Preparation of novel carrier having functions of inducing ferroptosis and delivering nucleic acid drug, and use thereof in tumor treatment

Abstract

Disclosed in the present invention are a novel carrier having the functions of inducing ferroptosis and delivering a nucleic acid drug, and use thereof in tumor treatment. The artesunate-protamine conjugate carrier of the present invention conjugates artesunate with protamine by means of an amide bond. The artesunate-protamine conjugate carrier prepared by the present invention has the characteristics of simple components and small toxic and side effects, and can induce ferroptosis of tumor cells. Moreover, said carrier has the function of delivering nucleic acids, and can be used for research in the anti-tumor field.

Description

Preparation of a novel carrier with the functions of inducing ferroptosis and delivering nucleic acid drugs and application thereof in tumor treatment TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and particularly relates to an artesunate-protamine conjugate for inducing ferroptosis and a preparation method thereof and application thereof in tumor treatment. BACKGROUND

[0002] Ferroptosis is a newly discovered form of programmed cell death, which is caused by lipid peroxidation due to accumulation of reactive oxygen species (ROS), and the accumulation of ROS exceeds the redox content of glutathione (GSH) and glutathione peroxidase 4 (GPX4). Compared with normal cells, tumor cells have more vigorous metabolism, mitochondrial dysfunction and more ROS accumulation, which increases the sensitivity of tumor cells to ferroptosis. The mechanism of ferroptosis mainly relates to iron metabolism disorder, imbalance of amino acid antioxidant system and accumulation of lipid peroxides.

[0003] Studies have shown that ferroptosis plays a key regulatory role in the occurrence, development and metastasis of various tumors, and induction of tumor cell ferroptosis can be used as an effective strategy for treating tumors. Currently discovered ferroptosis-inducing drugs mainly include three categories of cystine / glutamate antiporter inhibitors, glutathione peroxidase 4 (GPX4) inhibitors and Fenton reaction inducers, which can induce tumor cell ferroptosis through multiple pathways or targets.

[0004] Artesunate (ART) has the structural formula as shown in formula (1), and is a semi-synthetic derivative of artemisinin, which is a highly effective and low-toxicity antimalarial drug. In recent years, ART has attracted attention due to its inhibitory effect on cancer cell proliferation, invasion and migration. The anticancer mechanism of artemisinin drugs is different from that of traditional anticancer drugs. In tumor cells, the peroxyl group is cleaved to produce a large number of free radicals, which induce oxidative stress response, thereby leading to cell apoptosis, autophagy, ferroptosis and the like.

[0005] Gene therapy is a technology that uses genes to treat or prevent diseases, and is currently mainly aimed at genetic diseases, some cancers and viral infections, etc. Gene drugs can regulate genes related to protein expression, and can regulate both intracellular and extracellular and cell membrane proteins, while the success of gene therapy is mainly limited by its instability, immunogenicity, low cellular uptake efficiency, difficulty in endosome escape, and low efficiency of gene drug carrier delivery system. Therefore, the development of a carrier system for delivering gene drugs to target cells is crucial. At present, gene delivery carrier systems include viruses and non-viruses. Non-viral vectors are mainly composed of cationic polymers and cationic lipid nanoparticles, and have the advantages of reduced risk, low carcinogenicity or immunogenicity, easy manufacturing, etc., and are being widely studied in gene therapy. However, there is no report on a nano-carrier that can induce ferroptosis and deliver nucleic acid drugs to effectively increase the cytotoxicity of ART to tumor cells. SUMMARY

[0006] The present application provides a novel carrier with the functions of inducing ferroptosis and delivering nucleic acid drugs, and the conjugate carrier has the function of inducing ferroptosis.

[0007] The second object of the present application is to provide a preparation method of the artemether-protamine conjugate carrier.

[0008] The third object of the present application is to provide the use of the artemether-protamine conjugate carrier in the preparation of an anti-tumor drug.

