Albumin nanoparticles encapsulating iron death-related drugs and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine material preparation, and particularly relates to albumin nanoparticles loaded with ferroptosis related drugs and a preparation method thereof. The ferroptosis related drug composition comprises porphyrin drugs, unsaturated fatty acid drugs-cystamine and sulfosuccinimidyl oleate (SSO). The porphyrin drugs, the unsaturated fatty acid drugs-cystamine and the SSO are slowly added dropwise into an albumin solution under stirring, the drugs enter the hydrophobic cavities of the albumin, and uniform nanoparticles are spontaneously formed. The obtained nanoparticles are small in size and uniform, can efficiently deliver drugs through passive targeting mediated by the EPR effect and active targeting mediated by the Gp60-Caveola-SPARC pathway, improve stability, reduce accumulation at non-target sites and systemic toxicity, amplify the effect of ferroptosis after combination compared with the ferroptosis related drugs used alone, and thus produce a synergistic anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation technology, and relates to a method for constructing and applying green nanoparticles containing human serum albumin-encapsulated ferroptosis-related drugs. Specifically, it relates to a method for constructing and applying green nanoparticles containing human serum albumin-encapsulated ferroptosis-related drugs to antitumor applications. Background Technology

[0002] Cancer is a highly malignant, rapidly progressing disease with a poor prognosis, and is the second leading cause of death worldwide. Because tumor cells rely more on iron than normal cells to promote their high proliferation rate, the importance of ferroptosis in cancer treatment has been increasingly recognized. Ferroptosis, a strategy for regulating cell death in an iron-dependent manner mediated by ROS accumulation, lipid peroxidation, and GSH depletion, holds great potential in cancer treatment. Ferroptosis mainly occurs in two modes: one is the typical ferroptosis-inducing mode, which disrupts the membrane protection mechanisms of tumor cells, activating siderophores, primarily managed by the GPX4 pathway. GPX4 protein is considered the only glutathione peroxidase (GPX) family molecule capable of protecting biological membranes from hydrogen peroxidation. There are two main therapeutic approaches targeting the GPX4 pathway to induce ferroptosis. The first is the inactivation and depletion of GPX4 protein. For example, (1S,3R)-RSL3 (RSL3) can covalently bind to the active site of GPX4, thereby mediating GPX4-regulated siderophore. The second approach is to deplete intracellular GSH levels. Reduced glutathione, a tripeptide form of an antioxidant, can act as a co-molecule with GPX4, assisting GPX4 in eliminating lipid ROS. Another major mechanism involves leveraging intracellular iron availability and lipid peroxidation levels. A second, non-classical mechanism is initiated by increasing Fe2+ levels in tumor cells. For example, overactivation of HMOX1 protein, downregulation of iron transporter expression, and upregulation of transferrin expression can lead to Fe2+ overload in tumor cells. Some exogenous iron-containing substances (heme chloride, FeCl2, and (NH4)2Fe(SO4)2) can cause intracellular iron overload, effectively inducing ferroptosis. Therefore, a combination of consuming intracellular GSH to reduce GPX4 protein expression, exogenous iron causing intracellular iron overload, and exacerbating intracellular lipid peroxidation levels, along with HMOX1 activation, can maximize the therapeutic effect of ferroptosis. Due to the synergistic effect of GPX4 protein downregulation and intracellular Fe2+ overload, it effectively increases intracellular lipid ROS content, induces ferroptosis, and upregulates the expression of intracellular BID, AIF, and EndoG proteins, exhibiting excellent anti-tumor efficacy in vitro.

[0003] Simultaneously, the development and progression of malignant tumors are accompanied by dysfunction of the body's immune system and dysregulation of immune surveillance. Tumor immunotherapy, by actively or passively reactivating the immune system and restoring its immune surveillance function to control and eliminate tumors, represents a revolutionary breakthrough in the field of tumor treatment. During ferroptosis therapy, the significant downregulation of the GPX4 system at the tumor site leads to overexpression of the CD36 receptor on the surface of T cells. Excessive intake of fatty acids causes lipid peroxidation, resulting in ferroptosis of T cells, further promoting immune escape from tumor cells and affecting the therapeutic effect of ferroptosis. CD36 is a scavenger receptor expressed in various cell types. In the immune system, CD36 mediates the acquisition and presentation of dendritic cell antigens and supports regulatory T cell function. Studies have shown that targeting CD36 may be an effective strategy to improve the anti-tumor efficacy of CD8+ T cell immunotherapy, providing new ideas and possibilities for clinical treatment. CD36, through the uptake of fatty acids in the tumor environment, especially polyunsaturated fatty acids—arachidonic acid—leads to the accumulation of lipid peroxides in CD8+ T cells, increased iron ion content, increased ferroptosis, and decreased secretion of cytotoxic cytokines. By blocking CD36 expression or adding inhibitors of ferroptosis, the content of lipid peroxides in CD8+ T cells can be significantly reduced, the expression of cytotoxic secretory molecules can be increased, and the function of immune cells can be restored.

