A composite nanoparticle synergistically inducing ferroptosis and anti-tumor immune response and a preparation method thereof

Abstract

This invention belongs to the field of pharmaceutical technology and relates to a composite nanoparticle that synergistically induces ferroptosis and antitumor immune responses, as well as its preparation method. The invention first provides a dihydroartemisinin dimer prodrug or a pharmaceutically acceptable salt thereof with the following structure. Then, composite nanoparticles are prepared using the aforementioned dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt as the main drug component. The composite nanoparticles are prepared by covalently binding an albumin-manganese dioxide complex and ferritin, while simultaneously co-encapsulating the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt and a photosensitizer. The composite nanoparticles prepared by this invention can disrupt the reducing homeostasis of tumor cells by simultaneously enhancing ROS generation and GSH consumption. PDT and CDT synergistically enhance ROS generation, and manganese dioxide can also promote GSH consumption. These composite nanoparticles can significantly improve drug targeting and significantly enhance antitumor activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a composite nanoparticle that synergistically induces ferroptosis and anti-tumor immune response, and its preparation method. Background Technology

[0002] The redox balance maintained by tumor cells is a key factor contributing to the failure of many cancer treatments. Redox homeostasis, a dynamic equilibrium between the generation and clearance of reactive oxygen species (ROS), plays a crucial role in maintaining normal physiological function. Compared to normal cells, tumor cells are more sensitive to ROS accumulation. However, various intracellular antioxidant defense systems, especially the major antioxidant molecule glutathione (GSH), can weaken the therapeutic effects of ROS-enhanced therapies. Therefore, synergistically enhancing ROS accumulation and clearing GSH to disrupt redox homeostasis can effectively accelerate tumor cell death and can be considered a highly effective cancer treatment strategy.

[0003] Ferroprelation is an iron-dependent regulated death mechanism characterized by the abnormal accumulation of reactive oxygen species (ROS) and redox homeostasis imbalance. These changes trigger the production of lethal lipid peroxides (LPO) and lead to the inactivation of glutathione peroxidase 4 (GPX4). Excessive ROS production and GSH depletion can enhance the sensitivity of tumor cells to oxidative stress, thereby significantly amplifying the ferroptotic effect.

[0004] Chemokinetic therapy (CDT) and photodynamic therapy (PDT) are both important ROS-enhancing therapies. CDT, through Fenton and Fenton-like reactions, catalyzes the conversion of endogenous peroxidation into cytotoxic free radicals. PDT, as a non-invasive and spatiotemporally controllable treatment, utilizes photosensitizers to absorb laser energy in the presence of oxygen, thereby generating ROS. ROS generated by CDT and PDT can lead to GPX4 inactivation, triggering LPO accumulation, thereby damaging cell structure and function, and ultimately inducing ferroptosis. However, the efficacy of these therapies is limited by the hypoxic environment of the tumor microenvironment and high GSH expression. Furthermore, redox homeostasis imbalance exacerbates oxidative stress, thereby inducing immunogenic ferroptosis and immunogenic cell death, synergistically enhancing the anti-tumor immune response. The released damage-related molecular patterns can induce dendritic cell maturation, thereby activating T cells to enhance the anti-tumor immune response.

[0005] Nanomedicine delivery systems have been widely used in cancer treatment in recent years. They can integrate multimodal synergistic treatment strategies into a single nanomedicine delivery system, thereby overcoming the limitations of tumor heterogeneity on single therapies and achieving synergistic anti-tumor effects.

[0006] Dihydroartemisinin is an effective anti-tumor drug, but when used alone, its targeting is poor and its anti-tumor effect is limited. There are no reports on existing technologies that co-encapsulate dihydroartemisinin dimer prodrug and photosensitizer through the covalent binding of albumin-manganese dioxide complex and ferritin. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the first objective of the present invention is to provide a dihydroartemisinin dimer prodrug or a pharmaceutically acceptable salt thereof, wherein the dihydroartemisinin dimer prodrug is a dihydroartemisinin dimer linked by a reactive oxygen species (ROS) sensitive bond.