[0009] To achieve the above-mentioned objects, the present application provides the following technical solutions:

[0010] The artemether-protamine conjugate is obtained by coupling artemether and protamine through an amide bond; the carrier artemether-protamine conjugate (ART-PROTM) obtained by activating the carboxyl group of artemether and reacting with the amino group of protamine to form an amide bond can induce ferroptosis in tumor cells and further cause tumor cell death.

[0011] As a preferred embodiment of the present application, the molar ratio of artemether to protamine in the artemether-protamine conjugate is 1-10:1.

[0012] As a preferred embodiment of the present application, the molecular weight of protamine sulfate is about 5000 Da.

[0013] As a preferred embodiment of the present application, the artemether-protamine conjugate (ART-PROTM) is prepared by the following method:

[0014] (1) Artemether is dissolved in a DMF aqueous solution with a certain proportion, carboxyl activator is added, and the pH is adjusted to 5.5; after uniform magnetic stirring at room temperature, the pH is adjusted to 7-8 with a NaOH solution to obtain a clear solution I;

[0015] (2) Protamine sulfate is dispersed in an inorganic solution to obtain solution II;

[0016] (3) Solution I is slowly dropped into solution II, and the reaction is stirred at room temperature for 12-18 hours in the dark to obtain an artemether-protamine conjugate capable of inducing ferroptosis.

[0017] As a further preferred embodiment of the present application, the carboxyl activator is selected from 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS); the molar ratio of artemether, NHS and EDC·HCl is 1:1.5-3:1.5-3.

[0018] The preparation method of the artemether-protamine conjugate according to the present application comprises the following steps:

[0019] (1) Artemether is dissolved in a DMF aqueous solution, EDC·HCl and NHS are added, and the pH is adjusted to 5.5 with an acid, and the reaction is carried out at room temperature for 4 hours in the dark; the pH is adjusted to 7-8 to obtain a clear solution I.

[0020] (2) Protamine sulfate is dispersed in an inorganic solution to obtain solution II;

[0021] (3) Solution I is slowly dropped into solution II, and the reaction is stirred at room temperature for 12-18 hours in the dark to obtain an artemether-protamine conjugate capable of inducing ferroptosis. The reaction formula is as follows:

[0022] The volume ratio of DMF to water in step (1) is 1:1.

[0023] The amount of solvent used in step (1) is calculated as 0.1-0.2 mL of mixed solvent per milligram of artemether; preferably, 0.2 mL of mixed solvent per milligram of artemether.

[0024] The molar ratio of artemether, NHS and EDC·HCl in step (1) is 1:2:2.

[0025] The inorganic solvent in step (2) is at least one of water, phosphate buffer and physiological saline; preferably, a phosphate buffer (i.e. PBS buffer).

[0026] The inorganic solvent used in step (2) is 0.2-0.3 mL per mg of protamine sulfate; preferably 0.2 mL per mg of protamine sulfate.

[0027] The molar ratio of artesunate to protamine sulfate in step (3) is 1-10:1; preferably 9:1.

[0028] The preparation method of the iron death-inducing conjugate further comprises the step of further separating and purifying the obtained iron death-inducing conjugate after step (3), and the specific steps are as follows:

[0029] (4) Transfer the conjugate solution to a dialysis bag and dialyze with ultrapure water as dialysate to obtain purified iron death-inducing artesunate-protamine conjugate.

[0030] (5) Freeze-dry the conjugate in a test tube at-80℃ to-40℃ to obtain white loose solid.

[0031] The dialysis bag in step (4) is preferably a dialysis bag with a molecular weight cut-off of 3500 Da.

[0032] The dialysis time in step (4) is preferably more than 24 hours.

[0033] The artesunate-protamine conjugate as a carrier for delivering nucleic acid, wherein the nucleic acid is selected from deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), preferably plasmid DNA (pDNA), messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), CRISPR-Cas system components (such as sgRNA, Cas9 mRNA or protein), circular RNA, ribozyme, further preferably plasmid DNA or a complex of plasmid DNA and lipid particles.

[0034] As a further preferred embodiment of the present application, the nucleic acid is plasmid DNA or a complex of the plasmid DNA and cationic lipid nanoparticles for inhibiting tumor growth and / or metastasis and modulating tumor immune microenvironment.