[0004] As a natural biomaterial, human serum albumin (HSA) is an excellent nanomedicine carrier. It can encapsulate drugs using its functional groups and hydrophobic binding sites, possessing advantages such as non-hemolyticity, surface modifiability, biodegradability, good stability, long in vivo half-life, and safety and non-toxicity. Furthermore, it can efficiently deliver drugs through EPR-mediated passive targeting. Drugs are encapsulated in albumin's hydrophobic regions via electrostatic interactions, π-π conjugation, and hydrophobic interactions to form nanoparticles. Albumin prevents the nanosystem from being phagocytosed by the endothelial reticulum, enabling long-term circulation of the nanoformulation. This maximizes drug entry into cells, synergistically inducing ferroptosis and activating anti-tumor immunotherapy effects through multiple mechanisms. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a safe, non-toxic, stable, composite nano-sized albumin nanoparticle carrying ferroptosis-related drugs, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first technical objective of this invention is to provide an albumin nanoparticle loaded with a ferroptosis-related drug, wherein the ferroptosis-related drug and albumin are assembled into nanoparticles, and the ferroptosis-related drug and albumin are co-assembled into drug-loaded nanoparticles through electrostatic interactions, π-π stacking and hydrophobic interactions.

[0008] The ferroptosis-related drug composition includes a porphyrinated drug, an unsaturated fatty acid drug cystamine, and sulfosuccinimide oleate SSO, and the molar ratio of the porphyrinated drug, the unsaturated fatty acid drug cystamine, and SSO is 4:1:1 to 1:4:1.

[0009] The formation mechanism of the albumin-encapsulated ferroptosis-related drug green nanoparticles in this invention:

[0010] Through stirring technology, because albumin contains functional groups such as amino groups and hydrophobic binding sites, the selected porphyrinated drugs, unsaturated fatty acid drugs - cystamine and SSO, as hydrophobic small molecules, will approach the hydrophobic sites in the albumin molecule, ultimately forming an albumin nanoparticle with the ferroptosis-related drug as its core. The hydrophilic groups of the albumin molecule are exposed in the aqueous solution. At the same time, because the porphyrinated drugs, unsaturated fatty acid drugs - cystamine and SSO molecules contain multiple groups, including carboxyl groups and double bonds, as well as hydrophobic forces, they can form a compact nanoparticle with the albumin molecule.

[0011] Furthermore, the albumin nanoparticles are uniform in size, with a particle size of 100–200 nm and a zeta potential of -5 to -30 mV.

[0012] The second technical objective of this invention is to provide a method for preparing albumin nanoparticles loaded with ferroptosis-related drugs as described above, comprising the following steps:

[0013] 1) Prepare an aqueous solution of albumin with a volumetric concentration of 0.1–10 mg / ml to obtain solution A:

[0014] 2) Solution B is prepared by using an organic compound as a solvent to prepare a solution of porphyrin-based drugs with a volume mass concentration of 1–20 mg / ml; solution C is prepared by using an organic compound as a solvent to prepare a solution of unsaturated fatty acid drug-cystamine with a volume mass concentration of 4–40 mg / ml; and solution D is prepared by using an organic compound as a solvent to prepare a solution of sulfosuccinimide oleate SSO with a volume mass concentration of 0.5–15 mg / ml.

[0015] 3) Mix sterile double-distilled water with solution A and stir at 500-1000 rpm for 5-20 min to obtain solution E; mix solutions B, C and D and sonicate for 5-10 min to obtain solution F; then add solution F dropwise to solution E and mix, stir at 500-1000 rpm for 5-20 min, and let stand at room temperature for 10-30 min to obtain solution G;

[0016] 4) Transfer the solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 24-72 hours to remove free drugs and organic solvents, thereby obtaining the albumin nanoparticles loaded with ferroptosis-related drugs.