[0008] The ROS-sensitive bond is a thioketal bond, a monoselenobond, a diselenobond, a disulfide bond, or a thioether bond.

[0009] Furthermore, the dihydroartemisinin dimer prodrug is preferably a dihydroartemisinin dimer with the following structure:

[0010] A second objective of this invention is to provide a method for preparing the dihydroartemisinin dimer prodrug, the steps of which are as follows: First, mercaptoacetic acid, acetone, and hydrochloric acid are mixed and stirred for 2-3 hours. The precipitate is then collected by centrifugation. The product is washed sequentially with purified water, n-hexane, and methanol, and then purified to obtain ketithiothiol-carboxyl (TK-COOH).

[0011] TK-COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 4-(dimethylamino)pyridine (DMAP) were weighed and dissolved in N,N-dimethylformamide, and stirred in an ice bath for 1-2 hours. Then, dihydroartemisinin (DHA) was added to the reaction system, and the mixture was stirred at room temperature until the reaction was complete. The product, dihydroartemisinin dimer (Di-DHA), was obtained by separation and purification.

[0012]

[0013] A third objective of this invention is to provide composite nanoparticles that synergistically induce ferroptosis and antitumor immune responses. These composite nanoparticles are prepared by covalently binding albumin-manganese dioxide complex and ferritin, while simultaneously co-encapsulating dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt and a photosensitizer.

[0014] The photosensitizer is a porphyrin-based photosensitizer, selected from one or more of dihydroporphyrin e6, chlorophyll a, and pheophytin a, preferably dihydroporphyrin e6.

[0015] The mass ratio of the albumin-manganese dioxide complex to ferritin is 20:1 to 1:1; The mass ratio of dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt to photosensitizer is 10:1 to 1:10.

[0016] Furthermore, the mass ratio of the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt to the photosensitizer is 2-3:1; The mass-to-volume ratio of the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt to the albumin-manganese dioxide complex is 1.0-2.0 mg / ml, preferably 1.0-1.5 mg / ml.

[0017] The albumin-manganese dioxide complex was obtained by adding potassium permanganate solution to serum albumin solution and stirring. The mass ratio of serum albumin to potassium permanganate is 6-8:1; The volume ratio of serum albumin solution to potassium permanganate solution is 3-5:1.

[0018] In the albumin-manganese dioxide complex, the mass-volume concentration of serum albumin is 10-15 mg / mL, and the mass-volume concentration of potassium permanganate is 1.5-2.0 mg / mL.

[0019] The concentration of the photosensitizer is 0.5-5 μg / mL, and the concentration of the dihydroartemisinin prodrug is 2.5-25 μg / mL.

[0020] The composite nanoparticles were prepared by the following method: Weigh out the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt, photosensitizer, and NHS-activated carboxyl compound (such as bis(succinimide) polyethylene glycol NHS-PEG-NHS), dissolve them in an organic solvent, and slowly add them dropwise to a mixed solution of albumin-manganese dioxide complex and ferritin under stirring. Dialyze to remove the organic solvent to obtain composite nanoparticles.

[0021] The molecular weight of the NHS-PEG-NHS is 200-20000, preferably 2000-5000.

[0022] The mass ratio of dihydroartemisinin dimer prodrug to NHS-PEG-NHS is 1:3-5.

[0023] The organic solvent is dimethyl sulfoxide, acetone, or tetrahydrofuran.

[0024] The mass ratio of the dihydroartemisinin dimer prodrug to the albumin-manganese dioxide complex is 1:5 to 1:20.

[0025] The mass ratio of the albumin-manganese dioxide complex to bis(succinimide) polyethylene glycol NHS-PEG-NHS is 1:15 to 15:1.

