Artesunate prodrug and its preparation method and application

Abstract

The present invention belongs to the field of medical technology, and relates to artesunate prodrugs, preparation methods and applications thereof, and specifically to reduction-responsive artesunate prodrugs, preparation methods thereof and applications in the preparation of drug delivery systems. The structure of the artesunate prodrug is as follows, n=1-3. The artesunate prodrug of the present invention can be self-assembled into nanoparticles in combination with a photosensitizer, wherein the photosensitizer is one or more of dihydrochlorin e6, chlorophyll a, and pheophytin a, and the mass ratio of the artesunate prodrug to the photosensitizer is 10:1 to 1:10. The self-assembled nanoparticles of the artesunate prodrug combined with the photosensitizer Ce6 of the present invention can be used for cancer treatment. Under the stimulation of excessive glutathione (GSH) in the tumor microenvironment, the nanoparticles disintegrate, significantly alleviating the quenching effect caused by the aggregation of Ce6, and the reactive oxygen species (ROS) generated by the photosensitizer under laser irradiation and the ROS generated by ferroptosis induced by artesunate synergistically promote the death of tumor cells, thereby improving the combined therapeutic effect of ferroptosis and photodynamic therapy. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to an artesunate prodrug and a preparation method and application thereof, and particularly relates to a reduction-responsive artesunate prodrug and a preparation method thereof and application thereof in preparing a drug delivery system. Background Art

[0002] Artesunate (ART) is an artemisinin derivative extracted from Artemisia annua Linn., a plant of the Asteraceae family. Its chemical formula is C 19 H 28 O8, easily soluble in ethanol, acetone or chloroform, very slightly soluble in water.

[0003]

[0004] Currently, artesunate is widely used worldwide to treat mild to severe malaria. Increasing evidence suggests that, in addition to its antimalarial effects, artesunate also exhibits anticancer properties. Artesunate exerts anti-tumor effects by impairing angiogenesis, inhibiting cell invasion and migration, inducing cell cycle arrest, upregulating reactive oxygen species (ROS) levels, and modulating signal transduction pathways, thereby inducing apoptosis, differentiation, and autophagy in colorectal cancer cells and blocking immune evasion. Furthermore, artesunate has been shown to restore the sensitivity of various cancer types to chemotherapeutic drugs by modulating various signaling pathways. For example, it improves apoptosis in hepatocellular carcinoma by inhibiting the PI3K / Akt / mTOR pathway and increases the sensitivity of hepatocellular carcinoma cells to sorafenib by inhibiting the MEK / ERK pathway. Artesunate exerts its anti-colorectal cancer effects by inducing apoptosis, inhibiting cancer cell proliferation, invasion, and metastasis, inhibiting neovascularization, promoting ROS production, and combating inflammation and oxidative stress.

[0005] Colon cancer is a type of gastrointestinal cancer. In recent years, with the gradual improvement of living standards and changes in lifestyles worldwide, the incidence of colon cancer has shown an alarming upward trend. Approximately 1.36 million new patients are diagnosed with colorectal cancer each year, and the death toll exceeds 500,000. Because colon cancer is difficult to detect, approximately 50% to 60% of patients are diagnosed in the advanced stage, and 80% to 90% of patients will develop liver metastasis, resulting in a high mortality rate. Current treatments for colon cancer include surgical and non-surgical methods. Surgery is the most direct and effective, but it is difficult to achieve a complete cure and has a high recurrence rate. Among non-surgical tumor treatments, chemotherapy remains a common treatment for colon cancer. However, the use of single chemotherapy drugs has numerous toxic effects, a single target, and poor drug resistance.

[0006] In recent years, nanotechnology has been widely used in the treatment of colorectal cancer. However, most drugs are non-covalently encapsulated in nanocarriers, resulting in low drug loading, drug leakage, and carrier-related toxicity. Furthermore, complex preparation techniques are widely considered one of the major obstacles hindering the successful clinical translation of most conventional nanomedicines. Therefore, scientists are committed to developing simple and efficient nanodelivery systems for the treatment of colorectal cancer.

[0007] Because conventional nanomedicines have many shortcomings, researchers are committed to developing carrier-free prodrug self-assembling nanoparticles. These nanoparticles offer significant advantages, including ease of preparation, good reproducibility, high drug loading capacity, and negligible carrier-induced toxicity, providing a promising approach for cancer treatment.