[0017] Optionally, the porphyrinized drug is one or a combination of heme and dihydroporphyrin, and the solvent for the porphyrinized drug, the unsaturated fatty acid drug cystamine, and SSO is one or a combination of toluene, xylene, dimethyl sulfoxide, tetrahydrofuran, and chlorobenzene.

[0018] Optionally, the unsaturated fatty acid drug includes one or more combinations of oleic acid, linoleic acid, and linolenic acid, and the preparation method of linoleic acid-cystamine in the unsaturated fatty acid drug-cystamine includes the following steps:

[0019] Linoleic acid and catalyst 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride were dissolved in an organic solvent to obtain solution 1; cystamine was dissolved in an organic solvent to obtain solution 2; solution 2 was added dropwise to solution 1 and heated to react to obtain reaction solution; after adding silica gel powder to the reaction solution and evaporating to dryness, the solution was purified by silica gel column chromatography using a mixed solvent of dichloromethane and methanol as eluent to obtain linoleic acid-cystamine;

[0020] The organic solvent is one or a combination of ultra-dry pyridine, ultra-dry N,N-dimethylformamide, ultra-dry dichloromethane, and ethyl acetate.

[0021] Optionally, the molar ratio of the porphyrinated drug, the unsaturated fatty acid drug cystamine, and SSO is 4:1:1 to 1:4:1.

[0022] A third technical objective of this invention is to provide the application of albumin nanoparticles containing ferroptosis-related drugs as described above in pharmaceutical formulations.

[0023] Specifically, the application of albumin nanoparticles loaded with ferroptosis-related drugs described in this invention in anti-triple-negative breast cancer drugs.

[0024] As can be seen from the above technical solutions, this invention has discovered that multimodal ferroptosis has a synergistic effect in combined immunotherapy for tumors. In terms of efficacy, the tumor-suppressing effects of the three free drug groups are not as good as those of the nanoparticle group. The formulations with or without SSO show different effects on immunity, and the SSO-containing group has a strong immune effect and anti-tumor effect. Moreover, this invention is the first to discover that four ferroptosis-related drugs can self-assemble with albumin to form uniformly sized nanoparticles without the need for synthesis.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] (1) This invention utilizes a green formulation method, which has a simple preparation process and is easy to scale up for production.

[0027] (2) The nanoparticles formed by the present invention have small and uniform particle size and certain targeting properties, which is conducive to the accumulation of nanoparticles in tumor sites; and the large drug loading capacity and safe carrier are conducive to reducing the adverse reactions and toxicity of nanoparticles.

[0028] (3) Compared with the formulation group, any single free drug group of the present invention has a significant ability to be taken up by cells and induce apoptosis; and the green nanoparticles prepared by the present invention have good anti-tumor ability in vivo and in vitro.

[0029] In summary, the green nanoparticles disclosed in this invention utilize a green, synthesis-free preparation process to effectively reduce nanoparticle toxicity and improve their biosafety. In vitro and in vivo anti-tumor studies demonstrate that these nanoparticles possess a potent effect on ferroptosis, enabling multimodal regulation of ferroptosis for tumor treatment. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0031] Figure 1 In the diagram, A represents the particle size and potential of the green nanoparticles of this invention; B represents the in vitro hydroxyl radical detection of the green nanoparticles; and C represents the toxicity test results of the green nanoparticle formulation.

[0032] Figure 2 In the diagram, A represents the detection of the inhibition of GSH production in 4T1 cells by the green nanoparticles of the present invention; B represents the detection of the lipid peroxidation induced by the green nanoparticles of the present invention in 4T1 cells.

[0033] Figure 3 This is a diagram showing the results of ROS production induced by the green nanoparticles of this invention.

[0034] Figure 4 This is a diagram showing the results of 4T1 cell apoptosis induced by the green nanoparticles of this invention.