[0026] The photosensitizer is a porphyrin-based photosensitizer, preferably one or more of dihydroporphyrin e6, chlorophyll a, and pheophytin a, with dihydroporphyrin e6 being the most preferred.

[0027] The mass ratio of the albumin-manganese dioxide complex to ferritin is 30:1 to 2:1; The mass ratio of dihydroartemisinin prodrug to photosensitizer is 10:1 to 1:10.

[0028] Furthermore, the mass ratio of the dihydroartemisinin dimer prodrug to the photosensitizer is 2-3:1.

[0029] In the mixed solution of albumin-manganese dioxide complex and ferritin, the volume ratio of manganese dioxide complex solution to ferritin solution is 200-250:1.

[0030] The albumin-manganese dioxide complex is prepared as follows: Serum albumin and potassium permanganate were dissolved separately in deionized water. Then, under stirring, the potassium permanganate solution was added dropwise to the serum albumin solution. After continuous stirring, the mixture was dialyzed to obtain an albumin-manganese dioxide complex.

[0031] The serum albumin mentioned is selected from bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin (OVA).

[0032] The mass ratio of serum albumin to potassium permanganate is 6-8:1; The volume ratio of serum albumin solution to potassium permanganate solution is 3-5:1.

[0033] This invention provides the use of the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt or its composite nanoparticles in the preparation of antitumor drugs.

[0034] The tumors mentioned include: colorectal cancer, breast cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, or glioma, etc.

[0035] This invention provides the application of the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt or its composite nanoparticles in the preparation of a drug delivery system that synergistically induces ferroptosis and antitumor immune responses.

[0036] The present invention provides the use of the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt or its composite nanoparticles in the preparation of injection, oral or topical drug delivery systems.

[0037] This invention has the following advantages: 1. This invention is the first to synthesize a novel dihydroartemisinin dimer prodrug.

[0038] 2. The present invention uses dihydroartemisinin dimer prodrug as the main drug component, and the prepared composite nanoparticles can actively target tumors with high expression of transferrin receptor 1.

[0039] 3. The composite nanoparticles prepared in this invention have a photo-triggered effect. After laser irradiation, PDT consumes oxygen and generates ROS, which promotes the ROS responsiveness of the dimer and releases the drug. The oxygen generated by the reaction of manganese dioxide with endogenous hydrogen peroxide in the tumor can enhance the effect of PDT, and the generated manganese ions can synergistically promote the effect of CDT, thus synergistically improving the combined therapeutic effect of PDT and CDT.

[0040] 4. The composite nanoparticles prepared in this invention can disrupt the reducing homeostasis of tumor cells by simultaneously enhancing ROS generation and GSH consumption. PDT and CDT synergistically enhance ROS generation, while manganese dioxide can also promote GSH consumption. This invention integrates multiple mechanisms, proposes a novel anti-tumor treatment strategy, and develops an effective nanoplatform for enhancing tumor treatment efficacy. Attached Figure Description

[0041] Figure 1 The thioether-linked dihydroartemisinin dimer (Di-DHA) in Example 1 of this invention 1 H NMR spectrum.

[0042] Figure 2 ESI-MS spectrum of dihydroartemisinin dimer (Di-DHA) linked by thioether bonds in Example 1 of this invention.

[0043] Figure 3 The particle size and transmission electron microscope image of the composite nanoparticles in Example 2 of this invention.

[0044] Figure 4 In Example 3 of this invention, the decomposition of Di-DHA and the release of Ce6 in laser-controlled composite nanoparticles were demonstrated.

[0045] Figure 5 Cellular uptake diagram of the composite nanoparticles in Example 4 of this invention.

[0046] A: Average fluorescence intensity of FeBMnDC NPs taken up at different times.

[0047] B: Average fluorescence intensity of different formulations after 4 hours of uptake.

[0048] Figure 6 In vitro cytotoxicity experiment of the composite nanoparticles in Example 5 of this invention.

[0049] A: Cytotoxicity of NIH3T3 cells by different treatment groups.