[0008] Ferroptosis is an iron-dependent cell death process characterized by the massive accumulation of lethal lipid reactive oxygen species (ROS). This process is significantly influenced by ferrous ions, which mediate the Fenton reaction. The Fenton reaction converts excess hydrogen peroxide in the tumor microenvironment into hydroxyl radicals and oxygen. The generated hydroxyl radicals trigger a series of free radical chain reactions, ultimately leading to increased intracellular ROS levels and oxidative damage to cells. In addition, ferroptosis leads to the production of highly toxic phospholipid hydroperoxides (PLOOH) by consuming intracellular glutathione (GSH) and glutathione peroxidase 4 (GPX4). Artesunate can exert its anti-tumor effects through the ferroptosis mechanism. As a component of traditional Chinese medicine, it has fewer toxic side effects than chemical drugs.

[0009] After intravenous injection of artesunate, the blood concentration of artesunate decreases rapidly. 1 / 2 The drug release period is approximately 30 minutes. Artesunate is widely distributed in the body, with higher concentrations in the intestines, liver, and kidneys. It undergoes primary metabolic transformation in the body, with only small amounts excreted in urine and feces. Producing it as a prodrug and then preparing it into a nanoformulation can overcome issues such as artesunate's poor hydrophobicity, poor water solubility, low drug loading due to polymer encapsulation, drug leakage, and excipient-related toxicity.

[0010] Photodynamic therapy (PDT) involves the transfer of energy and electrons from a photosensitizer to functional intracellular macromolecules under light irradiation, generating ROS that can kill cancer cells. PDT has become a promising and developing cancer treatment modality due to its advantages such as strong controllability and low toxicity.

[0011] The combination of ferroptosis and PDT is an effective strategy to improve the efficiency of tumor treatment through the synergistic effect of ferroptosis drugs and photosensitizers. Therefore, the research and development of artesunate prodrug-photosensitizer self-assembled nanoparticles for enhancing the synergistic therapeutic effect of ferroptosis and photodynamic therapy is an important topic that needs to be studied urgently.

[0012] There is no report in the prior art on preparing artesunate into a prodrug and combining it with a photosensitizer to self-assemble into nanoparticles for anti-tumor use. Summary of the Invention

[0013] To overcome the shortcomings of the prior art, the present invention provides an artesunate prodrug that can be combined with a photosensitizer to self-assemble into nanoparticles, thereby achieving high drug loading, good stability, low toxicity and side effects, and targeted release at tumor sites. The artesunate prodrug is a reduction-responsive artesunate prodrug.

[0014] The present invention is achieved through the following technical solutions:

[0015] The present invention provides an artesunate prodrug or a pharmaceutically acceptable salt thereof having the following structure:

[0016]

[0017] Wherein, n=1-3.

[0018] The present invention preferably uses an artesunate prodrug or a pharmaceutically acceptable salt thereof having the following structure:

[0019]

[0020] The present invention provides a method for preparing the reduction-responsive artesunate prodrug or a pharmaceutically acceptable salt thereof. The method for preparing ASSFe comprises the following steps:

[0021] (1) Artesunate, 2-hydroxyethyl disulfide, 4-(dimethylamino)pyridine (DMAP) and dichloromethane are placed in a reaction vessel and stirred until the solid is completely dissolved; a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) is added dropwise to the reaction system under ice bath conditions. After the addition is complete, the mixture is stirred at room temperature until the reaction is complete, and the product ASSOH is obtained by separation and purification;

[0022] (2) Ferrocenecarboxylic acid is added to oxalyl chloride to prepare ferrocenylcarbonyl chloride; under ice bath conditions, a dichloromethane solution of ferrocenylcarbonyl chloride is added dropwise to a dichloromethane solution of ASSOH and N,N-diisopropylethylamine (DIPEA). After the addition is complete, the mixture is stirred at room temperature until the reaction is complete, and the product ASSFe is obtained by separation and purification.

[0023]

[0024] In the step (1), the molar ratio of artesunate, 2-hydroxyethyl disulfide, DMAP and EDCI is 1:(1-5):(0.5-3):(1-5).

[0025] In the step (2), the molar ratio of ASSOH, ferrocenecarboxylic acid, and DIPEA is 1:(1-5):(1-5).

[0026] The preparation method of ASSA comprises the following steps:

[0027] (1) Artesunate and EDCI were added to a reaction vessel, dissolved in dichloromethane, and stirred in an ice bath for 1-4 hours;

[0028] (2) Add 2-hydroxyethyl disulfide and DMAP to the reaction vessel of (1), continue to add dichloromethane, stir at room temperature for 1-24 hours, and separate and purify by column chromatography to obtain the prodrug ASSA.

[0029]

[0030] In the step (1), the molar ratio of artesunate to EDCI is 1:(1-5).

[0031] In the step (2), the molar ratio of 2-hydroxyethyl disulfide to DMAP is 1:(0.2-2).

