for 12 hours to allow for cell adhesion. After adhesion, Cy7 solution or Cy7-spRO nanoparticles (Cy7 concentration 10 μM) were added. Cells were incubated at 37°C for 0.5 hours and 2 hours, respectively. Cells were then washed with cold PBS, fixed, and finally analyzed using confocal microscopy to assess cell uptake of the various reagents. The results are shown in Figure 6. Quantitative analysis involved seeding 4T1 cells at a density of 2 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of 100 cells / well in 12-well plates and incubated for 12 hours to allow them to adhere. After cell adhesion, Cy7 solution or Cy7-sp RO nanoparticles were added, with a Cy7 concentration of 10 μM. After incubation at 37°C for 0.5 hours and 2 hours, the cells were washed, digested, and collected. Finally, flow cytometry was used to analyze the uptake of various reagents by the cells. The experimental results are shown in Figure 7.

[0072] The experimental results above indicate that cellular uptake of the formulation is time-dependent, and that Cy7-sp RO nanoparticles exhibit higher intracellular fluorescence intensity than cells treated with Cy7 solution. Therefore, Cy7-sp RO nanoparticles demonstrate higher cellular uptake efficiency than Cy7 solution.

[0073] Example 5: Cytotoxicity of Nanoparticles

[0074] The MTT assay was used to investigate the cytotoxicity of Orlistat solution, RSL3 solution, RO solution, and sp-RO nanoparticles against mouse breast cancer (4T1) cells. Cells in good condition were digested and diluted with culture medium to a concentration of 1 × 10⁻⁶. 4Cells were collected at a density of [number] cells / mL, and after homogenization, 200 μL of cell suspension was added to each well of a 96-well plate. The plates were incubated for 12 h to allow cell adhesion. After cell adhesion, 200 μL of medium containing Orlistat solution, RSL3 solution, RO solution, or sp-RO nanoparticles was added to each well. The control group was cultured in medium without the drug. The 96-well plate was then removed, and 25 μL of 5 mg / mL MTT solution was added to each well. After incubation for 4 h, the drug solution was discarded. The 96-well plate was inverted on filter paper to thoroughly absorb any remaining liquid. 200 μL of DMSO was added to each well, and the plate was shaken for 10 min to dissolve the blue-purple crystals. The absorbance of each well was measured at 490 nm using a microplate reader after zeroing. The cytotoxicity results are shown in Figure 8. In 4T1 cells, Orlistat solution at concentrations of 0-300 nM exhibited very weak cytotoxicity. Compared with the free drug alone, both the RO solution and sp-RO nanoparticles showed synergistic effects, with a more significant inhibitory effect on cell growth. sp-RO nanoparticles showed the most potent antitumor activity in vitro, which is attributed to their efficient cellular uptake and highly effective synergistic tumor killing.

[0075] Example 6: Validation of the ferroptosis pathway

[0076] Fer-1 was used as an inhibitor of ferroptosis, which affects cytotoxicity results by scavenging lipid peroxides, thus validating the occurrence of ferroptosis. Ferroptosis was verified using the MTT assay; healthy 4T1 cells were digested and diluted with culture medium to 1×10⁻⁶ cells. 4 Cells were collected at a density of [number] cells / mL, and after homogenization, 200 μL of cell suspension was added to each well of a 96-well plate. The plates were incubated for 12 h to allow cell adhesion. After cell adhesion, 200 μL of culture medium containing RSL3 solution, RO solution, or sp-RO nanoparticles was added to each well, followed by the addition of Fer-1 (0, 5, and 10 μM). The control group was cultured in culture medium without the drug. The 96-well plate was then removed, and 25 μL of 5 mg / mL MTT solution was added to each well. After incubation for 4 h, the drug solution was discarded. The 96-well plate was inverted on filter paper to thoroughly absorb any remaining liquid. 200 μL of LDMSO was added to each well, and the plate was shaken for 10 min to dissolve the blue-purple crystals. The absorbance of each well was measured at 490 nm using a microplate reader after zeroing.

[0077] The experimental results are shown in Figure 9. After 4T1 cells treated with RSL3 solution, RO solution, and sp-RO nanoparticles were co-treated with Fer-1, the cell survival rate was significantly improved, which verified that RSL3 and Orlistat induced ferroptosis to effectively kill tumor cells.

[0078] Example 7: Glutathione Detection

[0079] Healthy 4T1 cells were digested, diluted with culture medium, and then... 6 Cells were seeded at a density of 100 cells / dish and incubated in an incubator for 12 hours to allow them to adhere. After cell adhesion, the cells were cultured in medium containing Orlistat solution, RSL3 solution, RO solution, or sp-RO nanoparticles, 200 μL per well, with RSL3 concentration of 20 μM or Orlistat concentration of 100 μM. The control group was cultured in medium without the drug. After 4 hours of culture, the cells were washed three times with ice-cold PBS and collected. 4T1 cells were resuspended in 300 μL PBS and further disrupted by sonication. 100 μL of the disrupted cells were mixed with 100 μL of GSH probe precipitant, centrifuged at 3500 rpm for 10 min, and the supernatant was collected for detection according to the GSH detection kit instructions. The UV absorbance at 405 nm was measured using a multi-mode microplate reader.