[0050] B: Cytotoxicity of CT26 cells under different treatment groups in the absence of light.

[0051] C: Cytotoxicity of CT26 cells under different treatment groups under light conditions.

[0052] D: Cytotoxicity of FeBMnDC NPs to CT26 after pretreatment with several ferroptosis inhibitors.

[0053] Figure 7 The generation of ROS in CT26 cells by composite nanoparticles in Example 6 of this invention.

[0054] Figure 8 The GSH consumption of composite nanoparticles in CT26 cells in Example 7 of this invention.

[0055] Figure 9 The in vitro maturation of myeloid-derived dendritic cells induced by the composite nanoparticles in Example 8 of this invention.

[0056] Figure 10 Tumor growth curve of the composite nanoparticles in vivo anti-tumor experiment in Example 9 of this invention.

[0057] Figure 11 Tumor weight diagram of the in vivo anti-tumor experiment of composite nanoparticles in Example 9 of this invention.

[0058] Figure 12 Figure showing the change in body weight of mice in the in vivo anti-tumor experiment of the composite nanoparticles in Example 9 of this invention.

[0059] Figure 13 The H&E staining results of various major organs of mice in Example 9 of this invention.

[0060] Figure 14 The in vivo maturation of dendritic cells induced by composite nanoparticles in Example 10 of this invention.

[0061] Figure 15 The in vivo maturation of dendritic cells induced by the composite nanoparticles in Example 10 of this invention. Detailed Implementation

[0062] Example 1: Synthesis of Di-DHA, a thioether-linked dihydroartemisinin dimer prodrug (1) Synthesis of TK-COOH Mercaptoacetic acid (1 eq), acetone (2.1 eq), and hydrochloric acid (1.9 eq) were mixed and stirred for 2 hours. The precipitate was collected by centrifugation. The product was washed sequentially with purified water, n-hexane, and methanol, and then purified to obtain a white powder with a yield of 92%.

[0063] 1H NMR (600 MHz, DMSO) δ 12.61 (s, 2H), 3.36 (s, 4H), 1.54 (s, 6H); ESI-MS (m / z): 223.07 [MH] - . (2) Synthesis of Di-DHA TK-COOH (179.2 mg, 0.8 mmol), EDCI (337.4 mg, 1.76 mmol), and DMAP (214.72 mg, 1.76 mmol) were weighed and dissolved in N,N-dimethylformamide, and stirred in an ice bath for 1 hour. Then, DHA (500 mg, 1.76 mmol) was added to the reaction mixture, and the reaction was continued to be stirred at room temperature for 24 hours. The reaction mixture was finally purified by column chromatography using petroleum ether:ethyl acetate as the eluent in a 3:1 ratio. A white powder was obtained in 56% yield.

[0064] 1 H NMR (600 MHz, CDCl3) δ 5.77 (d, J = 9.8 Hz, 2H), 5.43 (s, 2H), 3.49 (dd, J = 4.6 Hz, 4H), 2.63-2.55 (m, 2H), 2.40-2.34 (m, 2H), 2.05-2.01 (m,2H), 1.92-1.87 (m, 2H), 1.81-1.70 (m, 4H), 1.66-1.63 (m, 2H), 1.62 (s, 6H),1.52-1.45 (m, 2H), 1.43 (s, 6H), 1.38 (dd, J =13.6, 3.6 Hz, 2H), 1.32-1.24(m, 4H), 1.05-0.99 (m, 2H), 0.96 (d, J = 6.1 Hz, 6H), 0.89 (d, J = 7.1 Hz, 6H); ESI-MS (m / z): 779.24 [M+Na] + . Example 2 Preparation of composite nanoparticles (1) Preparation of albumin-manganese dioxide complex Bovine serum albumin (250 mg) and potassium permanganate (35 mg) were dissolved in 15 mL and 5 mL of deionized water, respectively. Then, under stirring, the potassium permanganate solution was added dropwise to the bovine serum albumin solution, and after stirring for 4 hours, the mixture was dialyzed to obtain the albumin-manganese dioxide complex.