[0032] The artesunate prodrug of the present invention can be combined with a photosensitizer to self-assemble into nanoparticles, wherein the photosensitizer is one or more of chlorin e6, chlorophyll a, and pheophytin a, preferably chlorin e6;

[0033] The mass ratio of the artesunate prodrug to the photosensitizer is 10:1 to 1:10, preferably 10:1 to 3:1, and more preferably 4:1 to 5:1.

[0034] The self-assembled nanoparticles are prepared by the following method:

[0035] Artesunate prodrug, photosensitizer and PEG modifier are weighed, dissolved in DMSO solvent, slowly added dropwise into deionized water under stirring, and then the organic solvent is removed by dialysis to obtain the product.

[0036] The photosensitizer is a porphyrin photosensitizer, preferably one or more of dihydrochlorin e6, chlorophyll a, and pheophytin a.

[0037] The PEG modifier is one of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) or distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FA), and the molecular weight of PEG is 200-20000, preferably 2000-5000.

[0038] Preferably, the PEG modifier is DSPE-PEG 2k or DSPE-PEG 2k-FA.

[0039] The mass ratio of the artesunate prodrug to the photosensitizer is 10:1 to 1:10, preferably 10:1 to 3:1, and more preferably 4:1 to 5:1.

[0040] The mass ratio of the artesunate prodrug to the PEG modifier is 2:1 to 20:1, preferably 2:1 to 10:1, and more preferably 5:1 to 10:1.

[0041] The concentration of the artesunate prodrug is 1-20 mg / mL.

[0042] The present invention provides the use of the reduction-responsive artesunate prodrug or its pharmaceutically acceptable salt or their self-assembled nanoparticles in preparing a drug delivery system.

[0043] The present invention provides the use of the reduction-responsive artesunate prodrug or its pharmaceutically acceptable salt or their self-assembled nanoparticles in the preparation of anti-tumor drugs.

[0044] The present invention provides the use of the reduction-responsive artesunate prodrug or its pharmaceutically acceptable salt or their self-assembled nanoparticles in improving drug efficacy and reducing toxicity.

[0045] The present invention provides the use of the reduction-responsive artesunate prodrug or its pharmaceutically acceptable salt or their self-assembled nanoparticles in the preparation of an injection, oral administration or local administration system.

[0046] The present invention has the following beneficial effects compared to the prior art:

[0047] 1. The present invention synthesizes two artesunate prodrugs and prepares self-assembled nanoparticles of the artesunate prodrugs combined with the photosensitizer Ce6 for cancer treatment. Under the stimulation of excessive GSH in the tumor microenvironment, the nanoparticles disintegrate, significantly alleviating the quenching effect caused by the aggregation of Ce6. In addition, the ROS generated by the photosensitizer under laser irradiation and the ROS generated by artesunate-induced ferroptosis synergistically promote the death of tumor cells, thereby improving the combined therapeutic effect of ferroptosis and photodynamic therapy.

[0048] 2. The artesunate prodrug combined with photosensitizer self-assembled nanoparticles of the present invention achieve technical effects such as high drug loading, good stability, low toxic side effects and targeted release at the tumor site, meeting the urgent clinical demand for high-efficiency and low-toxicity preparations, providing a new strategy for the self-assembly of dimer prodrugs, and providing an effective nano-platform for the development of carrier-free dimer drug nano-delivery systems and ferroptosis-photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1This is the release graph of ART in Ce6@ASSA-FA nanoparticles in Example 3 in solutions with different GSH concentrations;

[0050] Figure 2 The release graph of Ce6 in Ce6@ASSA-FA nanoparticles in Example 3 in solutions with different GSH concentrations;

[0051] Figure 3 This is the CT26 cell uptake diagram of Ce6@ASSFe-FA nanoparticles in Example 3;

[0052] A: Cellular uptake experimental results of Ce6@ASSFe-FA and Ce6@ASSA-FA at 4 h

[0053] B: Cell uptake experimental results of Ce6@ASSFe-FA group at different time periods;

[0054] Figure 4 This is the CT26 cell uptake diagram of Ce6@ASSA-FA nanoparticles in Example 3;

[0055] Figure 5 The cytotoxicity of Ce6@ASSFe-FA and Ce6@ASSA-FA to CT26 cells in Example 3;

[0056] Fc: ferrocenecarboxylic acid L: laser irradiation;

[0057] Figure 6 The cytotoxicity of Ce6@ASSFe-FA and Ce6@ASSA-FA to HT29 cells in Example 3;

[0058] Fc: ferrocenecarboxylic acid; L: laser irradiation;

