Anti-tumor self-assembly targeted nano-drug as well as preparation method and application thereof

Abstract

The invention relates to an anti-tumor self-assembly targeted nano-drug as well as a preparation method and application thereof, and belongs to the technical field of nanotechnology. The self-assembled nano-drug is composed of hemin and dihydroartemisinin and is formed by self-assembling a prodrug with an ROS sensitive bond and hemin, the molar ratio of the prodrug to hemin is 1: 1-1: 10, and the prodrug molecule is formed by connecting a hemin drug molecule with an iron supplementing effect and an artemisinin derivative through the ROS sensitive bond. The invention has the advantages that: the EPR effect is utilized to passively transport to the tumor tissue, the responsive drug release in the tumor tissue is realized, the tumor cells are stimulated to generate ICD, the expression of PD-L1 protein is down-regulated, the immune escape is prevented, and the multi-mechanism tumor killing is realized, so that the immune response of the body is enhanced, the anti-tumor effect is exerted, and the drug effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of nanotechnology, specifically relating to an anti-tumor self-assembling targeted nanomedicine, its preparation method, and its application. Background Technology

[0002] Breast cancer remains one of the most common cancers worldwide, accounting for approximately 30% of cancers in women. Triple-negative breast cancer (TNBC), a type of "cold tumor," suffers from low expression of immune cells, resulting in an immunosuppressive tumor microenvironment. Therefore, compared to other subtypes, TNBC is more prone to recurrence and metastasis, leading to lower patient survival rates. For decades, chemotherapy and radiotherapy have been the primary treatments for breast cancer, but they have not been effective in suppressing metastatic TNBC, resulting in poor patient prognosis. Currently, immunotherapy, as an emerging treatment in the field of oncology, has broad application prospects. Based on immune editing, it alleviates the immunosuppressive state in the tumor microenvironment and is a therapeutic strategy that utilizes the body's immune system to kill tumor cells.

[0003] Tumor immunotherapy is a method that uses modulators or drugs to activate and regulate the immune system to fight disease. It is the fourth generation of effective cancer treatment strategies after surgery, chemotherapy, and radiotherapy. Immunogenic cell death (ICD) is a form of immunogenic apoptosis that typically leads to the release of pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs). ICD can transform dying or dead tumor cells into vaccines, induce the maturation of dendritic cells (DCs), and elicit CTL-mediated immune responses. ICDs play an indispensable role in tumor immunotherapy by disrupting the immunosuppressive microenvironment, stimulating the transformation of "cold" tumors into "hot" tumors, and activating the anti-cancer immune network. Due to the low immunogenicity of tumors and the immunosuppressive state of the tumor microenvironment (TME), immunotherapy is often not very effective. As a special form of cell death, ICDs can not only induce tumor cells to fully expose tumor antigens and transform from a low-immunogenic to a high-immunogenic state, thus realizing the transformation of "cold" tumors into "hot" tumors, but also reverse the tumor immunosuppressive microenvironment, which helps dendritic cells to phagocytose dying tumor cells and cross-present antigenic peptides on MHC-I to CD8, thereby increasing the infiltration of CTLs at the tumor site and driving the anti-tumor immune response. Existing studies have shown that chemotherapy, photodynamic therapy, photothermal therapy, and radiotherapy can induce intracellular tumor cells (ICDs) within the tumor microenvironment. Tumor-associated antigens released by ICDs on the surface of tumor cells are phagocytosed by tumor-associated cells (APCs) and presented to T cells. Simultaneously, they can promote the maturation of dendritic cells (DCs), activate cytokines (CTLs), and secrete cytokines related to innate and adaptive immunity, such as IFN-γ, TNF-α, and IL-6, triggering a potent immune stimulus. Therefore, ICD therapy may offer new hope for eliminating tumors and reducing recurrence rates.

[0004] Dihydroartemisinin (DHA) is an artemisinin derivative isolated from the traditional Chinese medicine Artemisia annua, possessing a specific internal peroxide bridge structure. Due to its safety and efficacy, DHA has been used for decades to treat malaria and fever. At the molecular level, DHA exerts cytotoxicity through the breaking of internal peroxide bridges and the generation of reactive oxygen species (ROS), inhibiting angiogenesis, inducing apoptosis, and regulating tumor-related genes to exert its anti-tumor proliferation effects.

[0005] Dihydroartemisinin (DHA), as an inducer of immunogenic cell death (ICD), has been shown to induce immunogenic cell death and modulate immune function to fight tumors and prevent metastasis. Due to its dual effects, DHA has long been considered an excellent anti-tumor agent. It is worth noting that Fe... 2+ DHA plays a crucial role in therapeutic efficacy due to the breaking of the internal peroxide bridges required for its development. This is because Fe... 2+ The extreme consumption of DHA in cancer cells hinders its ability to disrupt internal peroxide bridges and exert therapeutic effects. Another obstacle is DHA's poor water solubility and short circulating half-life, which significantly limits its application in anti-tumor therapy. Addressing iron deficiency, modifying DHA and constructing suitable delivery systems to increase its solubility may be a feasible approach to enhancing its anti-tumor effects. Summary of the Invention

[0006] This invention provides an anti-tumor self-assembling targeted nanomedicine, its preparation method, and its application. The nanoparticles are co-delivered with heme chloride and dihydroartemisinin prodrug. Two molecules of DHA are chemically coupled via a thioketal bond to form the prodrug, which, together with heme chloride, forms a hydrophobic core embedded within DSPE-mPEG2000, self-assembling into nanoparticles in water. Dihydroartemisinin, as an ICD inducer, releases DAMPs to promote DC uptake, processing, and antigen presentation, activates T cell infiltration at tumor sites, reduces the proportion of Tregs, and remodels the tumor immune microenvironment. When combined with heme chloride, heme chloride, an iron-based porphyrin molecule, is a purified form of endogenous heme in vitro. In the cytoplasm, it is decomposed by heme oxygenase (HO-1) to provide sufficient Fe. 2+ On the one hand, it stimulates the ICD effect; on the other hand, it downregulates the PD-L1 protein on the surface of tumor cells, causing dual stress in mitochondria and endoplasmic reticulum, generating strong ICD signals, improving the tumor immunosuppressive microenvironment, reducing tumor immune escape, and stimulating the body to produce a strong immune anti-tumor effect.