[0065] (2) Preparation of composite nanoparticles Weigh 1.25 mg of dihydroartemisinin dimer prodrug, 0.5 mg of dihydroporphyrin e6 and 5 mg of NHS-PEG-NHS, dissolve them in dimethyl sulfoxide, and slowly add them dropwise to a mixed solution of 1 mL of albumin-manganese dioxide complex and 5 μL of ferritin with a concentration of 64 mg / mL in step (1) under stirring. After continuous stirring, dialyze to remove the organic solvent to obtain composite nanoparticles FeBMnDC.

[0066] The results showed that the prepared composite nanoparticles had a particle size of 170.9 ± 2.2 nm and a PDI of 0.129 ± 0.028.

[0067] Example 3: In vitro release experiment of the composite nanoparticles prepared in Example 2 The composite nanoparticles prepared in Example 2, subjected to laser irradiation (650 nm, 50 mW / cm², 0.5 h on / off cycling, for 4 h), were loaded into an ultrafiltration tube and dialyzed with PBS buffer containing 0.05% Tween 80. The dialysis tubes were treated under laser irradiation, and samples were collected at predetermined time points. The samples were diluted with 4 volumes of acetonitrile, vortexed, and centrifuged. The concentrations of Di-DHA and Ce6 in the supernatant were analyzed using an HPLC system. The results are shown in [Figure number missing]. Figure 4 .

[0068] The results showed that drug release from nanoparticles is phototriggered and depends on ROS generated by PDT. Under laser irradiation, the degradation rate and release rate of Di-DHA can both reach more than 50% after 3 hours.

[0069] Example 4: Cellular uptake experiment of the composite nanoparticles prepared in Example 2 The ferritin-free formulation BMnDC and the manganese dioxide-free and ferritin-free formulation BDC were prepared according to the method of Example 2.

[0070] The uptake of the prepared composite nanoparticles in CT26 cells was determined by flow cytometry.

[0071] CT26 cells were administered at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL into 12-well plates and incubated for 24 h to allow adherence. After adherence, the nanoparticles prepared in Example 2 were added, and the cells were incubated at 37°C for predetermined times of 0.5, 1, 2, and 4 hours. Cells were then washed, collected, and dispersed in PBS. Flow cytometry was used to determine the uptake of various formulations by the cells. A formulation containing manganese dioxide and ferritin, BDC, was used as a control. Results are as follows: Figure 5 -A is shown.

[0072] CT26 cells were administered at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL into 12-well plates and incubated for 24 h to allow adherence. After cell adherence, different samples were added according to the groups, and the plates were incubated at 37°C for a predetermined period of 4 hours. Cells were then washed, collected, and dispersed in PBS. Flow cytometry was used to determine the uptake of various reagents by the cells. Results are as follows: Figure 5 -B is shown.

[0073] Groups: Group 1: BDC formulation without manganese dioxide and ferritin; Group 2: Ce6; Group 3: BMnDC; Group 4: FeBMnDC; Group 5: FeBMnDC added after ferritin pretreatment.

[0074] Depend on Figure 5 The fluorescence intensity results measured by flow cytometry in -A show that the uptake of nanoparticles by cells is time-dependent, and the uptake gradually increases with time.

[0075] The uptake results of different treatment groups showed that the cells treated with nanoparticles had higher fluorescence intensity compared with free Ce6. The formulation containing ferritin had a greater uptake than the formulation without ferritin. At the same time, the uptake of the formulation pre-saturated with ferritin was significantly reduced, which proved that the presence of ferritin in nanoparticles can enhance the targeting of the formulation.