[0035] As a still further preferred embodiment of the present application, the nucleic acid is a plasmid containing granulocyte-macrophage colony-stimulating factor gene or a complex of the plasmid containing granulocyte-macrophage colony-stimulating factor gene and cationic lipid nanoparticles.

[0036] The cationic lipid nanoparticles are prepared from phospholipid, cholesterol and octadecylamine, preferably from 15-30 parts by weight of phospholipid, 0.5-1 part by weight of cholesterol and 0.5-1 part by weight of octadecylamine, and more preferably from 20 parts by weight of phospholipid, 1 part by weight of cholesterol and 1 part by weight of octadecylamine.

[0037] As a preferred embodiment of the present application, the cationic lipid nanoparticles are prepared by the following method: phospholipid 40 mg, cholesterol 2 mg and octadecylamine 2 mg are dissolved in 5 ml of ethanol, vortexed for 10 min until completely dissolved, rotary evaporated for 30 min (vacuum degree slowly rises, speed: 50 rpm, temperature: 45-50 DEG C), placed in a vacuum desiccator overnight, added with 5 ml of H2O, vortexed, and the film was dispersed in water, the film was hydrated at 50 DEG C for 30 min, then transferred to a 10 ml EP tube, ultrasonicated by a cell disrupter for 10 min (300 w, 1 s interval), filtered through a 0.22 mu m filter membrane, and the particle size and potential were measured.

[0038] A nano-complex with the function of inducing ferroptosis and delivering nucleic acid, wherein the nucleic acid nano-complex is a stable nanoparticle formed by electrostatic interaction between the artemether-protamine conjugate and plasmid DNA; or the nucleic acid nano-complex is a stable nanoparticle formed by electrostatic interaction between the artemether-protamine conjugate, plasmid DNA and cationic lipid nanoparticles, and the particle size is 100-300 nm.

[0039] The preparation method of the nano-complex with the function of inducing ferroptosis and delivering nucleic acid, wherein the artemether-protamine conjugate carrier aqueous solution is mixed with plasmid DNA such as granulocyte-macrophage colony-stimulating factor (pGM-CSF) aqueous solution by oscillation to obtain a binary nucleic acid nano-complex; or the artemether-protamine conjugate carrier aqueous solution is mixed with plasmid DNA such as granulocyte-macrophage colony-stimulating factor (pGM-CSF) and cationic lipid nanoparticles by oscillation to obtain a ternary nucleic acid nano-complex. The particle size of the nano-complex is 100-300 nm, and the average particle size is 210 nm, indicating that the carrier and DNA form a stable nanoparticle through electrostatic interaction, which is beneficial to the delivery of nucleic acid.

[0040] The artemether-protamine conjugate and the nano-complex with the function of inducing ferroptosis and delivering nucleic acid according to the present application are used for preparing an antitumor drug. The artemether-protamine conjugate as a carrier is used for preparing an antitumor drug or as an active part to prepare a targeted antitumor drug. The artemether-protamine conjugate according to the present application has a strong inhibitory effect on the proliferation of mouse colon cancer CT26 cells in the in-vitro antitumor activity evaluation.

[0041] A pharmaceutical composition comprising the artesunate-protamine conjugate or the nanocomposite with the function of inducing ferroptosis and delivering nucleic acid. Advantages:

[0042] The present application creatively selects protamine as a basic carrier, and combines artesunate with the function of inducing ferroptosis through chemical reaction by an amide bond to obtain an artesunate-protamine conjugate, which not only can play the function of artesunate in inducing ferroptosis, but also can play the nucleic acid delivery function of protamine. The nanocomposite particles formed by self-assembly of the conjugate for nucleic acid drugs have high transfection activity, and can be used for delivery of nucleic acid drugs, so that the prepared nanocomposite can not only induce ferroptosis, but also deliver nucleic acid drugs to play an anti-tumor activity. The artesunate-protamine conjugate has a strong inhibitory effect on the proliferation, migration and invasion of mouse colon cancer CT26 cells and the migration and invasion of human colon cancer HCT116 cells in the in-vitro anti-tumor activity evaluation.