[0007] The technical solution adopted in this invention is to link ROS-sensitive prodrugs with reactive oxygen species responsive bonds and thioketal bonds, and then self-assemble the prodrugs with heme chloride and distearate phosphatidyl acetamide-methoxy polyethylene glycol 2000 in water to form ROS-responsive nanoparticles.

[0008] The ROS-sensitive prodrug DSD described in this invention is two molecules of dihydroartemisinin.

[0009] The monomer chemical structure of the ROS-sensitive prodrug described in this invention is shown below:

[0010] or .

[0011] The ROS-responsive bond in the ROS-sensitive prodrug of this invention is a thioacetal bond, and the synthetic steps of the thioacetal linker TK are as follows:

[0012] 1.25 mL (1.43 g, 13.5 mmol) of 3-mercaptopropionic acid was transferred to 2 mL of anhydrous acetone (1.57 g, 27 mmol), and then 0.67 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 4 h. The mixture was then filtered to obtain a white solid, which was washed four times with ice-cold hexane and ice-cold deionized water to obtain crude thioacetate. The crude product was dissolved in ethyl acetate in a certain proportion and then cooled and recrystallized to obtain thioacetate.

[0013] .

[0014] The method for preparing ROS-sensitive prodrugs according to the present invention is as follows:

[0015] Accurately weigh 212 mg (0.84 mmol) of TK and dissolve it in 15 mL of dichloromethane. Then add 313 mg (2.02 mmol) of EDC and 24 mg (0.2 mmol) of DMAP. Stir in an ice bath for 20 min, then add 477 mg (1.68 mmol) of DHA and react at room temperature for 72 h. After the reaction is complete, concentrate the reaction system by rotary evaporation. Use a mixed solution of dichloromethane and ethyl acetate (40:1) as the eluent and separate and purify the target compound by silica gel column chromatography. Remove the eluent by rotary evaporation, dissolve in methanol, dilute with a large amount of purified water, and freeze dry to obtain a white solid powder, DSD.

[0016] .

[0017] The hemin chloride described in this invention is a commonly used iron supplement compound for the human body, and its chemical structure is as follows:

[0018] .

[0019] A method for preparing a self-assembled targeted nanomedicine with anti-tumor properties, comprising the following steps:

[0020] Accurately weigh heme chloride into a round-bottom flask, then dissolve it in 5 mL of DMSO to obtain a heme chloride stock solution, which is stored at -20 ℃. Accurately weigh the prodrug and distearate phosphatidylacetamide-methoxy polyethylene glycol 2000, mix them with 200 μL of heme chloride stock solution, and after thorough dissolution, add the solution dropwise to an appropriate volume of pure water. Stir vigorously for 10 min, then place the solution in an ultrasonic cell disruptor at 60 W for 3 s, with a 4 s interval, and sonicate for 10 min. Finally, place the sonicated sample in a centrifuge at 5000 rpm for 10 min to remove insoluble free drug, obtaining self-assembled nanoparticles.

[0021] The organic solvent used in this invention to dissolve ROS-sensitive prodrugs and heme chloride is one or more of dimethyl sulfoxide, ethanol, methanol, acetone, and tetrahydrofuran.

[0022] This invention relates to the application of self-assembled targeted nanomedicines in the preparation of tumor therapeutic drugs.

[0023] This invention involves mixing heme chloride, dihydroartemisinin prodrug molecules, and DSPE-mPEG2000 in a specific ratio, and preparing ROS-responsive self-assembled nanoparticles using an antisolvent-precipitation method. The synergistic antitumor effect of the two drugs is significantly enhanced. Dihydroartemisinin is an artemisinin derivative with active hydroxyl groups, and heme chloride is an in vitro purified form of natural heme. This invention exhibits no toxicity to normal cells and achieves a synergistic antitumor effect greater than the sum of its parts ("1+1>2").

[0024] This invention utilizes heme chloride, distearate phosphatidyl acetamide-methoxy polyethylene glycol 2000, and prodrug molecules to self-assemble in water to form carrier-free nanoparticles. This enhances drug solubility and bioavailability, as well as biological stability and retention. The nanoparticles are used in anti-tumor applications. Through enhanced permeability and retention (EPR) effects, the nanoparticles enhance the accumulation of therapeutic drugs at the tumor site, and ROS-responsive release achieves targeted release within the tumor microenvironment. DHA and Hemin are released; Hemin reacts with intracellular GSH (highly expressed in tumor cells) to produce Fe... 2+ Inducible internal peroxide bridges of DHA in Fe 2+ The presence of fragmentation generates toxic carbon-centered free radicals (ROS). The excessive production of ROS also leads to apoptosis. Importantly, the combined effect of Hemin / DHA stimulates oxidative stress, resulting in high immune activation of the ICD. It can promote dendritic cell (DC) maturation, enhance CD4 and CD8 T cell infiltration, and promote macrophage polarization. Furthermore, DHA is also used as an immunomodulator to suppress Treg cells and stimulate beneficial aspects of the host immune response. DHA can enhance HO-1 activity, further promoting Fe from Hemin. 2+The release of [the virus / material]. Therefore, this invention provides a tumor treatment strategy for potential clinical applications.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) The nanoparticles constructed in this invention are a novel concept, namely the self-assembled nanoparticles provided by this invention. The combined use of dihydroartemisinin and heme chloride has the advantage that it can be transported to tumor tissues via EPR and can also achieve responsive drug release in tumor tissues, thereby achieving multi-mechanism tumor killing.

[0027] (2) The self-assembled nanoparticles have a particle size of 90-100 nm, are uniform in size, and can exist stably in blood.

[0028] (3) It exhibits ROS responsiveness. Under normal physiological conditions, the self-assembled nanoparticles maintain their nanostructure and slowly release drugs; in an oxidizing environment, the self-assembled nanoparticles rapidly disintegrate, and the cumulative release of heme chloride and dihydroartemisinin increases significantly, proving that the self-assembled nanoparticles have good ROS responsiveness.