[0035] Figure 5 The following graphs show the in vivo antitumor pharmacodynamic results of the green nanoparticles of this invention: A is the tumor inhibition curve; B is the mouse body weight change curve; C is the tumor photograph; and D is the survival curve. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0038] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0039] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0040] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0041] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0042] Example 1

[0043] A method for preparing green nanoparticles containing human serum albumin-loaded with ferroptosis-related drugs includes the following steps:

[0044] 1) Prepare an aqueous solution of albumin with a volume mass concentration of 10 mg / ml to obtain solution A;

[0045] 2) A solution of heme with a volume-to-volume concentration of 19.56 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution B; a solution of linoleic acid-cystamine with a volume-to-volume concentration of 24.84 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution C; and a solution of sulfosuccinimide oleate (SSO) with a volume-to-volume concentration of 14.46 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution D.

[0046] 3) Mix 900 μl of sterile double-distilled water with 100 μl of solution A, stir at 800 rpm for 10 min to obtain solution E; mix 30 μl, 30 μl, and 40 μl of solutions B, C, and D, sonicate for 5 min to obtain solution F; add solution F dropwise to solution E and mix, stir at 800 rpm for 10 min, let stand at room temperature for 30 min to obtain solution G;

[0047] 4) Transfer solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 72 h to remove free drugs and organic solvents, thereby obtaining albumin green nanoparticles loaded with ferroptosis-related drugs.

[0048] Example 2

[0049] A method for preparing green nanoparticles containing human serum albumin-loaded with ferroptosis-related drugs includes the following steps:

[0050] 1) Prepare an aqueous solution of albumin with a volume mass concentration of 10 mg / ml to obtain solution A;

[0051] 2) A solution of dihydroporphyrin with a volume mass concentration of 17.63 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain B; a solution of linoleic acid-cystamine with a volume mass concentration of 24.84 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain C; and a solution of sulfosuccinimide oleate (SSO) with a volume mass concentration of 14.46 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain D.

[0052] 3) Mix 900 μl of sterile double-distilled water with 100 μl of solution A, stir at 800 rpm for 10 min to obtain solution E; mix 30 μl, 30 μl, and 40 μl of solutions B, C, and D, sonicate for 5 min to obtain solution F; add solution F dropwise to solution E and mix, stir at 800 rpm for 10 min, let stand at room temperature for 30 min to obtain solution G;

[0053] 4) Transfer solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 72 h to remove free drugs and organic solvents, thereby obtaining albumin green nanoparticles loaded with ferroptosis-related drugs.

[0054] Example 3

[0055] A method for preparing green nanoparticles containing human serum albumin-loaded with ferroptosis-related drugs includes the following steps:

[0056] 1) Prepare an aqueous solution of albumin with a volume mass concentration of 10 mg / ml to obtain solution A;

[0057] 2) A solution of heme with a volume-to-volume concentration of 19.56 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution B; a solution of oleic acid-cystamine with a volume-to-volume concentration of 22.56 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution C; and a solution of sulfosuccinimide oleate (SSO) with a volume-to-volume concentration of 14.46 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution D.

[0058] 3) Mix 900 μl of sterile double-distilled water with 100 μl of solution A, stir at 800 rpm for 10 min to obtain solution E; mix 30 μl, 30 μl, and 40 μl of solutions B, C, and D, sonicate for 5 min to obtain solution F; add solution F dropwise to solution E and mix, stir at 800 rpm for 10 min, let stand at room temperature for 30 min to obtain solution G;

[0059] 4) Transfer solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 72 h to remove free drugs and organic solvents, thereby obtaining albumin green nanoparticles loaded with ferroptosis-related drugs.

[0060] Example 4

[0061] A method for preparing green nanoparticles containing human serum albumin-loaded with ferroptosis-related drugs includes the following steps:

[0062] 1) Prepare an aqueous solution of albumin with a volume mass concentration of 10 mg / ml to obtain solution A;

[0063] 2) A solution of heme with a volume-to-volume concentration of 19.56 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution B; a solution of linoleic acid-cystamine with a volume-to-volume concentration of 26.73 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution C; and a solution of sulfosuccinimide oleate (SSO) with a volume-to-volume concentration of 14.46 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain solution D.

[0064] 3) Mix 900 μl of sterile double-distilled water with 100 μl of solution A, stir at 800 rpm for 10 min to obtain solution E; mix 30 μl, 30 μl, and 40 μl of solutions B, C, and D, sonicate for 5 min to obtain solution F; add solution F dropwise to solution E and mix, stir at 800 rpm for 10 min, let stand at room temperature for 30 min to obtain solution G;

[0065] 4) Transfer solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 72 h to remove free drugs and organic solvents, thereby obtaining albumin green nanoparticles loaded with ferroptosis-related drugs.