[0059] Figure 7 The cytotoxicity of Ce6@ASSFe-FA and Ce6@ASSA-FA to HCT116 in Example 3;

[0060] Fc: ferrocenecarboxylic acid; L: laser irradiation;

[0061] Figure 8 Safety investigation of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3;

[0062] Fc: ferrocenecarboxylic acid;

[0063] Figure 9 This is the situation of Ce6@ASSFe-FA generating ROS in CT26 cells in Example 3;

[0064] Control: blank culture medium control group; Fc: ferrocenecarboxylic acid; L: laser irradiation; Fer-1: Ferrostatin-1;

[0065] Figure 10 This is the situation of Ce6@ASSA-FA generating ROS in CT26 cells in Example 3;

[0066] Control: blank culture medium control group; Fc: ferrocenecarboxylic acid; L: laser irradiation; Fer-1: Ferrostatin-1;

[0067] Figure 11 Figure 3 is a graph showing the tumor growth curve of the in vivo anti-tumor experiment of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3; L: laser irradiation;

[0068] Figure 12 Figure 3 shows the tumor images of the in vivo anti-tumor experiments of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3;

[0069] Figure 13 The tumor weight curve of the in vivo anti-tumor experiment of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3; L: laser irradiation;

[0070] Figure 14 This is a graph showing the changes in mouse body weight during the in vivo anti-tumor experiment of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3; L: laser irradiation. DETAILED DESCRIPTION

[0071] Example 1 Synthesis of Reduction-Responsive Artesunate-Ferrocene Prodrug ASSFe

[0072] (1) Synthesis of ASSOH

[0073] Artesunate (1 g, 2.60 mmol, 1 eq), 2-hydroxyethyl disulfide (0.80 g, 5.20 mmol, 2 eq), DMAP (0.16 g, 1.30 mmol, 0.5 eq), and 15 mL of DCM were added to a 50 mL two-necked flask and stirred at room temperature until completely dissolved. 5 mL of a DCM solution of EDCI (0.60 g, 3.12 mmol, 1.2 eq) was slowly added dropwise to the reaction system. After the addition was complete, the reaction was stirred at room temperature and monitored by TLC to terminate the reaction. The reaction solution was washed once with 30 mL of 50% aqueous citric acid and then once with 30 mL of saturated aqueous NaCl. The organic layer was dried over anhydrous MgSO₄. The MgSO₄ solid was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to yield 2.30 g of a colorless semisolid. Column chromatography was performed for separation and purification. 1.5 g of column chromatography silica gel was wet-mixed with the sample and 5 g of column chromatography silica gel was wet-packed into the column. The eluent was petroleum ether (PE) and ethyl acetate (EA) in a ratio of 3:1. 0.87 g of a colorless oil was obtained with a yield of 64.4%. ESI-MS (m / z): 543.09 [M+Na] + .

[0074] (2) Synthesis of ASSFe

[0075] Ferrocenecarboxylic acid (0.77 g, 3.34 mmol, 2 eq) was added to 5 mL of oxalyl chloride and stirred at room temperature for 3 h. The excess oxalyl chloride was evaporated under reduced pressure and the residue was dissolved in 5 mL of dichloromethane for later use. ASSOH (0.87 g, 1.67 mmol, 1 eq), DIPEA (0.43 g, 3.34 mmol, 2 eq), and 10 mL of dichloromethane were placed in a 50 mL three-necked flask and stirred under ice. The dichloromethane solution of ferrocenecarboxylic acid was slowly added dropwise to the reaction system. After the addition was complete, the mixture was stirred at room temperature and monitored by TLC to terminate the reaction. The reaction solution was washed sequentially with 30 mL of 20% aqueous sodium bicarbonate solution, 30 mL of 50% aqueous citric acid solution, and 30 mL of saturated aqueous NaCl solution. The organic layer was dried over anhydrous MgSO₄. The MgSO₄ solid was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to yield 2.01 g of a black solid. Column chromatography separation and purification: 2.5 g of column chromatography silica gel was wet-mixed with the sample, 7 g of column chromatography silica gel was wet-packed into the column, and the eluent was PE:EA=3:1. 0.51 g of a yellow solid was obtained, with a yield of 42.1%. 1H NMR (400MHz, CDCl3) δ6.21(d,J=7.1Hz,1H),5.30(s,1H),4.92(d,J=1.96Hz,2H),4.88(td,J=12.2,3.1Hz,1H),4.64(td,J=12.1,3.7Hz,1H),4 .56(d,J=2Hz,2H),4.37(s,5H),4.33(td,J=12.1,3.8Hz,1H),3.85(ddd,J=12.4,11.3,4.2Hz,1H),3.35(dt,J=12.2,2.7Hz,1H),3.10(dt,J=1 2.2,2.7Hz,1H),2.95(td,J=12.0,3.7Hz,1H),2.81-2.73(m,2H),2.69 -2.57(m,3H),2.23(h,J=6.9Hz,1H),1.91-1.82(m,1H),1.76(dt,J=12. 8,6.9Hz,1H),1.70-1.45(m,7H),1.34(s,3H),1.31-1.25(m,1H),1.05-0.97(m,1H),0.90(dd,J=7.8,6.6Hz,6H); ESI-MS(m / z):755.09[M+Na] + .