[0029] (4) The dihydroartemisinin and heme chloride contained in the self-assembled nanoparticles can achieve a cascade reaction and activate the immunotherapy anti-tumor effect of ICD. The tumor volume and weight of mice that received the self-assembled nanoparticles were significantly smaller than those of mice that received dihydroartemisinin and heme chloride alone. Attached Figure Description

[0030] Figure 1 This is the TK mass spectrum of the thioketone linker in Example 1 of the present invention;

[0031] Figure 2 This is the nuclear magnetic resonance spectrum of the thioketone linker in Example 1 of the present invention;

[0032] Figure 3 This is the mass spectrum of the prodrug DSD in Example 2 of this invention;

[0033] Figure 4 This is the nuclear magnetic resonance spectrum of DHA in Embodiment 2 of the present invention;

[0034] Figure 5 This is the DSD nuclear magnetic resonance spectrum in Embodiment 2 of the present invention;

[0035] Figure 6 This is a particle size and potential diagram of Hemin@DSD NPs in Example 4 of the present invention;

[0036] Figure 7 This is a transmission electron microscope image of Hemin@DSD NPs in Embodiment 4 of the present invention;

[0037] Figure 8These are stability graphs of different formulations in Example 4 of this invention.

[0038] Figure 9 These are plasma stability graphs of different formulations in Example 4 of this invention;

[0039] Figure 10 This is a cell diagram from the hemolysis experiment in Example 4 of this invention;

[0040] Figure 11 This is a graph showing the in vitro drug release of different formulations at different H2O2 concentrations in Example 4 of the present invention;

[0041] Figure 12 This is a transmission electron microscope image of Hemin@DSD NPsROS response in Example 4 of the present invention.

[0042] Figure 13 This is a graph showing the effect of different formulations on the activity level of 4T1 cells in Example 4 of this invention;

[0043] Figure 14 This is a graph showing the effect of different formulations on ATP levels in 4T1 cells in Example 4 of this invention;

[0044] Figure 15 This is a graph showing the effect of different formulations on HMGB1 levels in 4T1 cells in Example 4 of this invention;

[0045] Figure 16 This is a graph showing the effect of different formulations on calreticulin exposure in 4T1 cells in Example 4 of this invention;

[0046] Figure 17 This is a time diagram of the uptake of Hemin@DSD NPs by 4T1 cells in Example 13 of the present invention;

[0047] Figure 18 This is a graph showing the effect of different formulations on the maturation of dendritic cells in 4T1 cells in Example 14 of the present invention;

[0048] Figure 19 This is a graph showing the effect of different formulations on the expression of PD-L1 protein in 4T1 cells in Example 15 of the present invention;

[0049] Figure 20 These are in vivo antitumor experimental images of different formulations in Example 16 of the present invention, where a, b, and c are tumor volume, mouse weight, and actual tumor photographs, respectively. Detailed Implementation

[0050] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0053] Reagents (kits) used for performance testing of nanomedicines included: TFR1 assay kit, CCK-8 assay kit, ATP assay kit, HMGB1 assay kit, and BCA protein assay kit, purchased from Beyotime International (Shanghai, China). C11-BODIPY581 / 591 cells were purchased from Thermo Fisher Scientific. 3T3 and 4T1 cells were purchased from Wuhan Pronosei Life Sciences Co., Ltd. Female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Unless otherwise specified, all reagents were analytical grade and ready for use without further purification.

[0054] Materials characterization: Transmission electron microscopy (TEM) imaging was performed on a JEM-2100 field emission transmission electron microscope (Hitachi, Japan). Particle size and potential measurements of the nanomedicine were performed on a nanoparticle size potentiometer (Zetasizer Nano ZS90, Malvern Panaco). UV-Vis absorption spectra were recorded using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies). Absorbance in CCK-8 assays was measured using a microplate reader (EP0CH2, China). Flow cytometry analysis was performed using a Cytoflex (A00-1-1102). Cell images were measured using an FV3000 laser confocal microscope.

[0055] The present invention is further illustrated below by way of examples, but the present invention is not limited to the examples described herein.

[0056] Example 1: Synthesis of the thioketone linker (TK).

[0057] 1.25 mL (1.43 g, 13.5 mmol) of 3-mercaptopropionic acid was transferred to 2 mL of anhydrous acetone (1.57 g, 27 mmol), followed by the addition of 0.67 mL of trifluoroacetic acid. The mixture was stirred in an ice bath for 4 h. The reaction mixture was then filtered to obtain a white solid, which was washed four times with ice-cold hexane and ice-cold deionized water, respectively, to obtain crude thioacetate. The crude product was dissolved in ethyl acetate in a specific ratio and recrystallized upon cooling to obtain thioacetate. The results are as follows: Figure 1 The mass spectrum of TK shows that the peak at m / z = 275.04051 is the molecular ion peak of TK ([M+Na]+), indicating the successful preparation of the thioacetate linker. The TK structure can be further verified by nuclear magnetic resonance spectroscopy. Figure 1 It can be seen that TK has four chemical structures: -COOH, -CH3, -S-CH2-, and -CH2-COOH, containing four different proton peaks. Figure 2 In the results, the proton peak with a chemical shift of 3.3 ppm corresponds to the solvent water peak. The proton peaks with chemical shifts of 1.45 ppm, 2.48 ppm, 2.67 ppm, and 12.21 ppm correspond to four different chemical structures in the thioacetate: -CH3, -CH2-COOH, -S-CH2-, and -COOH, respectively, proving the successful synthesis of the thioacetate bond.

[0058]

[0059] Example 2: Synthesis of ROS-responsive prodrug DSD.