[0076] Example 5: In vitro cytotoxicity experiment of the composite nanoparticles prepared in Example 2 The cytotoxicity of the drug on CT26 cells and NIH-3T3 cells was investigated using the CCK8 assay. Cells were digested and diluted with culture medium to a concentration of 1 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well, thoroughly mixed, and then seeded into 96-well plates. The plates were incubated for 24 hours to allow cell adhesion. After cell adhesion, different concentrations of different reagents were used to treat the cells, with each concentration applied in quadruplicate, for a total of three replicates. The plates were then incubated together with the cells. After 4 hours of incubation, the medium was replaced with fresh medium, and the cells were either irradiated with a 650 nm laser (300 mW / cm²) for 1 minute or left untreated. After another 20 hours of incubation, CCK-8 reagent was added to the medium, and the plates were incubated for 30 minutes. The absorbance was then measured at 450 nm using a microplate reader.

[0077] In addition, preliminary validation of ferroptosis was conducted. Cells were pretreated with the ferroptosis inhibitors GSH (5 mmol / L), DFO (100 μmol / L), and Fer1 (10 μmol / L), and the cytotoxicity of FeBMnDC nanoparticles under light irradiation was then assessed. Results are shown below. Figure 6 .

[0078] Figure 6-A indicates that the prepared nanoparticles have low toxicity to normal cells and good safety.

[0079] Figure 6 -B results indicate that the formulation exhibits weak toxicity to CT26 cells under non-light conditions.

[0080] Figure 6 -C results showed that the formulation had significant cytotoxicity against CT26 cells under light conditions.

[0081] Figure 6 The results showed that the ferroptosis inhibitor could significantly suppress the cytotoxic effects of the formulation, demonstrating that ferroptosis may be one of the ways in which the formulation induces cell death.

[0082] As can be seen, FeBMnDC nanoparticles exhibit strong cytotoxicity under laser irradiation, demonstrating that multiple synergistic mechanisms within the nanoparticles can jointly achieve tumor suppression. Treatment with ferroptosis inhibitors significantly reduced the cytotoxicity of the nanoparticles, providing preliminary evidence that the prepared nanoparticles can induce ferroptosis.

[0083] Example 6: Generation of intracellular reactive oxygen species (ROS) in CT26 cells by the composite nanoparticles prepared in Example 2. The generation of ROS by the prepared FeBMnDC nanoparticles in CT26 cells was determined by flow cytometry. CT26 cells were cultured at 2×10⁻⁶ cells per cell line. 5 Cells were seeded at a density of [number] cells / mL into 12-well plates and incubated for 24 hours to allow adherence. After adherence, the respective drug formulations prepared with culture medium were added: Ce6 at a concentration of 2.5 μg / mL and DHA at a concentration of 12.5 μg / mL. After incubation at 37 °C for 4 hours, the cells were washed, and ROS in the cells were labeled with the probe DCFH-DA (20 μM). After incubation for 30 minutes, the cells were washed with PBS, and each sample was either irradiated with a 650 nm laser (300 mW / cm²) for 5 minutes or left unirradiated. After cell digestion, the cells were collected and dispersed in PBS. The amount of ROS generated by the cells in each drug formulation was determined by flow cytometry. The experimental results are as follows: Figure 7 As shown.

[0084] Experimental results showed that the FeBMnDC nanoparticle group produced the most ROS compared with the control groups, which proves that the formulation can fully synergize with CDT and PDT to enhance ROS production and thus induce ferroptosis.

[0085] Example 7: GSH consumption of the composite nanoparticles prepared in Example 2 in CT26 cells. GSH consumption was detected using a GSH assay kit. CT26 cells were seeded in 6-well plates and cultured overnight. After treatment with the experimental reagent for 4 hours, the cells were irradiated with a 650 nm laser (300 mW / cm²) and cultured for another 20 hours before cell harvesting. Cells were sonicated, centrifuged, and the supernatant was collected. Reagents were then added according to the kit instructions, and the absorbance at 405 nm was recorded. The experimental results are shown below. Figure 8 As shown.