[0066] Example 5

[0067] A method for preparing green nanoparticles containing human serum albumin-loaded with ferroptosis-related drugs includes the following steps:

[0068] 1) Prepare an aqueous solution of albumin with a volume mass concentration of 10 mg / ml to obtain solution A;

[0069] 2) A solution of dihydroporphyrin with a volume mass concentration of 17.63 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain B; a solution of oleic acid-cystamine with a volume mass concentration of 22.56 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain C; and a solution of sulfosuccinimide oleate (SSO) with a volume mass concentration of 14.46 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain D.

[0070] 3) Mix 900 μl of sterile double-distilled water with 100 μl of solution A, stir at 800 rpm for 10 min to obtain solution E; mix 30 μl, 30 μl, and 40 μl of solutions B, C, and D, sonicate for 5 min to obtain solution F; add solution F dropwise to solution E and mix, stir at 800 rpm for 10 min, let stand at room temperature for 30 min to obtain solution G;

[0071] 4) Transfer solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 72 h to remove free drugs and organic solvents, thereby obtaining albumin green nanoparticles loaded with ferroptosis-related drugs.

[0072] Example 6

[0073] A method for preparing green nanoparticles containing human serum albumin-loaded with ferroptosis-related drugs includes the following steps:

[0074] 1) Prepare an aqueous solution of albumin with a volume mass concentration of 10 mg / ml to obtain solution A;

[0075] 2) A solution of dihydroporphyrin with a volume mass concentration of 17.63 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain B; a solution of linoleic acid-cystamine with a volume mass concentration of 26.73 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain C; and a solution of sulfosuccinimide oleate (SSO) with a volume mass concentration of 14.46 mg / ml was prepared using dimethyl sulfoxide as a solvent to obtain D.

[0076] 3) Mix 900 μl of sterile double-distilled water with 100 μl of solution A, stir at 800 rpm for 10 min to obtain solution E; mix 30 μl, 30 μl, and 40 μl of solutions B, C, and D, sonicate for 5 min to obtain solution F; add solution F dropwise to solution E and mix, stir at 800 rpm for 10 min, let stand at room temperature for 30 min to obtain solution G;

[0077] 4) Transfer solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 72 h to remove free drugs and organic solvents, thereby obtaining albumin green nanoparticles loaded with ferroptosis-related drugs.

[0078] To further demonstrate the beneficial effects of the present invention and to better understand it, using the green nanoparticles of human serum albumin-encapsulated with ferroptosis-related drugs prepared in Example 1 as an example, the following experiments further illustrate the technical features disclosed in the present invention, but should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above-described invention, without inventive effort, are also considered to fall within the protection scope of the present invention.

[0079] Experimental Example 1: Particle size potential, in vitro hydroxyl radical generation, and formulation toxicity detection of green nanoparticles containing ferroptosis-related drugs encapsulated in human serum albumin.

[0080] To evaluate the morphology of the green nanoparticles, particle size and potential were investigated, and the results are as follows: Figure 1 As shown in Figure A, the nanoparticle has a particle size of 147 nm and a potential of -15.74.

[0081] To evaluate the in vitro hydroxyl radical generation performance of green nanoparticles containing human serum albumin-loaded ferroptosis-related drugs, the hydroxyl radical generation performance of the green nanoparticles was detected using stromal monophosphate (MB) as a probe. MB degradation and H₂O₂ decomposition of MB were negligible. With decreasing pH, the Fenton activity of the green nanoparticles significantly increased, and the generated hydroxyl radicals reacted with MB, leading to MB degradation and a significant decrease in its UV absorption at 664 nm. The results are as follows: Figure 1 As shown in B, the green nanoparticles exhibit excellent hydroxyl radical generation properties and can respond to the acidic tumor microenvironment, thus avoiding damage to normal tissues.

[0082] The cytotoxicity of green nanoparticles to 4T1 cells was investigated using the MTT assay. Results are as follows: Figure 1 As shown in C, the SSO solution had almost no inhibitory effect on 4T1 cell proliferation, while the other drugs showed a concentration-dependent effect. Both formulations showed similar effects due to the weak effect of SSO, and both exhibited strong anti-tumor effects through ferroptosis.