[0060] TK (212 mg, 0.84 mmol) was accurately weighed and dissolved in 15 mL of dichloromethane. EDC (313 mg, 2.02 mmol) and DMAP (24 mg, 0.2 mmol) were then added. The mixture was stirred in an ice bath for 20 min, followed by the addition of DHA (477 mg, 1.68 mmol). The reaction proceeded at room temperature for 72 h. After the reaction was complete, the reaction mixture was concentrated by rotary evaporation. Using a dichloromethane:ethyl acetate mixture of 40:1 as the eluent, the target compound was purified by silica gel column chromatography. The eluent was removed by rotary evaporation, and the compound was dissolved in methanol, diluted with a large amount of purified water, and freeze-dried to obtain a white solid powder, DSD. The results are as follows: Figure 3 The peak m / z=807.34106 in the mass spectrum of DSD is the molecular ion peak of DSD ([M+Na]+), which proves the successful synthesis of the ROS-sensitive prodrug DSD. Figure 4 The proton peak with a chemical shift of 4.98 ppm is the characteristic peak of DHA, specifically the -OH group. When two molecules of DHA undergo esterification with TK, as... Figure 5The characteristic proton peaks of DHA (4.98 ppm -OH) and TK (12.21 ppm -COOH) disappeared, forming a two-molecule ester bond (-COO-). Simultaneously, a slight shift in the chemical shift of the TK characteristic proton peak was observed, from 1.45 ppm (-CH3), 2.48 ppm (-CH2-COOH), and 2.67 ppm (-S-CH2-) to 1.52 ppm (-CH3), 2.69 ppm (-CH2-COOH), and 2.79 ppm (-S-CH2-). Furthermore, the chemical shift of the DHA proton characteristic peak was observed to shift slightly from 5.42 ppm (R1O-CH-OR2) and 6.24 ppm (-CH-OR3) to 5.54 ppm (R1O-CH-OR2) and 5.68 ppm (-CH-OR3). These findings demonstrate the successful synthesis of the ROS-responsive prodrug DSD.

[0061]

[0062] Example 3: Optimization of the preparation of self-assembled nanoparticles.

[0063] A precise amount of heme chloride was weighed into a bottle and dissolved in DMSO to obtain a heme chloride stock solution, which was stored at -20 ℃. The prodrug and DSPE-mPEG2000 were precisely weighed and mixed with 200 μL of heme chloride stock solution in different proportions. After thorough dissolution, the mixture was added dropwise to an appropriate volume of pure water and stirred vigorously for 10 min. The mixture was then placed in an ultrasonic cell disruptor and sonicated at 60 W (3 s operation, 4 s interval) for 10 min. Finally, the sonicated samples were placed in a centrifuge (5000 rpm, 10 min) to remove insoluble free drug, yielding nanoparticles with different drug ratios.

[0064] Nanoparticles were prepared according to the method in Example 3 by setting different feed ratios of hemin chloride to prodrug molecules. The particle size, PDI, drug loading, and encapsulation efficiency were used as evaluation indicators. Different feed ratios of Hemin:Prodrug:DSPE-mPEG were set. 2000 The effects of different drug ratios (4:1:4, 4:2:4, and 1:1:1) on the self-assembly of nanoparticles in water were further investigated, as shown in Table 1. When the drug ratio was 1:1:1, the prepared nanoparticles had a larger particle size and a PDI greater than 0.3. When the drug ratios were 4:1:4 and 4:2:4, the nanoparticle sizes were 119.45 nm and 121.77 nm, respectively, and the PDIs were both less than 0.3. Although the nanoparticles with a drug ratio of 4:1:4 had more stable particle size and PDI, the encapsulation efficiency and drug loading of the prodrug molecules were lower. However, when the drug ratio was 4:1:4, the nanoparticle size and PDI were more stable, the encapsulation efficiency and drug loading of the prodrug molecules were lower. 2000When the feed ratio is 4:2:4, the resulting nanoparticles have uniform particle size and PDI distribution, and the synergistic effect of the two drugs is optimal. Therefore, when preparing nanoparticles, a formulation with a Hemin:Prodrug feed ratio of 2:1 can produce the best anti-tumor effect.

[0065] Table 1. Results of Drug Ratio Screening

[0066]

[0067] Drug-to-carrier ratio:

[0068] DSPE-mPEG2000 combines the amphiphilic properties of phospholipids with the hydrophilic polymer properties of PEG, and its application in drug carriers such as long-circulating nanoparticles, long-circulating liposomes, and polymeric micelles has seen rapid development in recent years. DSPE-mPEG2000 can improve the encapsulation stability, plasma stability, and drug loading of nanoparticles, reduce the probability of clearance by the reticuloendothelial system, and increase their residence time in the bloodstream, allowing more nanoparticles to enter tumor tissues through enhanced permeability and retention. As shown in Table 2, three different ratios of drug to carrier were set: high, medium, and low. As the ratio of DSPE-mPEG2000 was gradually increased, the particle size and PDI of the formed nanoparticles gradually decreased, the nanoparticles became more stable, and the encapsulation efficiency of the drug gradually increased. When the Hemin:Prodrug:DSPE-mPEG2000 feed ratio was 4:2:4, the particle size of Hemin@DSD NPs was 118.58 nm, the PDI was 0.062, the drug loading of Hemin was 69.46%, and the encapsulation efficiency was 98.65%. The drug loading of DSD was 39.23%, and the encapsulation efficiency was 67.62%. The encapsulation efficiency of the prodrug was significantly lower than that of Hemin. This may be related to the extremely poor water solubility of DHA. The poor water solubility of DHA prevents it from acting as a hydrophobic core for encapsulation within DSPE-mPEG2000; instead, it precipitates directly, packaging the DHA as a prodrug. This significantly improves the water solubility of DHA, resulting in a lower encapsulation efficiency. Therefore, the optimal dosage ratio of Hemin:Prodrug:DSPE-mPEG2000 in the final formulation was 4:2:4.

[0069] Table 2. Results of Drug / Carrier Ratio Screening

[0070]

[0071] Example 4: Preparation of self-assembled nanoparticles.