[0043] The present application also provides a preparation method of the artesunate-protamine conjugate, which is simple, has mild reaction conditions, and is simple in post-treatment; it has been verified that the prepared conjugate as a carrier can combine with DNA to form a nanocomposite. The binary nucleic acid nanocomposite is obtained by adding a plasmid DNA aqueous solution to the artesunate-protamine conjugate carrier aqueous solution prepared by the above preparation method and oscillating and mixing. The average particle size of the carrier / DNA complex is 210 nm, indicating that the carrier and DNA form stable nanoparticles through electrostatic interaction, which is beneficial to the delivery of nucleic acid. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is a reaction for preparing an artesunate-protamine conjugate

[0045] Figure 2 is an infrared spectrum of the artesunate-protamine conjugate carrier (ART-PROTM, AP) prepared in Example 1. 1 H-NMR spectrum.

[0046] Figure 3 is an infrared spectrum of the artesunate-protamine conjugate carrier (AP) prepared in Example 1 and protamine sulfate (PROTM).

[0047] Figure 4 is a UV spectrum of the artesunate-protamine conjugate carrier (AP), free artesunate (ART) and protamine sulfate (PROTM) prepared in Example 1.

[0048] Figure 5 is a nanometer particle size distribution graph of the AP / DNA complex prepared in Example 2.

[0049] Figure 6 is the effect of artemisinin-polyphenylxin conjugate carrier (AP) and free artemisinin (ART) prepared in Example 1 on cell viability. A: Effect of ART on the viability of mouse colon cancer CT26 cells; B: Effect of AP on the viability of mouse colon cancer CT26 cells; C: Effect of ferroptosis inhibitors deferoxamine (DFO), ferrostatin-1 (FER-1) and iron ions on the viability of CT26 cells treated with ART / ARTPROM

[0050] Figure 7 is the expression of solute carrier family 7 member 11 (SCL7A11) and glutathione peroxidase 4 (GPX4) in mouse colon cancer CT26 treated with artemisinin- polyphenylxin conjugate carrier (AP) and free artemisinin (ART). 1. Control; 2. Artemisinin (ART), 3: Artemisinin-polyphenylxin conjugate (AP); 4. Lipid nanoparticles (LNP); 5. AP / pGM-CSF; 6. AP / pGM-CSF / LNP; 7: AP / pGM-CSF / LNP + Ferrostatin 1 (FER-1)

[0051] Figure 8 is the effect of different concentrations of ART and artemisinin-polyphenylxin conjugate carrier (AP) prepared in Example 1 on the reactive oxygen species (ROS) level of CT26 cells.

[0052] Figure 9 is a photograph of the CT26 mouse tumors peeled off (A) and the change in tumor volume of colon cancer-bearing mice in each group within 14 days of administration (B). n = 8, *P < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001

[0053] Figure 10 is the tumor weight of CT26 colon cancer-bearing mice in each treatment group 14 days after administration. n = 8, *P < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

[0054] Figure 11 is the content of liver and kidney biochemical indicators in the blood of CT26 colon cancer-bearing mice in each treatment group.

[0055] A: ALT content in the blood of mice in each group; B: AST (aspartate aminotransferase) content in the blood of mice in each group; C: BUN (blood urea nitrogen) content in the blood of mice in each group; D: CREA (creatinine) content in the blood of mice in each group, n = 3.

[0056] Figure 12 Lipid nanoparticle (LNP), artemether-protamine conjugate carrier (AP), AP-LNP carrier binding granulocyte-macrophage colony-stimulating factor plasmid (pGM-CSF) DNA gel retardation assay. 1. Marker plasmid; 2. 4 pg DNA + 32 pg LNP; 3. 4 pg DNA + 32 pg LNP + 12 pg AP; 4. 4 pg DNA + 32 pg LNP + 18 pg AP; 5. 32 pg LNP + 18 pg AP; 6. 4 pg DNA + 64 pg LNP.