[0076] Example 2 Synthesis of Reduction-Responsive Artesunate Dimer Prodrug ASSA

[0077] Artesunate (1 g, 2.60 mmol, 1 eq), 2-hydroxyethyl disulfide (0.20 g, 1.30 mmol, 0.5 eq), DMAP (0.16 g, 1.30 mmol, 0.5 eq), and 15 mL of DCM were added to a 50 mL two-necked flask and stirred at room temperature until completely dissolved. 5 mL of a DCM solution of EDCI (0.60 g, 3.12 mmol, 1.2 eq) was slowly added dropwise to the reaction system. After the addition was complete, the reaction was stirred at room temperature and monitored by TLC to terminate the reaction. The reaction solution was washed once with 30 mL of 50% aqueous citric acid and then once with 30 mL of saturated aqueous NaCl. The organic layer was dried over anhydrous MgSO₄. The MgSO₄ solid was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to yield 2.02 g of a colorless semisolid. Column chromatography separation and purification: 1.2 g of column chromatography silica gel was wet-mixed with the sample, 5 g of column chromatography silica gel was wet-packed into the column, and the eluent was PE:EA=2:1. 1.65 g of a colorless oil was obtained, with a yield of 71.4%. 1H NMR (600MHz, CDCl3) δ5.79(d,J=9.8Hz,2H),5.43(s,2H),4.35(t,J=6.6Hz,4H),2.92(t,J=6.6Hz,4H),2.76-2.72(m,4H),2 .71-2.60(m,4H),2.60-2.53(m,2H),2.37(td,J=14.0,3.9Hz,2H),2.03(ddd,J=14.6,4.9,2.9Hz,2H),1.91-1.86(m,2H),1. 78(dt,J=13.5,3.8Hz,2H),1.72(dq,J=13.5,3.4Hz,2H),1.62(dt,J=13.8,4.5Hz,2H),1.52-1.45(m,2H),1.43(s,6H),1.40 -1.28(m,6H),1.02(td,J=12.6,12.1,3.6Hz,2H),0.96(d,J=6.2Hz,6H),0.86(d,J=7.2Hz,6H); ESI-MS(m / z):543.09[M+Na] + .

[0078] Example 3 Preparation of Reduction-Responsive Artesunate Prodrug Self-Assembled Nanoparticles

[0079] Artesunate prodrug, Ce6 and DSPE-PEG at different mass ratios were added 2k -FA was dissolved in 0.1 mL of DMSO, and the solution was slowly added dropwise to 1 mL of deionized water under stirring. Artesunate prodrug and Ce6 spontaneously formed uniform nanoparticles, which were then dialyzed in deionized water at room temperature to remove the organic solvent in the nanoformulation, obtaining a nanosolution free of organic solvent.

[0080] Self-assembled nanoparticles (Ce6@ASSFe-FA) were prepared by using artesunate-ferrocene prodrug ASSFe and Ce6 as described above, wherein artesunate prodrug was combined with DSPE-PEG 2k -FA mass ratio is: 5:1.

[0081] The particle size, particle size distribution, Zeta potential, encapsulation efficiency and drug loading of self-assembled nanoparticles prepared with different mass ratios of artesunate-ferrocene prodrug ASSFe and Ce6 are shown in Table 1:

[0082] Table 1 Particle size, encapsulation efficiency and drug loading of artesunate prodrug self-assembled nanoparticles

[0083]

[0084] The results showed that the particle size of artesunate prodrug and Ce6 self-assembled nanoparticles were both below 200 nm, and both exhibited high permeability and retention (EPR effect). When the ASSFe:Ce6 ratio was 4:1-5:1, the encapsulation efficiency of Ce6@ASSFe nanoparticles exceeded 90%, and the drug loading was around 85%. When the ASSFe:Ce6 ratio was 4:1, the particle size and PDI were both smaller, while the encapsulation efficiency and drug loading were greater. Therefore, a mass ratio of ASSFe to Ce6 of 4:1 was preferred.