[0072] A precise amount of heme chloride was weighed into a bottle and dissolved in DMSO to obtain a heme chloride stock solution, which was stored at -20 ℃. The prodrug and DSPE-mPEG2000 were precisely weighed and mixed with 200 μL of heme chloride stock solution in different proportions. After being fully dissolved, the solution was added dropwise to an appropriate volume of pure water and stirred vigorously for 10 min. Then, the solution was placed in an ultrasonic cell disruptor and sonicated at 60 W (3 s working, 4 s interval) for 10 min. Finally, the ultrasonically treated sample was placed in a centrifuge (5000 rpm, 10 min) to remove insoluble free drug and obtain self-assembled nanoparticles.

[0073] like Figure 6 As shown in AB, the prepared nanoparticles have a particle size of approximately 100 nm and a negative charge, which effectively prevents them from being eliminated by the human body and provides stronger penetration. The morphology of Hemin@DSD NPs was observed using transmission electron microscopy. The Hemin@DSD NPs nanoparticles are uniform in shape and spherical in appearance. Figure 7 As shown.

[0074] Example 5: Stability of self-assembled rice grains.

[0075] This example examines the storage time and circulating fate of nanoparticles by studying their stability under conditions of storage and plasma, using PBS solution at 4 °C to simulate storage conditions. Figure 8 As shown, the particle size of Hemin@DSD NPs increased slightly over 7 days, but remained below 150 nm, with a PDI of less than 0.3. Body fluid conditions were simulated using a solution containing 10% FBS at 37 °C. Figure 9 As shown, within 48 hours, although the particle size of Hemin@DSD NPs fluctuated slightly over time, it remained within 140 nm, and the PDI was less than 0.3. These results indicate that Hemin@DSD NPs have good stability, can be stored for a long time, and maintain stable particle size and structural integrity in body fluid circulation, making them easily taken up by tumor tissue and achieving the EPR effect.

[0076] Example 6: Safety study of self-assembled rice grains.

[0077] The in vivo safety of the obtained nanoparticles was preliminarily evaluated through a hemolysis experiment. Physiological saline was used as a negative control, and purified water as a positive control. Solutions with different concentrations of nanoparticles were prepared. Since Hemin solution is red, it was used as a control to minimize color error. Figure 10 It can be observed that regardless of the concentration of the nanoparticle solution, after adding 2% red blood cell suspension, the red blood cells all settle to the bottom. The red color of the solution is due to the color of Hemin itself and is not related to hemolysis, indicating that Hemin@DSD NPss has good biocompatibility.

[0078] Example 7: In vitro drug release of self-assembled nanoparticles under different conditions.

[0079] This embodiment employs dialysis to investigate the in vitro drug release of nanoparticles. H2O2 is used to simulate the high ROS environment of tumor tissue, and different H2O2 concentrations are set at pH 7.4 to explore the release properties of the ROS-responsive nanoparticles Hemin@DSD NPs. Figure 11 As shown, under different concentrations of H2O2, the cumulative release rate of free Hemin reached 90% after 2 h, indicating that the release of Hemin is not affected by the dialysis bag retention and meets the leakage conditions, making it suitable for subsequent nanoparticle release studies. Under different concentrations of H2O2, the release of Hemin@DSD NPs after 24 h was positively correlated with the H2O2 concentration. Under 0, 5, and 10 mM H2O2 conditions, the cumulative release rate of Hemin@DSD NPs after 24 h was approximately 30%, 50%, and 80%, respectively, indicating that Hemin@DSD NPs rapidly disintegrate and release drugs quickly under ROS conditions. This demonstrates that Hemin@DSD NPs are ROS-responsive, stable in body fluid circulation, and can precisely release drugs at tumor sites.

[0080] Example 8: Investigation of ROS responsiveness of nanoparticles.

[0081] The prodrug DSD prepared in this embodiment is ROS-responsive, causing internal TK fragmentation. To further verify the ROS-responsiveness of the prepared nanoparticles, the nanoparticles were incubated with a 10 mM H2O2 aqueous solution, and the morphology of the nanoparticles was observed by TEM. Figure 12 After incubation with H2O2, the complete nanostructure could not be observed within the field of view, indicating that the nanostructure completely disintegrated and the drug was released rapidly. In summary, Hemin@DSD NPs have ROS responsiveness. After being taken up by tumor tissue, the nanostructure is destroyed, and the internal drug is released, thus exerting its anti-tumor effect.

[0082] Example 9: Cytotoxicity of different formulations on mouse 4T1 cells.

[0083] Healthy 4T1 cells were digested and resuspended, and seeded at a density of 3500 cells per well in 96-well plates for 24 h. The culture medium was then discarded, and incomplete culture medium containing free Hemin, DHA, Hemin@DSD NPs, and Hemin@DCD NPs was added. After culturing for 24 h in a cell culture incubator, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured for another 4 h. The culture medium was then discarded, and 200 μL of formazan dissolved in DMSO was added to each well. The cells were then gently shaken on a shaker in the dark for 15 min until the formazan was completely dissolved. Finally, the absorbance was measured at 570 nm using a microplate reader. The cell viability was calculated by subtracting the absorbance of the blank control wells from the absorbance of each well using the following formula:

[0084]

[0085] Where Abs (treated), Abs (untreated), and Abs (blank) represent the absorbance of the drug-treated wells, control wells, and blank wells, respectively. The MTT assay was used to investigate the effects of each single drug and formulation on 4T1 cell viability, and the results are as follows: Figure 13 As shown, DHA has relatively low cytotoxicity; it only induces cell death at high concentrations, while low concentrations are almost non-toxic. At high concentrations, DHA has a greater impact on cell viability than DSD. This may be because DSD requires a response to intracellular ROS to break bonds and release DHA, while TK is a weak ROS-responsive bond. The breaking of the TK bond may be the rate-limiting step for DSD to exert its effect, while naked DHA can exert its effect rapidly after entering the cell, resulting in higher toxicity at high concentrations. In contrast, the TK in DSD can respond to ROS breaking in tumor cells, reducing steric hindrance and further promoting ester bond hydrolysis, releasing DHA to exert its effect. At low concentrations, the effects of both DHA and DSD on cell viability are above 80%, and they can be considered safe for cells. Nanoparticle formulations improve the poor water solubility of DHA, and Hemin@DSD NPs nanoparticles have a greater impact on cell viability, showing stronger cytotoxicity; and due to the synergistic effect of Hemin and DSD, Hemin@DSD NPs are more toxic than Hemin+DHA, and the difference is significant, proving that Hemin@DSD NPs have superior tumor killing ability.