[0080] As shown in Figure 10, incubation with formulations containing RSL3 (RSL3 solution, RO solution, and sp-RO nanoparticles) resulted in a decrease in intracellular GSH levels in 4T1 cells compared to the control group. The sp-RO nanoparticles showed a significant advantage over the other formulations in terms of GSH consumption.

[0081] Example 8: Malondialdehyde Detection

[0082] Healthy 4T1 cells were digested, diluted with culture medium, and then... 6 Cells were seeded at a density of 100 cells / dish and incubated in an incubator for 12 hours to allow them to adhere. After cell adhesion, the cells were cultured in medium containing Orlistat solution, RSL3 solution, RO solution, or sp-RO nanoparticles, 200 μL per well, with RSL3 concentration of 20 μM or Orlistat concentration of 100 μM. The control group was cultured in medium without the drug. After 4 hours of culture, the cells were washed three times with ice-cold PBS and collected. 4T1 cells were resuspended in 300 μL of extraction buffer and further destroyed by sonication. The cells were centrifuged at 8000 rpm for 10 min, and the supernatant was collected for detection according to the MDA assay kit instructions. The UV absorbance at 532 nm and 600 nm was measured using a multi-mode microplate reader.

[0083] As shown in Figure 11, incubation with formulations containing RSL3 (RSL3 solution, RO solution, and sp-RO nanoparticles) resulted in increased MDA levels in 4T1 cells compared to the control group. The sp-RO nanoparticles showed a significant advantage in MDA generation compared to the other formulations.

[0084] Example 9: Intracellular ROS Measurement

[0085] The DCFH-DA probe was used to detect intracellular ROS levels. In fact, DCFH-DA is a general indicator of oxidative stress and does not show fluorescence, but intracellular esterases and ROS can oxidize non-fluorescent DCFH-DA to DCFH and fluorescent DCF. First, 4T1 cells were... 4 4T1 cells were seeded at a density of 100 cells / well in 24-well plates for 12 h. Then, 4T1 cells were cultured in medium containing Orlistat solution, RSL3 solution, RO solution, or sp-RO nanoparticles, 200 μL per well, with RSL3 at a concentration of 20 μM or Orlistat at a concentration of 100 μM. The control group was cultured in medium without the drug. After co-incubation with 4T1 cells for 4 h, the cells were incubated with DCFH-DA (10 μM) for 30 min, and the fluorescence of DCF was detected by confocal microscopy. Furthermore, 4T1 cells were treated and collected using the same method. The cells were resuspended in PBS (pH 7.4), and the fluorescence of intracellular ROS was detected by flow cytometry.

[0086] As shown in Figures 12-13, compared with the single solution, the combined treatment group of RO solution and sp-RO nanoparticles induced higher levels of ROS. Among them, the sp-RO nanoparticle light irradiation group caused the most significant ROS production.

[0087] Example 10: In vitro protein imprinting experiment

[0088] The expression of GPX4 and FASN proteins in cells was investigated using proteoblotting. Culture dishes containing confluent 4T1 cells were treated with blank medium, Orlistat solution, RSL3 solution, RO solution, or sp-RO nanoparticles. Cells were then lysed using RIPA lysis buffer. Protein concentrations in cells were determined using the BCA assay (Invitrogen, CA). Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF. Primary antibody was added and the cells were incubated overnight at 4°C. After incubation with secondary antibody, ECL Western blotting substrate was added to visualize the protein bands.

[0089] As shown in Figure 14, the groups containing RSL3 significantly downregulated GPX4 expression levels; the groups containing Orlistat significantly downregulated FASN expression levels. Furthermore, sp-RO nanoparticles exhibited a clear advantage in downregulating the expression of both GPX4 and FASN.

[0090] Example 11: Pharmacokinetic Study of Nanoparticles

[0091] SD rats weighing 180-220g were randomly divided into groups and fasted for 12 hours before drug administration, with free access to water. Cy7 solution and Cy7-sp RO nanoparticles (both at a dose of 2 mg / kg Cy7) were administered intravenously. Blood samples were collected from the orbital sinus at specified time points, and plasma was obtained. Cy7 was then extracted using a protein precipitation method, and the pharmacokinetic behavior of each formulation was detected using an ELISA reader (excitation 750nm, emission 773nm). The experimental results are shown in Figure 15. Due to its short half-life, the Cy7 solution was rapidly metabolized and cleared. Compared to the solution, the nanoparticles exhibited a significantly prolonged circulation time, laying the foundation for efficient drug accumulation at the tumor site.

[0092] Example 12: Nanoparticle Organization and Distribution Experiment

[0093] 4T1 cell suspension was subcutaneously inoculated into the ventral side of BALB / c mice. When the tumor volume reached 300 mm², the cells were cultured. 3 At the same time, Cy7 solution and Cy7-sp RO nanoparticles were administered via tail vein injection (both at a dose of 2 mg / kg Cy7). Mice were anesthetized at 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration for in vivo imaging analysis. The results are shown in Figure 16. The time points with the strongest in vivo fluorescence signals in each group were selected for fluorescence intensity analysis of isolated tissues and organs, and the results are shown in Figures 17-18.