[0085] Change the PEG modifier to DSPE-PEG 2k Self-assembled nanoparticles of artesunate-ferrocene prodrug ASSFe and Ce6 were prepared, and their particle size, particle size distribution, encapsulation efficiency and drug loading did not change significantly.

[0086] Artesunate dimer prodrug ASSA and Ce6 were self-assembled into nanoparticles (Ce6@ASSA-FA), with the mass ratio of ASSA to Ce6 being 4:1. 2k -FA was used as a PEG modifier, and self-assembled nanoparticles were prepared according to the above method. The obtained nanoparticles had a particle size of 152.0 nm, PDI=0.028, an encapsulation efficiency of 92.56%, and a drug loading of 84.45%.

[0087] Example 4 In vitro release experiment of the nanoparticles prepared in Example 3

[0088] 1 mL of Ce6@ASSA-FA nanoparticles prepared in Example 3 was added to 100 mL of release medium containing different concentrations of GSH, the release medium being phosphate buffer containing 20% ​​ethanol. Samples were taken out at predetermined time points and the release of ART and Ce6 was analyzed by high performance liquid chromatography. The results are shown in Figure 1 and 2 .

[0089] like Figure 1 and 2 As shown in Figure 3, the release of ART and Ce6 from Ce6@ASSA-FA nanoparticles was dependent on the GSH concentration.

[0090] Similarly, the release of ART and Ce6 from Ce6@ASSFe-FA nanoparticles was GSH concentration-dependent.

[0091] Example 5 Cellular uptake experiment of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3

[0092] Flow cytometry was used to determine the uptake of the prepared Ce6@ASSFe (Ce6@ASSA) and Ce6@ASSFe-FA (Ce6@ASSA-FA) nanoparticles in CT26 cells. 5 Cells were seeded into 12-well plates at a density of 50 cells / mL and incubated in an incubator for 24 hours to allow them to adhere to the wall. After the cells adhered to the wall, Ce6 solution, Ce6@ASSFe (Ce6@ASSA), Ce6@ASSFe-FA (Ce6@ASSA-FA), Ce6@ASSFe-FA (Ce6@ASSA-FA) nanoparticles and FA-saturated Ce6@ASSFe-FA (Ce6@ASSA-FA) nanoparticles were added. The concentration of Ce6 was 2μM. After incubation at 37°C for 4 hours, the cells were washed, collected and dispersed in PBS, and the cell uptake of various preparations was measured by flow cytometry. The experimental results are shown in Figure 2. Figure 3 and 4 shown.

[0093] Depend on Figure 3 It can be seen that in the 4-hour experimental group, the cells treated with nanoparticles had a higher intracellular fluorescence intensity than the cells treated with free Ce6. Therefore, the prepared Ce6@ASSFe-FA nanoparticles have a higher cellular uptake efficiency than non-targeted preparations and free Ce6. At the same time, after the folate receptors on the cell surface are saturated, the uptake capacity of Ce6@ASSFe-FA nanoparticles is reduced, which also shows that the targeting of the preparation is good. At the same time, there is no significant difference in the uptake capacity of Ce6@ASSA-FA and Ce6@ASSFe-FA nanoparticles, which shows that changing the connecting group of the disulfide bond has no effect on the formation of self-assembled nanoparticles. Exploring the uptake of Ce6@ASSFe-FA at 0.5h, 1h, 2h and 4h, it can be seen that the cell uptake of the preparation is time-dependent. As time increases, the uptake gradually increases, and there are significant differences between different times.

[0094] Depend on Figure 4 As can be seen, in the 4-hour experimental group, cells treated with nanoparticles had higher intracellular fluorescence intensity than cells treated with free Ce6. Therefore, the prepared Ce6@ASSA-FA nanoparticles have a higher cellular uptake efficiency than non-targeted preparations and free Ce6. At the same time, after the cell surface folate receptors are saturated, the uptake capacity of Ce6@ASSA-FA nanoparticles decreases, which also indicates that the preparation has good targeting.

[0095] Example 6 Cytotoxicity experiments of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3

[0096] The CCK8 method was used to investigate the cytotoxicity of drugs to CT26 cells, HT29 cells, and HCT116 cells. After digestion, the cells were diluted with culture medium to a cell density of 5000 cells / mL. After mixing evenly, 100 μL of cell suspension was added to each well of a 96-well plate and placed in an incubator for 24 hours to allow it to adhere. After the cells adhered, each group of drugs was added. This experiment used 1640 and 5A culture media to prepare and dilute drug solutions and nanoparticle preparations. 100 μL of drug was added to each well of cells, and 4 replicates were made for each concentration, and the process was repeated 3 times. The control group did not add the drug to be tested, but only 100 μL of culture medium was added, and the cells were incubated in an incubator. After incubation for 4 hours, fresh culture medium was replaced, and the laser irradiation experimental group was involved. Each well was irradiated with laser (660 nm, 300 mW cm -2 ) for 1 min. After 20 hours, the 96-well plate was taken out and 100 μL of 10% CCK8 was added to each well. The plate was placed in an incubator and incubated for a certain period of time. The absorbance of each well was measured at 450 nm using a microplate reader. Figure 5-7 .