[0086] Example 10: Effects of different formulations on ATP levels in tumor cells.

[0087] 4T1 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 6Cells were seeded at a density of 10 cells / well in 6-well plates and cultured until 80% confluence. The nanoparticles prepared in Example 2, along with the free drug, a mixture of the free drug and free lactoferrin, were added to each well, with a DHA concentration of 37.5 μg / mL (based on diluted DHA content). The final nanoparticle solution was also added to each well, with a DHA concentration of 37.5 μg / mL (based on diluted DHA content). Three replicates were set up, with 200 μL per well. After 24 hours of incubation, cells were collected and centrifuged. The supernatant was used for detection, and cells were lysed using RIPA cell lysis buffer (Saiwell, China). After lysis, cells were centrifuged at 12000g for 10 minutes at 4°C, and the supernatant was collected. To eliminate errors caused by variations in protein content during sample preparation, protein concentration was determined according to the BCA protein assay kit instructions, and ATP concentration was determined according to the ATP assay kit instructions.

[0088] The results are as follows Figure 14 As shown, the release of ATP from dying cells into the extracellular space constitutes one of the main markers of ICD. It can be seen that free dihydroartemisinin can induce the release of ATP into the extracellular space. The effect of the formulation group is significantly better than that of the free dihydroartemisinin group, which proves that the formulation induces ICD more significantly. The anti-tumor pathway of inducing ICD through ferroptosis has obvious advantages.

[0089] Example 11: Effects of different formulations on HMGB1 levels in tumor cells.

[0090] 4T1 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 6 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured until 80% confluence. The nanoparticles prepared in Example 2, along with the free drug, a mixture of the free drug and free lactoferrin solution, were added to each well. The DHA concentration was diluted to 37.5 μg / mL. Three replicates were set up, with 200 μL added to each well. After 24 hours of incubation, the cell samples were centrifuged (500×g, 5 minutes) to collect the supernatant. Simultaneously, cells were lysed using RIPA cell lysis buffer (Saiwell, China). After lysis, the cells were centrifuged at 12000g for 10 minutes at 4°C, and the supernatant was collected. To eliminate errors caused by variations in protein content during sample preparation, protein concentration was determined according to the BCA protein assay kit instructions. HMGB1 concentration in the cell sample supernatant was determined according to the HMGB1 assay kit instructions.

[0091] The results are as follows Figure 15As shown, the release of high-mobility group box 1 (HMBG1) is also one of the main markers of ICD. It can be seen that free dihydroartemisinin can induce the release of HMBG1 into the extracellular space, while there is no significant difference between the free heme group and the control group. The effect of the preparation group is significantly better than that of the free dihydroartemisinin group, indicating that the combined use of the two drugs significantly enhances the anti-tumor effect.

[0092] Example 12: Effects of different formulations on the exposure level of calreticulin in tumor cells.

[0093] Healthy 4T1 cells were digested, resuspended, and seeded at a density of 2 × 10⁵ cells per well in 12-well plates and cultured for 24 h. The culture medium was then discarded, and incomplete culture medium containing free Hemin, DHA, Hemin@DHA, and Hemin@DSD NPs was added. Untreated blank 4T1 cells were used as a control, and the cells were cultured in a cell culture incubator for 24 h. The drug-containing culture medium was then discarded, and the cells were washed three times with pre-chilled PBS. 200 μL of trypsin was added, and the cells were digested at 37 °C. After 3 min, 1 mL of complete culture medium was added to terminate the digestion reaction. The cell pellet was collected by centrifugation, resuspended in PBS, and repeatedly pipetted and aspirated. The cell pellet was then collected by centrifugation. 200 μL of CRT antibody dilution buffer was added, and the cells were incubated at room temperature on a horizontal shaker for 3 h. The antibody was recovered, washed three times with pre-chilled PBS, and 200 μL of Cy5-labeled secondary antibody dilution buffer was added. The cells were incubated at room temperature on a horizontal shaker for 30 min. After incubation, the secondary antibody was recovered, and the cells were washed three times with pre-cooled PBS. Finally, the cells were resuspended in 200 μL of PBS and analyzed using flow cytometry. The intracellular fluorescence intensity was analyzed using FlowJo software.

[0094] Figure 16 To quantitatively investigate CRT translocation by flow cytometry, the results showed that both Hemin and DHA can induce CRT translocation on the cell membrane surface. Strong fluorescence signals were collected from Hemin@DSD NPs, indicating that nanoparticles can induce significant CRT translocation.

[0095] Example 13: 4T1 cell uptake of nanomedicine.

[0096] The therapeutic efficacy of nanomedicines depends on rapid intracellular uptake and effective accumulation. Therefore, the cellular uptake of Cy5-labeled NPs (the preparation method for C6-labeled NPs was the same as in Example 4, except that DHA was replaced with Cy5-labeled DHA) was evaluated using laser scanning confocal microscopy (LSCM). Figure 17 As shown, the intensity of red fluorescence gradually increases over time, indicating that cellular uptake is time-dependent.

[0097] Example 14: Effects of different formulations on the maturation of dendritic cells.

[0098] Healthy 4T1 cells were digested, resuspended, and seeded at a density of 5.10⁵ cells per well in 6-well plates and cultured for 24 h. The culture medium was then discarded, and incomplete culture medium containing free Hemin, DHA, Hemin@DHA, Hemin@DSD NPs, and Hemin@DCDNPs was added. Untreated blank 4T1 cells were used as a control, and the cells were cultured in a cell culture incubator for 24 h. Simultaneously, healthy DC2.4 cells were digested, resuspended, and seeded at a density of 2.10⁵ cells per well in 12-well plates and cultured for 24 h. The DC2.4 cell culture medium was then discarded, and 4T1 cell supernatant was added to the DC2.4 cells for co-culture. Lipopolysaccharide (LPS) was used as an inducer for DC maturation, and a positive control group was set up to observe the effect of nanoparticle-induced DC2.4 cell maturation.