[0083] Experiment Example 2: Detection of Intracellular GSH and Lipid Peroxidation Levels

[0084] 4T1 cells were seeded at a density of 160 x 10⁵ cells / well in 100 mm culture dishes. After overnight culture, different diluted drugs were added to the culture medium for incubation. Drug concentrations were kept consistent across all groups, with hemin at 30 μg / ml. After 24 h of incubation, cells were washed with PBS, sonicated, and intracellular GSH / GSSG and MDA concentrations were measured using a GSH and oxidized glutathione (GSSG) assay kit and an MDA assay kit.

[0085] As a key biomarker of ferroptosis, the accumulation of lipid peroxides in tumor cells determines the lethality of ferroptosis, with malondialdehyde (MDA), a lipid oxidation end product, showing a significant increase during ferroptosis. Conversely, glycosaminoglycans (GSH), a major nutrient and marker of the antioxidant system involved in multiple aspects of tumor cell redox processes, is consumed by cytotoxic hydroxyl radicals generated by the Fenton reaction during ferroptosis. Results are as follows... Figure 2 As shown, due to superior cellular uptake, green nanoparticles exhibit more efficient MDA generation and GSH consumption capabilities than free hemin.

[0086] Experiment Example 3: Detection of Cellular ROS Production Performance

[0087] DCFH-DA was used as a probe to detect the generation of intracellular hydroxyl radicals. After entering the cell, DCFH-DA is hydrolyzed by intracellular esterases to DCFH, which is easily oxidized by reactive oxygen species (ROS) to generate green fluorescent DCF. The level of ROS generation can be assessed by detecting the intracellular fluorescence intensity. 4T1 cells were seeded in 6-well plates and cultured overnight. The cells were then incubated with media containing Hemin and nanoparticles, respectively. The concentration of Hemin was kept consistent across all groups at 20 μg / mL. After 24 h of incubation, the original medium was discarded, and fresh medium containing DCFH-DA (4.0 μM) was added to each well. The cells were incubated in the dark for 20 min. After washing the cells with PBS, they were photographed using a fluorescence microscope.

[0088] The results are as follows Figure 3 As shown, SSO, as an immune-related drug, does not have a significant impact on the cell's ability to generate ROS; LA-Cys, due to the disulfide bonds in its cystamine, consumes GSH and disrupts the intracellular oxidative balance, resulting in a significant increase in intracellular ROS; hemin generates ROS due to the accumulation of intracellular iron caused by its internal iron ions and activated heme oxygenase 1; at the same time, compared with free hemin, green nanoparticles have a stronger uptake efficiency, which helps to improve their ROS generation performance.

[0089] Experiment Example 4: Apoptosis Detection

[0090] 4T1 cells were seeded in 6-well plates (1.5 x 10⁵ cells / well) and incubated for one day. The old culture medium was aspirated, and different drugs diluted with the medium were added to incubate the cells. A blank control group without drugs was also set up. The cells were cultured for another 24 hours. The old culture medium was aspirated, and the cells were washed, digested, and collected. The cells were stained and washed according to the apoptosis staining kit method. Finally, the cells were analyzed by flow cytometry.

[0091] The results are as follows Figure 4 As shown, green nanoparticles have a strong ability to induce apoptosis, with an apoptosis rate of 53.8%. Compared with the free drug group, the apoptosis ability of the formulation group is more prominent, indicating that green nanoparticles have stronger uptake efficiency and enhanced effect.

[0092] Experimental Example 5: In vivo antitumor pharmacodynamics study

[0093] A 4T1 breast cancer model was established in BALB / c mice and randomly divided into six groups: a control group receiving intravenous injection of saline (NS group), a control group receiving Hemin solution (Hemin group), a linoleic acid-cystamine solution group (LA-Cys group), an SSO solution group (SSO group), a group receiving linoleic acid-cystamine / Hemin NPs without SSO (LH group), and a group receiving linoleic acid-cystamine / Hemin / SSO NPs with SSO (LHS group). The mice were administered the drugs five times at intervals for a total of eight days. Every two days, the mice were weighed, and the length and diameter of the tumor tissue were recorded, along with the tumor volume and survival time. One week after the last administration, the tumors and organs (heart, liver, spleen, lung, and kidney) were dissected. All tumors were weighed and photographed.