[0097] The results showed that after laser irradiation, the toxicity of Ce6@ASSFe-FA nanoparticles was significantly higher than that of Ce6@ASSA-FA, which demonstrated that adding Fe to the delivery system is more conducive to achieving the synergistic effect of ferroptosis and photodynamics.

[0098] Example 7 Safety Investigation of Ce6@ASSFe-FA and Ce6@ASSA-FA in Example 3

[0099] Mouse embryonic fibroblasts (3T3 cells) were used to investigate the safety of Ce6@ASSFe-FA and Ce6@ASSA-FA nanoparticles. The 3T3 cells were digested and diluted with DMEM medium to a cell density of 5000 cells / mL. After blowing evenly, 100 μL of the cell suspension was added to each well of a 96-well plate and incubated in an incubator for 24 hours to allow the cells to adhere. After the cells adhered, each group of drugs was added. 100 μL of the test solution was added to each well, and 5 replicates were made for each concentration, and the same was repeated 3 times. In the control group, no test solution was added, and a single 100 μL culture medium was added, and the cells were incubated in an incubator for 24 hours. The 96-well plate was removed, 100 μL of 10% CCK8 was added to each well, and the plates were incubated in an incubator for a certain period of time. The absorbance of each well was measured at 450 nm using a microplate reader.

[0100] The results are as follows Figure 8 As shown, the drugs in each group showed negligible cytotoxicity, proving that both Ce6@ASSA-FA nanoparticles and Ce6@ASSFe-FA nanoparticles have certain selectivity, low toxicity to normal cells, and high cytotoxicity in tumor cells.

[0101] Example 8: ROS generation in CT26 cells by Ce6@ASSFe and Ce6@ASSFe-FA in Example 3

[0102] Flow cytometry was used to measure the ROS generation in CT26 cells by each drug group. CT26 cells were cultured at 2×10 5 Cells were seeded into 12-well plates at a density of 10 cells / mL and incubated in an incubator for 24 hours to allow them to adhere. After the cells adhered, the drugs prepared in the culture medium were added. The concentration of ART was 5 μM and the concentration of Ce6 was 1 μM. After incubation at 37°C for 4 hours, the cells were washed and ROS in the cells were labeled with 5 μM DCFDA. After incubation for 30 minutes, each sample was laser irradiated for 5 minutes. After digestion, the cells were collected and dispersed in PBS. The amount of ROS generated by the cells in each drug group was measured by flow cytometry. The experimental results are shown in the figure below. Figure 9 As shown in the figure, compared with each experimental group, the Ce6@ASSFe-FA nanoparticle group produced the most ROS, which proved that the preparation more fully exerted the synergistic therapeutic effects of ferroptosis and photodynamic therapy.

[0103] Flow cytometry was used to measure the ROS generation of Ce6@ASSA and Ce6@ASSA-FA nanoparticles in CT26 cells. 5 Cells were seeded into 12-well plates at a density of 100 cells / mL and incubated in an incubator for 24 hours to allow them to adhere. After the cells adhered, the drugs prepared in the culture medium were added. The concentration of ART was 10 μM and the concentration of Ce6 was 2 μM. After incubation at 37°C for 4 hours, the cells were washed and ROS in the cells were labeled with 20 μM DCFDA. After incubation for 30 minutes, each sample was laser irradiated for 5 minutes. After digestion, the cells were collected and dispersed in PBS. The amount of ROS generated by the cells in each drug group was measured by flow cytometry. The experimental results are shown in the figure below. Figure 10 shown.

[0104] Compared with other experimental groups, Ce6@ASSFe-FA and Ce6@ASSA-FA nanoparticle groups produced the most ROS, which proved that the preparation fully exerted the synergistic therapeutic effects of ferroptosis and photodynamic therapy.

[0105] Example 9 In vivo antitumor experiments of Ce6@ASSA-FA and Ce6@ASSFe-FA

[0106] CT26 cell suspension (1×10 6 cells / 100 μL) were inoculated subcutaneously on the right side of the back of female mice. 3The mice were randomly divided into 5 groups and given normal saline, ART solution, Ce6@ASSA-FA nanoparticles and Ce6@ASSFe-FA nanoparticles respectively. The drugs were given once every other day for 5 times in a row. The ART concentration was 20mg / kg and the Ce6 concentration was 5mg / kg. The group that needed light exposure was given laser light 8 hours after the drug was given. The survival status of the mice was observed every day, and the weight and tumor volume were measured. The results are shown in the figure below. Figure 11-14 shown.