[0099] After 24 h of culture, the cell supernatant was discarded, and the cells were washed three times with pre-chilled PBS. 200 μL of trypsin was added, and the cells were digested at 37°C for 3 min. Afterward, 1 mL of complete culture medium was added to terminate the digestion reaction. The cells were then centrifuged at 1000 rpm for 3 min, resuspended in PBS, and centrifuged again at 1000 rpm for 3 min to obtain the lower cell layer. Subsequently, 200 μL of Fc receptor blocker was added, and the cells were incubated at 4°C for 10 min. After incubation, the cells were washed three times with pre-chilled PBS. Then, according to the antibody instructions, the Brilliant Violet 421™-CD80, APC-CD86, and PE-CD11c antibodies were diluted and incubated at 4°C in the dark for 30 min. The antibodies were recovered, and the cells were washed three times with pre-chilled PBS. Finally, the cells were resuspended in 200 μL of PBS and analyzed using flow cytometry. The fluorescence intensity of the cells was analyzed using FlowJo software.

[0100] like Figure 18Using CD11c-positive cells as the research object, CD80 and CD86 are co-stimulatory molecules on the surface of dendritic cells (DCs). When DCs mature, they exhibit double positivity for both CD80 and CD86. When Hemin and DHA are administered alone, the level of DAMPs produced is insufficient to induce DC maturation, and no cell population in the double-positive region can be observed. However, due to the synergistic effect of Hemin and DHA, when the two are administered together, they can stimulate the production of sufficient levels of DAMPs to promote DC maturation. Furthermore, when cancer cells were stimulated using the Hemin@DSD NPs formulation, the maturation rate of DCs was significantly higher than that of other groups. This is because the Hemin@DSD NPs formulation can induce cancer cells to produce the highest level of DAMPs. The results show that under the action of Hemin@DSD NPs, the exposure of CRT on the cell membrane surface, the release of HMGB1 and ATP in 4T1 cells were significantly higher than those in other groups. This can maximize the recruitment of DCs and complete the recognition of surface receptors and antigen presentation, promote the phagocytosis, processing and presentation of TAAs by DCs, and induce DCs to mature and participate in systemic adaptive immunity.

[0101] Example 15: Effects of different formulations on the expression of PD-L1 protein in 4T1 cells.

[0102] Healthy 4T1 cells were digested, resuspended, and seeded at a density of 5.10⁵ cells per well in 6-well plates and cultured for 24 h. The culture medium was then discarded, and incomplete culture medium containing free Hemin, DHA, Hemin@DHA, Hemin@DSD NPs, and Hemin@DCDNPs was added. Untreated blank 4T1 cells were used as a control, and the cells were cultured in a cell culture incubator for 24 h. The drug-containing culture medium was then discarded, and the cells were washed three times with pre-chilled PBS. 50 μL of RIPA (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added, and the cells were lysed on ice for 20 min. The cell lysate was then collected in a 1.5 mL EP tube using a cell scraper and further lysed using an ultrasonic probe at 100 W for 3 s. Finally, the protein supernatant was obtained by centrifugation at 12000 rpm, 4℃. After quantifying the collected protein supernatant using a BCA protein quantification kit, the proteins from each group were diluted to the same concentration using RIPA lysis buffer. Loading buffer was added, and the proteins were boiled in a water bath for 5 min to ensure complete denaturation and development of the three-dimensional structure. After cooling to room temperature, the protein samples were stored at -80 ℃. A 10% gel electrophoresis kit was used to prepare PAGE gels, with 5 μL of protein loaded per well. 4 μL and 2 μL of marker were added to each side to indicate protein molecular weight. Electrophoresis was performed at 200 V for 40 min. The rapid transfer buffer was then diluted 8 times with pure water, and an equal volume of ethanol was added and mixed thoroughly to construct a "sandwich" structure. Transfer was performed at 400 mA for 25 min. After transfer, the PVDF membrane was rinsed once with TBST, and then blocked with rapid blocking buffer for 20 min. After blocking, the membrane was rinsed with TBST for 10 min, and this process was repeated three times. Following the antibody manufacturer's instructions, the PD-L1 polyclonal antibody and GAPDH monoclonal antibody were appropriately diluted and incubated overnight at 4 ℃. The antibody was recovered the next day, and the membrane was rinsed with TBST for 10 min, repeated three times. Then, the HRP-labeled secondary antibody was appropriately diluted with TBST and incubated at room temperature for 1 h. Finally, the secondary antibody was recovered, and the membrane was rinsed with TBST for 10 min, repeated three times. ECL luminescence working solution was prepared, and 200 μL of developing working solution was added to the membrane to make the liquid evenly cover the entire membrane surface. The membrane was exposed using a developing instrument and the band image was captured.

[0103] pass Figure 19 Western blot analysis was performed to examine the total intracellular PD-L1 protein content, and the results were as follows: Figure 3-9As shown in Figures B and C, after administration of Hemin@DSDNPs, the total intracellular PD-L1 level decreased significantly, with semi-quantitative analysis revealing a reduction to approximately one-third of the normal level. The nanoparticles induced immunogenic cell death in 4T1 cells while simultaneously downregulating PD-L1 on the cell surface, reducing the binding of cancer cells to T cells, and preventing 4T1 immune escape.

[0104] Example 16: In vivo antitumor experiments of different formulations.