[0057] Figure 13 Flow cytometry assay for pGM-CSF expression in CT26 cells after AP / pGM-CSF / LNP delivery of GM-CSF plasmid to mouse CT26 cells. 1. Control; 2. DNA:LNP:AP = 4 pg:48 pg:12 pg; 3. DNA:LNP:AP = 4 pg:64 pg:12 pg.

[0058] Figure 14 Western-blot assay for GM-CSF expression in CT26 cells after AP / pGM-CSF / LNP delivery of GM-CSF plasmid to mouse CT26 cells. 1. Control; 2. DNA:LNP:AP = 4 pg:48 pg:12 pg; 3. DNA:LNP:AP = 4 pg:64 pg:12 pg.

[0059] Figure 15 Transwell migration and invasion assay of mouse CT26 (A) and human HCT16 (B) cells. 1. Control; 2. Artemether (ART); 3. Artemether-protamine conjugate carrier (ART-PRO™, AP); 4. Lipid nanoparticle (LNP); 5. AP-pGM-CSF; 6. AP / pGM-CSF / LNP; 7. AP / pGM-CSF / LNP + Ferrostatin 1 (Fer-1). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

[0060] Figure 16 Reduced glutathione (GSH) and lipid peroxidation malondialdehyde (MDA) assay. 1. Control; 2. Artemether (ART); 3. Artemether-protamine conjugate carrier (ART-PRO™, AP); 4. Lipid nanoparticle (LNP); 5. AP / pGM-CSF; 6. AP / pGM-CSF / LNP; 7. AP / pGM-CSF / LNP + Ferrostatin 1 (Fer-1). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0061] The application will be further described in detail below with reference to examples. Unless otherwise specified, the reagents and raw materials used in the application are commercially available, and the methods and devices are commonly used in the art.

[0062] The protamine designed in the embodiments of the application is salmon protamine, which is purchased from Shenguo Bioengineering Co., Ltd.

[0063] Example 1

[0064] The preparation of the artemether-protamine conjugate with the function of inducing ferroptosis is as follows:

[0065] (1) 10 mg of artemether (ART) was added to 2 ml of a mixed solution of DMF and water (V / V = 1:1), 10 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 6 mg of N-hydroxysuccinimide (NHS) were added and uniformly mixed, and the pH was adjusted to 5.5, and the reaction was carried out at room temperature in the dark for 4 hours. The pH was adjusted to 7.5 to obtain a colorless clear solution I;

[0066] (2) 15 mg of protamine sulfate was dispersed in 3 mL of PBS phosphate buffer (pH = 7.4, 1x buffer solution) to obtain solution II;

[0067] (3) Solution I was slowly added to solution II, and the reaction was carried out at room temperature in the dark for 12 hours to obtain an artemether-protamine conjugate carrier solution capable of inducing ferroptosis;

[0068] (4) The conjugate solution was transferred to a 3500 Da dialysis bag, and ultra-pure water was used as the dialysate to dialyze at room temperature for 24 hours to obtain the purified artemether-protamine conjugate capable of inducing ferroptosis.

[0069] (5) The conjugate was placed in a test tube and freeze-dried at -80℃ to -40℃ to obtain a white loose solid.

[0070] The artemether-protamine conjugate with the function of inducing ferroptosis was prepared by the method. 1 The structure of the artemether-protamine conjugate carrier with the function of inducing ferroptosis was characterized by H-NMR method, as shown in FIG. 2. The characteristic absorption peaks of artemether appeared at δ = 5.7 ppm (-O-CH(CH)-O-) and δ = 5.5 ppm (-O-CH), and the carboxyl part of ART disappeared, which represented the formation of an amide bond.

[0071] A small amount of protamine sulfate (PROTM) and artemether-protamine conjugate (ART-PROTM) carrier were mixed with potassium bromide, respectively, and pressed into a thin sheet for infrared analysis, and the scanning range was 500-4000 cm-1 As shown in Fig. 3, compared with PROTM, the free amino characteristic absorption peak of ART-PROTM carrier at 3300-3500 cm -1 is weakened.