[0086] The results showed that, compared with the control groups, the FeBMnDC nanoparticle group after light exposure had the most significant GSH scavenging effect. This may be related to the higher uptake of FeBMnDC nanoparticles by cells, the reaction of manganese dioxide with GSH consuming GSH, and the ROS generated by light exposure further inhibiting GSH expression.

[0087] Example 8: Study on the maturation of bone marrow-derived dendritic cells (BMDC) in vitro using composite nanoparticles prepared in Example 2. Dendritic cells (DCs) are considered the most efficient antigen-presenting cells, playing a crucial role in antigen presentation and T cell activation. Under sterile conditions, bone marrow-derived dendritic cells (BMDCs) were isolated from the femur and tibia of 6-8 week old BALB / c mice. CT26 cells were treated with free Ce6 and various nanoparticle groups (Ce6: 2.5 μg / mL, DHA: 12.5 μg / mL) for 4 hours. The culture medium was then replaced with fresh medium, and the cells were cultured for another 20 hours with or without irradiation using a 650 nm laser (300 mW / cm²) for 5 minutes. The culture medium from the treated CT26 cells was collected and added to BMDC culture dishes. After co-culturing for 24 hours, the treated dendritic cells (DCs) were collected, washed with PBS, and stained with fluorescently labeled antibodies (anti-CD11c-APC, anti-CD80-BV421, and anti-CD86-PE·Cy7) for FCM analysis. The experimental results are as follows: Figure 9 As shown.

[0088] The results showed that tumor cells treated with light and FeBMnDC nanoparticles could significantly promote the maturation of DC cells, thus demonstrating their excellent immune activation potential.

[0089] Example 9: In vivo antitumor experiment of the composite nanoparticles prepared in Example 2 1 × 10 6CT26 cells were subcutaneously injected into female BALB / c mice to establish a tumor-bearing mouse model. When the tumor volume reached approximately 100 mm³, the mice were randomly divided into groups of five. Each group received a tail vein injection of PBS, a physical mixture of Ce6 and DHA, BDC, BMnDC, or FeBMnDC nanoparticles, respectively. The Ce6 / DHA doses were 5 / 25 mg / kg, administered intravenously every three days for three consecutive treatments. The tumors in each treatment group were then irradiated with a 650 nm laser for 5 minutes. Mouse weight and tumor size were recorded every two days. Tumor volume was calculated using the formula v = 0.5 × length × width. 2 Calculations were performed. On day 14, mice were sacrificed, and tumors were isolated and weighed. The results of the mouse tumor growth curve are shown below. Figure 10 The tumor results are as follows Figure 11 The results of the mouse weight change are as follows: Figure 12 As shown, the H&E staining results of various major organs of the mouse are as follows: Figure 13 As shown.

[0090] The results showed that FeBMnDC nanoparticles exhibited the strongest antitumor activity under light irradiation, with slow tumor growth and smaller tumor weight. No significant changes in body weight were observed in any group of mice during treatment. These results demonstrate that the nanoparticles, while possessing significant antitumor effects, have no obvious toxicity to the body, making them a safe and effective antitumor drug delivery system.

[0091] Example 10: In vivo DC maturation study of the composite nanoparticles prepared in Example 2. The maturation of dendritic cells (DCs) is crucial for tumor antigen presentation, a process essential for initiating adaptive immunity. DCs in lymph nodes were analyzed by flow cytometry (FCM). Mice were grouped and treated via tail vein, as described in Example 9. On the third day after treatment, tumor-draining lymph nodes were harvested from each group of mice. The tissues were digested with type IV collagenase at 37°C for 1 hour, and then filtered through a 70 μm filter to obtain a single-cell suspension. Subsequently, the collected cells were incubated with CD16 / CD32 blocking antibodies for 20 minutes, followed by staining with BD Horizon immobilizable active staining agent 780 at 4°C for 15 minutes. After staining with anti-CD45, CD11c, CD80, and CD86 antibodies, mature dendritic cells in the lymph nodes were analyzed by FCM. The results are as follows: Figure 14 and 15 As shown.