[0105] 4T1 cell suspension (1×10⁶ cells / 100 μL) was inoculated under the fourth mammary fat pad of female Balb / C mice. When the tumor volume reached 150 mm³, tumor-bearing mice were randomly divided into groups of five, receiving saline, free Hemin, free DHA, free DSD, Hemin+DHA, or Hemin@DSD NPs, respectively. Administered every 3 days for 6 consecutive days. The dosage, calculated based on dihydroartemisinin, was 10 mg / kg. Using 4T1-Balb / C tumor-bearing mice as an animal experimental model, tumor volume growth and body weight changes were the main indicators for evaluation. Results are as follows: Figure 20 A. Compared with the saline group, free DHA did not show a significant tumor-suppressing effect. This may be because the administered concentration of DHA was much lower than the IC50 value of 4T1, thus failing to achieve a tumor-suppressing effect. Both free Hemin and Hemin@DHA inhibited tumor growth, with inhibition rates of approximately 37.5%, 45.9%, and 50%, respectively, indicating that the combined administration of Hemin and DHA effectively enhanced the anti-tumor effect. Hemin@DSD NPs showed the best anti-tumor effect; during the administration period, no significant tumor growth was observed in the Hemin@DSD NPs group, with an inhibition rate reaching 91.7%. This indicates that ROS-responsive nanoparticles can fully release DHA, exerting a synergistic effect between Hemin and DHA, resulting in significant tumor inhibition. After administration, mice were sacrificed, tumor tissue was dissected and photographed. Figure 20As shown in -C, consistent with the tumor volume results, it indicates that DHA alone cannot produce a tumor-suppressive effect. This suggests that the DAMPs signal generated by DHA at low concentrations is weak and cannot induce ICD. Although DHA can downregulate PD-L1 and alleviate immunosuppression, breast cancer is a "cold tumor" with poor immunogenicity. Therefore, without the ICD effect to transform it into a "hot tumor," the tumor-suppressive effect of DHA is very poor. However, the combined administration of DHA and Hemin significantly inhibits tumor growth. This indicates that Hemin stimulates immunogenic death of tumor cells, generating DAMPs signals and transforming the "cold tumor" into a "hot tumor." DHA, on the one hand, synergizes with the ICD effect of Hemin, and on the other hand, alleviates the strong immunosuppression caused by Hemin, downregulates PD-L1 expression, and promotes the immune system to play an anti-tumor role, resulting in a significantly enhanced tumor-suppressive effect. Among them, Hemin@DSD NPs showed the best antitumor effect and was significantly different from the other groups. This indicates that ROS-responsive nanoparticles can achieve long-term circulation in vivo, reach high nanoparticle accumulation at the tumor site, and respond to the high ROS level of tumor cells, release the drug completely, and exert the body's immune antitumor effect.

[0106] Changes in body weight of tumor-bearing mice in each group during drug administration are as follows: Figure 20 As shown in Figure B, the body weight of mice in the saline group and different drug administration groups did not decrease significantly, indicating that no significant systemic toxicity was caused in any group, and the biosafety was good.

Claims

1. A self-assembling targeted nanomedicine with anti-tumor properties, characterized in that: ROS-sensitive prodrugs were linked by reactive oxygen species-responsive thioketal bonds, and the prodrugs were then self-assembled with heme chloride and distearate phosphatidyl acetamide-methoxy polyethylene glycol 2000 in water to form ROS-responsive nanoparticles.

2. The anti-tumor self-assembling targeted nanomedicine according to claim 1, characterized in that: The ROS-sensitive prodrug DSD is two molecules of dihydroartemisinin.

3. The anti-tumor self-assembly targeted nanomedicine according to claim 2, characterized in that: The monomer chemical structure of the ROS-sensitive prodrug is shown below: or .

4. A self-assembled, targeted nanomedicine with anti-tumor properties according to claim 1 or 2, characterized in that: The ROS-responsive bond in the aforementioned ROS-sensitive prodrug is a thioacetal bond, and the synthetic steps of the thioacetal linker TK are as follows: 1.25 mL (1.43 g, 13.5 mmol) of 3-mercaptopropionic acid was transferred to 2 mL of anhydrous acetone (1.57 g, 27 mmol), and then 0.67 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 4 h. The mixture was then filtered to obtain a white solid, which was washed four times with ice-cold hexane and ice-cold deionized water to obtain crude thioacetate. The crude product was dissolved in ethyl acetate in a certain proportion and then cooled and recrystallized to obtain thioacetate. 。 5. A self-assembled, targeted nanomedicine with anti-tumor properties according to claim 1 or 2, characterized in that: The method for preparing the ROS-sensitive prodrug is as follows: Accurately weigh 212 mg (0.84 mmol) of TK and dissolve it in 15 mL of dichloromethane. Then add 313 mg (2.02 mmol) of EDC and 24 mg (0.2 mmol) of DMAP. Stir in an ice bath for 20 min, then add 477 mg (1.68 mmol) of DHA and react at room temperature for 72 h. After the reaction is complete, concentrate the reaction system by rotary evaporation. Use a mixed solution of dichloromethane and ethyl acetate (40:1) as the eluent and separate and purify the target compound by silica gel column chromatography. Remove the eluent by rotary evaporation, dissolve in methanol, dilute with a large amount of purified water, and freeze-dry to obtain a white solid powder, DSD. 。 6. The anti-tumor self-assembly targeted nanomedicine according to claim 1, characterized in that: Hemin chloride is a commonly used iron supplement compound for the human body, and its chemical structure is as follows: 。 7. A method for preparing a self-assembled, targeted nanomedicine with anti-tumor properties as described in any one of claims 1-6, characterized in that, The steps are as follows: Accurately weigh heme chloride into a round-bottom flask, then dissolve it in 5 mL of DMSO to obtain a heme chloride stock solution, which is stored at -20 ℃. Accurately weigh the prodrug and distearate phosphatidylacetamide-methoxy polyethylene glycol 2000, mix them with 200 μL of heme chloride stock solution, and after thorough dissolution, add the solution dropwise to an appropriate volume of pure water. Stir vigorously for 10 min, then place the solution in an ultrasonic cell disruptor at 60 W for 3 s, with a 4 s interval, and sonicate for 10 min. Finally, place the sonicated sample in a centrifuge at 5000 rpm for 10 min to remove insoluble free drug, obtaining self-assembled nanoparticles.

8. The method for preparing a self-assembled, targeted nanomedicine with anti-tumor properties according to claim 7, characterized in that: The organic solvent for dissolving the ROS-sensitive prodrug and heme chloride is one or more of dimethyl sulfoxide, ethanol, methanol, acetone, and tetrahydrofuran.

9. The application of the self-assembled targeted nanomedicine as described in claim 1 or 2 in the preparation of tumor therapeutic drugs.

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