[0072] In addition, in order to qualitatively determine the effective combination of ART-PROTM carrier, the ultraviolet absorption spectra of ART, PROTM and ART-PROTM were scanned respectively, and the absorption spectrum curves were drawn, as shown in Fig. 4. ART-PROTM carrier has absorption peaks of ART and PROTM at 238 nm. In summary, it is preliminarily proved that we successfully synthesized ART-PROTM carrier.

[0073] The plasmid DNA used is GM-CSF-pcDNA3.1(+), with a size of 5866 bp.

[0074] A certain amount of plasmid DNA aqueous solution was slowly added to a certain amount of ART-PROTM carrier aqueous solution, and oscillated while adding. After the addition was completed, it was immediately oscillated for 5 minutes, and then stood for 30 minutes. The mass ratio of ART-PROTM carrier material to plasmid DNA was controlled to be 0.5-5:1 (W / W), and the concentration of plasmid DNA was controlled to be 0.5 mg / mL. There is electrostatic interaction between the carrier and DNA, which can self-assemble into nanoparticles in water. Dynamic light scattering method (DLS) was used to determine the particle size of the carrier / DNA complex in ultrapure water. When the mass ratio of carrier / DNA was 1:1, it was relatively stable, and the average hydration particle size of the carrier / DNA complex in water was 210 nm, as shown in Fig. 5.

[0075] Example 3: CCK-8 method was used to detect the effect of ART-PROTM carrier on the proliferation of CT26 tumor cells.

[0076] CCK-8 method was used to evaluate the proliferation inhibition effect of free artesunate ART and ART-PROTM carrier on colon cancer CT26 cells.

[0077] Logarithmic growth phase cells were trypsinized, and after the digestion was stopped, the cells were collected by centrifugation, diluted with an appropriate amount of culture medium, and the cell number was calculated. The counted cell suspension was added to a 96-well plate at 4×10 3 cells per well, and the volume of cell suspension per well was 100 μL. It was placed in a 37°C, 5% CO2 incubator to allow the cells to adhere.

[0078] When the cells under the lens grow to 75% to 80%, different concentrations of 0, 30, 50, 100, 150, 200 μg / mL of free ART and AP solution are used for treatment (3 holes are set for each concentration), and then incubated at 37°C, 5% CO2 for 24 hours and 48 hours respectively.

[0079] CCK-8 solution with a volume of 10 μL is added to each well, and incubated for 3 hours; the absorbance value of each well is measured at 450 nm wavelength by enzyme-linked immunoassay instrument.

[0080] The results are shown in FIGS. 6A and 6B, ART and carrier AP can significantly inhibit the proliferation of CT26 cells.

[0081] Example 4: CCK-8 method for detecting the effect of iron chelator DFO and ferroptosis inhibitor Fer-1 on artemether-protamine complex (ART-PROTM, AP) carrier treated colon cancer CT26 cells.

[0082] We respectively use iron chelator DFO, ferroptosis inhibitor Fer-1 to treat iron ions and ART and AP to treat colon cancer CT26 cells, and observe the recovery effect of each inhibitor molecule on the decrease of cell viability caused by ART under the same treatment conditions, and find that FER-1 has more obvious recovery effect on cell viability. Subsequently, the effects of FER-1 and Fe 2+ The effect of iron ions alone or in combination on the growth of ART / ART-PROTM treated CT26 cells. The results are shown in FIG. 5C: Fe 2+ ART and ART-PROTM treated CT26 cells can be further inhibited, and this inhibition is partially restored after Fer-1 and DFO treatment, and the results are shown in 6C.

[0083] Example 5:

[0084] The artemether-protamine complex (ART-PROTM, AP) carrier obtained by the method of Example 1 can induce ferroptosis, and its effect on intracellular ROS level is detected by flow cytometry, and the specific process is as follows:

[0085] The ROS level in the cell is reflected by the active oxygen probe DCFH-DA. CT26 cells are used as a model, and CT26 cells are inoculated into a 6-well plate (5×10 5Cells were incubated with free ART (50, 100, 150 μg / mL, respectively) and AP (75, 150, 225 μg / mL, respectively) for 24 hours, respectively, and then washed with PBS phosphate buffer (pH 7.4, IX). DCFH-DA was incubated with cells at a final concentration of 10 μM for 30 minutes, and the cells were washed with PBS three times. Finally, the cells were digested and dispersed in 500 μL of PBS for flow cytometry detection.