[0094] The above results indicate that, compared with Cy7 solution, Cy7-sp RO nanoparticles significantly increased fluorescence intensity in tumor tissues and reached maximum accumulation at 4 h.

[0095] Example 13: In vivo antitumor experiment of nanoparticles

[0096] 4T1 cell suspension (10 7 100 μL of cells per 100 μL were subcutaneously injected into the ventral side of female BALB / c mice. The tumors were allowed to grow to 100 mm². 3 Mice were randomly divided into groups of five, and administered physiological saline, Orlistat solution, RSL3 solution, RO solution, or sp-RO nanoparticles prepared in Example 1, respectively. Administered once every other day for five consecutive days, with a dose of 5 mg / kg of RSL3. Mice were observed for survival, weighed, and tumor volume measured daily. One day after the last administration, mice were sacrificed, and organs and tumors were harvested for further analysis and evaluation. Major organs (heart, liver, spleen, lung, and kidney) were collected and fixed with 4% tissue fixative for H&E staining.

[0097] Figures 19-21 show the in vitro tumor images, tumor growth curves, and tumor bearing rates of the 4T1 tumor model mice after treatment. There was no significant difference in tumor volume between the Orlistat solution group and the saline group, while the RO solution group showed a slight increase compared to the single solution, indicating a synergistic anti-tumor effect between RSL3 and Orlistat. Furthermore, as expected, sp-RO nanoparticles with longer tumor-specific accumulation exhibited the strongest tumor-inhibiting effect, demonstrating their excellent in vivo tumor-killing properties.

[0098] As shown in Figure 22, H&E staining also revealed the potent tumor-killing effect of sp-RO nanoparticles. A preliminary investigation into its therapeutic safety was also conducted. Throughout the treatment process, weight changes were negligible (Figure 23). As shown in Figures 24-25, no significant abnormalities were observed in liver and kidney blood indicators after multiple treatments.

[0099] Example 14: Western blot experiment on tumor tissue

[0100] Western blot was used to investigate the expression levels of GPX4 and FASN in tumor tissues. Approximately 100 mg of tumor tissue treated with different formulations was weighed, and the total cellular protein concentration was quantified using the BCA method. Equal volumes of protein samples were subjected to SDS-PAGE electrophoresis and electrophoretic transfer. The membranes were then blocked with 5% skim milk for 1 hour. Next, PVDF membranes were incubated overnight at 4°C with rabbit monoclonal antibodies against GPX4, FASN, and β-actin, respectively. After incubation with secondary antibodies at 37°C for 1 hour, ECL chromogenic buffer was added to visualize the protein bands.

[0101] As shown in Figure 26, the expression of GPX4 and FASN in the sp-RO nanoparticle group was significantly inhibited, which is attributed to the better stability, prolonged blood circulation and enhanced tumor-specific accumulation of sp-RO nanoparticles.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid nanomodulator that sensitizes ferroptosis, characterized in that, The lipid nanomodulator is co-assembled from the GPX4 inhibitor RSL3 and the FASN inhibitor Orlistat via intermolecular forces and modified with the PEG modifier DSPE-SS-PEG. 2K The molar ratio of the GPX4 inhibitor RSL3 and the FASN inhibitor Orlistat is 1:5; the sum of the GPX4 inhibitor and the FASN inhibitor is mixed with the PEG modifier DSPE-SS-PEG. 2K The mass ratio is 10:90 to 90:

10.

2. The lipid nanomodulator for sensitizing ferroptosis as described in claim 1, characterized in that, The intermolecular forces include π-π stacking forces, hydrophobic forces, and hydrogen bonds.

3. The method for preparing the lipid nanomodulator that sensitizes ferroptosis according to any one of claims 1-2, characterized in that, The process includes the following steps: dissolving the GPX4 inhibitor and the FASN inhibitor separately in an organic solvent, slowly adding the mixed solution dropwise to deionized water, and spontaneously forming co-assembled nanoparticles under stirring; adding an organic solvent containing the PEG modifier dropwise to the co-assembled nanoparticles under stirring; and removing the organic solvent to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The organic solvent is one or a combination of two or more of anhydrous ethanol, tetrahydrofuran, and dimethyl sulfoxide.

5. The use of the lipid nanomodulator that sensitizes ferroptosis as described in any one of claims 1-2, or the nanoassembly prepared by the preparation method described in claim 3 or 4, in the preparation of anti-breast cancer drugs.

6. The application of the lipid nanomodulator with sensitizing ferroptosis as described in any one of claims 1-2, and the nanoassembly prepared by the preparation method of claim 3 or 4, in the preparation of a drug delivery system.

7. The use of the lipid nanomodulator that sensitizes ferroptosis according to any one of claims 1-2 or the nanoassembly prepared by the preparation method according to claim 3 or 4 in the preparation of injectable and oral administration.

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