[0092] Experimental results show that the prepared nanoparticles can induce DC maturation not only in vitro under light conditions, but also in mice. The proportion of DC maturation induced by the nanoparticles in mice is about 1.9 times that of the PBS group, indicating that the nanoparticles can effectively enhance the body's anti-tumor immune response.

Claims

1. A dihydroartemisinin dimer prodrug or a pharmaceutically acceptable salt thereof having the following structure: 。 2. The method for preparing the dihydroartemisinin dimer prodrug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, First, mercaptoacetic acid, acetone, and hydrochloric acid are mixed, stirred, and centrifuged to collect the precipitate. The product is washed sequentially with purified water, n-hexane, and methanol, and then purified to obtain ketethiocarboxyl acetal. Ketoethiocarboxyl acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-(dimethylamino)pyridine are weighed and dissolved in N,N-dimethylformamide. The mixture is stirred under ice bath conditions, and then dihydroartemisinin is added to the reaction system. The mixture is stirred at room temperature until the reaction is complete, and then separated and purified to obtain the final product.

3. Composite nanoparticles comprising the dihydroartemisinin dimer prodrug of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The composite nanoparticles are prepared by covalently binding albumin-manganese dioxide complex and ferritin, while simultaneously co-encapsulating dihydroartemisinin dimer prodrug and photosensitizer.

4. The composite nanoparticles according to claim 3, characterized in that, The photosensitizer is a porphyrin-based photosensitizer, selected from one or more of dihydroporphyrin e6, chlorophyll a, and pheophytin a, preferably dihydroporphyrin e6; the mass ratio of the dihydroartemisinin dimer prodrug to the photosensitizer is 10:1 to 1:10, preferably 2-3:

1.

5. The composite nanoparticles of the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt as described in claim 3, characterized in that, The mass-to-volume ratio of the dihydroartemisinin dimer prodrug and the albumin-manganese dioxide complex is 1.0-2.0 mg / mL, preferably 1.0-1.5 mg / mL.

6. The composite nanoparticles of the dihydroartemisinin dimer prodrug or a pharmaceutically acceptable salt thereof as described in claim 3, characterized in that, The albumin-manganese dioxide complex is obtained by adding potassium permanganate solution to serum albumin solution and stirring; the mass ratio of serum albumin to potassium permanganate is 6-8:1; the volume ratio of serum albumin solution to potassium permanganate solution is 3-5:

1.

7. A method for preparing composite nanoparticles of the dihydroartemisinin dimer prodrug or its pharmaceutically acceptable salt as described in any one of claims 3-6, characterized in that, Weigh out the dihydroartemisinin dimer prodrug, photosensitizer, and NHS-activated carboxyl compound, dissolve them in an organic solvent, and slowly add them dropwise to a mixed solution of albumin-manganese dioxide complex and ferritin under stirring. Dialyze to remove the organic solvent to obtain composite nanoparticles. The NHS-activated carboxyl compound is preferably bis(succinimide) polyethylene glycol, and the mass ratio of dihydroartemisinin dimer prodrug to NHS-PEG-NHS is 1:3-5.

8. The use of the dihydroartemisinin dimer prodrug of claim 1 or a pharmaceutically acceptable salt thereof, or the composite nanoparticles of any one of claims 3-6, in the preparation of antitumor drugs.

9. The use of the dihydroartemisinin dimer prodrug of claim 1 or a pharmaceutically acceptable salt thereof, or the composite nanoparticles of any one of claims 3-6, in the preparation of a drug delivery system that synergistically induces ferroptosis and antitumor immune responses.

10. The use of the dihydroartemisinin dimer prodrug of claim 1 or a pharmaceutically acceptable salt thereof, or the composite nanoparticles of any one of claims 3-6, in the preparation of injectable, oral, or topical drug delivery systems.

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