[0086] The results are shown in Figure 7: both ART and ART-PRO™ produced higher levels of ROS in cells than the basal level of cells. The DCF fluorescence value in CT26 cells increased with increasing ART concentration, and the DCF fluorescence value increased significantly after ART-PRO™ treatment, indicating that ART and ART-PRO™ can significantly activate the accumulation of intracellular ROS, but there is no significant difference between ART and ART-PRO™ in the accumulation of ROS, indicating that the combination of ART and PROTAM does not affect the production of ROS.

[0087] Example 6 In vivo study of tumor growth inhibition in tumor-bearing mice

[0088] Preparation of cationic lipid nanoparticles: phospholipid 40 mg, cholesterol 2 mg, and octadecylamine 2 mg were dissolved in 5 ml of ethanol, vortexed for 10 min until completely dissolved, and rotary evaporated for 30 min (vacuum degree slowly increased, speed: 50 rpm, temperature: 45-50°C), placed in a vacuum desiccator overnight, added with 5 ml of H2O, vortexed to disperse the film in water, and the film was hydrated at 50°C for 30 min, then transferred to a 10 ml EP tube, sonicated for 10 min (300 w, 1 s interval) with a cell disruptor, filtered through a 0.22 μm filter, and the particle size and potential were measured.

[0089] A certain amount of plasmid DNA aqueous solution, ART-PRO™ carrier aqueous solution, and cationic lipid nanoparticles were uniformly mixed and allowed to stand for 30 minutes; the mass ratio of ART-PRO™ carrier material to plasmid DNA (GM-CSF-pcDNA3.1(+)) and cationic lipid nanoparticles was controlled to be 1:8:4.5 (W / W / W), and the concentration of plasmid DNA was controlled to be 0.5 mg / mL. There is an electrostatic interaction between the carrier and the DNA, which will self-assemble into nanoparticles in water. The particle size of the carrier / DNA complex in ultrapure water was determined by dynamic light scattering (DLS), and the average hydrated particle size of the complex in water was 210 nm.

[0090] According to the experimental grouping, the tumors were injected with normal saline (NaCl); ART (intratumoral injection), ART-PRO™ (AP) (intratumoral injection), AP / pGM-CSF (intratumoral injection), AP / pGM-CSF / LNP (NPs) (intratumoral injection), BMS-1 (intravenous injection), AP / pGM-CSF / LNP (NPs) (intratumoral injection) combined with BMS-1 (NPs+BMS-1, intravenous injection), once every two days, and the tumor volume was allowed to grow to 70mm 3 The drug administration was started from the left and right sides, and a total of six administrations were performed. The tumor volume change was recorded from the first administration, and the mice were euthanized after the orbital blood sampling on day 14. The tumor tissue and various organs (heart, liver, spleen, lung, and kidney) were subsequently used for the subsequent experiments. The tumor volume of each group of mice was measured and calculated every day, and all data were statistically analyzed. The results are shown in Figures 8-9. As can be seen from the figures, the tumor in the control group grew rapidly, and each treatment group had a significant therapeutic effect. After the first administration, the tumor volume of each group injected with normal saline (NaCl); ART (intratumoral injection), ART-PRO™ (AP) (intratumoral injection), AP / pGM-CSF (intratumoral injection), AP / pGM-CSF / LNP (NPs) (intratumoral injection), BMS-1 (intravenous injection), AP / pGM-CSF / LNP (NPs) (intratumoral injection) combined with BMS-1 (NPs+BMS-1, intravenous injection) was 71.04±21.79mm 3 , 65.61±28.38mm 3 , 86.24±9.49mm 3 , 77.74±5.71mm3, 81.97±9.26mm3, 71.01±11.46mm 3 , 77.78±9.11mm 3 , 518.40±309.13mm 3 , 196.96±126.22mm 3 , 679.92±259.58mm 3 , 581.87±241.68mm 3 , 518.40±309.13mm 3 , 196.96±126.22mm 3The treatment effect of the AP / pGM-CSF / LNP (APGL NPs) group was significantly different from that of the AP treatment alone. The treatment effect of the control group (NC) was significantly different from that of each of the AP / pGM-CSF (intratumoral injection), AP / pGM-CSF / LNP (NPs) (intratumoral injection), BMS-1 (intravenous injection), and AP / pGM-CSF / LNP (intratumoral injection) combined with BMS-1 (NPs+BMS-1, intravenous injection) treatment groups. There was a significant difference in the tumor weight of the colon cancer (COC) tumor-bearing mice of each group on day 14 after administration (Figure 10).