[0094] The results are as follows Figure 5 As shown in A, compared to other groups, the LHS group showed the highest inhibitory efficacy against tumors in the final formulation, and tumor growth was significantly inhibited during treatment. Figure 5 C shows tumor images of mice in each group after treatment. Low-molecular-weight hydrophobic drugs such as SSO, LA-Cys, and hemin have poor anti-tumor effects due to poor water solubility, poor bioavailability, rapid clearance from the body, and low tumor accumulation. Compared with free drugs, LHS can significantly inhibit tumor growth due to enhanced tumor accumulation and the combined use of multiple drugs. Furthermore, such as... Figure 5 As shown in B and 5D, during the treatment period, the difference in body weight among the mice in each group was negligible compared with the NS group, indicating that LHS has good safety and significantly prolongs the survival of mice.

[0095] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An albumin nanoparticle encapsulating a ferroptosis-related drug, characterized in that, The ferroptosis-related drugs and albumin are assembled into nanoparticles, and the ferroptosis-related drugs and albumin are co-assembled into drug-loaded nanoparticles through electrostatic interactions, π-π stacking and hydrophobic interactions. The ferroptosis-related drug is composed of a porphyrinated drug, an unsaturated fatty acid drug - cystamine, and sulfosuccinimide oleate SSO, and the molar ratio of the porphyrinated drug, the unsaturated fatty acid drug - cystamine, and SSO is 4:1:1 to 1:4:1; the porphyrinated drug is heme, and the unsaturated fatty acid drug is linoleic acid.

2. The albumin nanoparticles carrying ferroptosis-related drugs according to claim 1, characterized in that, The albumin nanoparticles are uniform in size, with a particle size of 100~200nm and a zeta potential of -5~-30mV.

3. A method for preparing albumin nanoparticles loaded with ferroptosis-related drugs as described in claim 1, characterized in that, Includes the following steps: 1) Prepare an aqueous solution of albumin with a volumetric concentration of 0.1~10 mg / ml to obtain solution A: 2) Solution B is prepared by using an organic compound as a solvent to prepare a solution of porphyrin-based drugs with a volume mass concentration of 1-20 mg / ml; solution C is prepared by using an organic compound as a solvent to prepare a solution of unsaturated fatty acid drug-cystamine with a volume mass concentration of 4-40 mg / ml; and solution D is prepared by using an organic compound as a solvent to prepare a solution of sulfosuccinimide oleate SSO with a volume mass concentration of 0.5-15 mg / ml. 3) Mix sterile double-distilled water with solution A and stir at 500-1000 rpm for 5-20 minutes to obtain solution E; Mix solutions B, C, and D, and sonicate for 5-10 minutes to obtain solution F; then add solution F dropwise to solution E and mix, stir at 500-1000 rpm for 5-20 minutes, and let stand at room temperature for 10-30 minutes to obtain solution G; 4) Transfer the solution G to a dialysis bag with a capacity of 3500 kDa or higher, and dialyze with ultrapure water for 24-72 hours to remove free drugs and organic solvents, thereby obtaining the albumin nanoparticles loaded with ferroptosis-related drugs.

4. The method for preparing albumin nanoparticles loaded with ferroptosis-related drugs according to claim 3, characterized in that, The organic compound solvent for the porphyrinated drugs, the unsaturated fatty acid drug cystamine, and SSO is dimethyl sulfoxide.

5. The method for preparing albumin nanoparticles loaded with ferroptosis-related drugs according to claim 3, characterized in that, The method for preparing linoleic acid-cystamine from the unsaturated fatty acid drug cystamine includes the following steps: Linoleic acid and catalyst 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride were dissolved in an organic solvent to obtain solution 1; cystamine was dissolved in an organic solvent to obtain solution 2; solution 2 was added dropwise to solution 1 and heated to obtain reaction solution; after adding silica gel powder to the reaction solution and evaporating to dryness, the solution was purified by silica gel column chromatography using a mixed solvent of dichloromethane and methanol as eluent to obtain linoleic acid-cystamine; The organic solvent is one or a combination of ultra-dry pyridine, ultra-dry N,N-dimethylformamide, ultra-dry dichloromethane, and ethyl acetate.

6. The use of albumin nanoparticles loaded with ferroptosis-related drugs as described in claim 1 or albumin nanoparticles loaded with ferroptosis-related drugs prepared by the method described in claim 3 in the preparation of anti-triple-negative breast cancer drugs.

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