[0107] The results showed that tumor growth in mice treated with saline and ART was virtually identical, with no significant differences. However, the Ce6@ASSA-FA and Ce6@ASSFe-FA nanoparticle groups exhibited stronger antitumor activity under laser irradiation, with slower tumor growth. The Ce6@ASSFe-FA nanoparticle group exhibited superior tumor suppression compared to the Ce6@ASSA-FA nanoparticle group. The mice in each experimental group also experienced a slight increase in body weight. There were no significant abnormalities in the function of major organs in any of the groups. These results demonstrate that the nanoparticles exhibit significant antitumor effects without causing significant nonspecific toxicity, making them a safe and effective antitumor drug delivery system.

Claims

1. An artesunate prodrug or a pharmaceutically acceptable salt thereof having the following structure: n=1-3。 2. An artesunate prodrug or a pharmaceutically acceptable salt thereof having the following structure: 。 3. The method for preparing the artesunate prodrug or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The ASSFe is prepared by the following method: (1) Place artesunate, 2-hydroxyethyl disulfide, 4-(dimethylamino)pyridine and dichloromethane in a reaction vessel and stir until the solid is completely dissolved; add a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the reaction system dropwise under ice bath conditions. After the addition is complete, stir at room temperature until the reaction is complete, separate and purify to obtain the product ASSOH; (2) Add oxalyl chloride to ferrocenecarboxylic acid to prepare ferrocenecarboxylic acid chloride; under ice bath conditions, add dichloromethane solution of ferrocenecarboxylic acid chloride dropwise to ASSOH and N , N -Diisopropylethylamine in dichloromethane solution, after completion of the addition, stirred at room temperature until the reaction is complete, separated and purified to obtain the product ASSFe; The ASSA is prepared by the following method: (1) Add artesunate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to a reaction vessel, add dichloromethane to dissolve, and stir under ice bath conditions for 1-4 hours; (2) Add 2-hydroxyethyl disulfide and 4-(dimethylamino)pyridine to the reaction vessel (1), continue to add dichloromethane, stir at room temperature for 1-24 hours, separate and purify to obtain the prodrug ASSA.

4. The self-assembled nanoparticles of artesunate prodrug or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The self-assembled nanoparticles contain artesunate prodrug, a photosensitizer and a PEG modifier, and the mass ratio of the artesunate prodrug to the photosensitizer is 10:1 to 1:

10.

5. The self-assembled nanoparticles of artesunate prodrug or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The mass ratio of the artesunate prodrug to the photosensitizer is 10:1 to 3:

1.

6. The self-assembled nanoparticles of artesunate prodrug or pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The mass ratio of the artesunate prodrug to the photosensitizer is 4:1 to 5:

1.

7. The self-assembling nanoparticles according to any one of claims 4 to 6, characterized in that The photosensitizer is a porphyrin photosensitizer; the PEG modifier is one of distearoylphosphatidylethanolamine-polyethylene glycol or distearoylphosphatidylethanolamine-polyethylene glycol-folic acid, and the molecular weight of PEG is 200-20000.

8. The self-assembling nanoparticles according to any one of claims 4 to 6, characterized in that The photosensitizer is one or more of dihydrochlorin e6, chlorophyll a, and pheophytin a.

9. The self-assembled nanoparticles according to claim 7, characterized in that The molecular weight of PEG is 2000-5000.

10. The method for preparing self-assembled nanoparticles according to any one of claims 4 to 9, characterized in that: Artesunate prodrug, photosensitizer and PEG modifier are weighed, dissolved in DMSO solvent, slowly added dropwise into deionized water under stirring, and then the organic solvent is removed by dialysis to obtain the product.

11. Use of the artesunate prodrug or pharmaceutically acceptable salt thereof according to claim 1 or 2, or the self-assembled nanoparticles according to any one of claims 4 to 9 in the preparation of a drug delivery system.

12. Use of the artesunate prodrug or pharmaceutically acceptable salt thereof according to claim 1 or 2, or the self-assembled nanoparticles according to any one of claims 4 to 9 in the preparation of an anti-tumor drug.

13. Use of the artesunate prodrug or pharmaceutically acceptable salt thereof according to claim 1 or 2, or the self-assembled nanoparticles according to any one of claims 4 to 9, in the preparation of an injection, oral administration or topical administration system.

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