[0091] Example 7 Detection of serum biochemical indicators of tumor-bearing mice

[0092] Serum biochemical indicators: 1. ALT (alanine aminotransferase); 2. AST (aspartate aminotransferase); 3. BUN (blood urea nitrogen); 4. CREA (creatinine) content in blood. ALT (alanine aminotransferase) and AST (aspartate aminotransferase) are two important serum transaminase indicators for liver function detection, mainly distributed in liver cells. When the liver is damaged or the liver function is abnormal, the permeability of liver cells increases, and the transaminase in the cells enters the blood, causing the levels of ALT and AST in the blood to rise, indicating liver disease signals, and are two important indicators for liver function detection. BUN (blood urea) is another nitrogen-containing compound in blood plasma in addition to protein, which is filtered by the glomerulus and excreted outside the body, and is one of the indicators for evaluating glomerular filtration function. CREA (creatinine) is a product of muscle metabolism in the human body, mainly filtered by the glomerulus and excreted outside the body. When the kidney is diseased, BUN and CREA produced by metabolism in the body cannot be normally excreted outside the body and enter the blood circulation, thereby causing abnormal elevation of the content of the two substances in the blood. Therefore, BUN and CREA are two important indicators for testing kidney function. Collect mouse whole blood, centrifuge at 1000 rpm for 15 minutes, and test the content of ALT (alanine aminotransferase), AST (aspartate aminotransferase), BUN (blood urea nitrogen), and CREA (creatinine) in serum (n = 3). The results are shown in Figure 11. In the ALT biochemical indicator detection, the determination results of the physiological saline (NaCl) group, the ART intratumoral injection group, the ART-PRO™ intratumoral injection (AP) group, the AP / pGM-CSF intratumoral injection group, the AP / pGM-CSF / LNP intratumoral injection (NPs) group, the BMS-1 intravenous injection group, and the AP / pGM-CSF / LNP intratumoral injection combined with BMS-1 intravenous injection (NPs+BMS-1) group were 54.95 ± 9.21 U / L, 67.34 ± 18.51 U / L, 54.23 ± 16.14 U / L, 69.90 ± 24.61 U / L, 51.51 ± 7.63 U / L, 45.64 ± 11.60 U / L, and 68.71 ± 10.63 U / L, respectively. The normal range of ALT in mouse blood is 10.06-96.47 U / L. The ALT levels of mice in each group were within the normal range, indicating that the prepared AP / pGM-CSF / LNP (NPs) nanofomulation and AP / pGM-CSF / LNP combined with BMS-1 have good safety.

[0093] In the AST biochemical index detection, the determination results of each group of normal saline (NaCl), ART intratumoral injection, ART-PRO™ intratumoral injection (AP), AP / pGM-CSF intratumoral injection, AP / pGM-CSF / LNP intratumoral injection (NPs), BMS-1 intravenous injection, and AP / pGM-CSF / LNP intratumoral injection combined with BMS-1 intravenous injection (APGLNPs) were 183.60±11.39 U / L, 210.80±10.57 U / L, 174.79±48.43 U / L, 174.82±25.17 U / L, 175.83±19.36 U / L, 173.46±55.95 U / L, and 165.15±54.55 U / L, respectively. The normal range of AST in the blood of mice was 36.31-235.48 U / L. The ALT levels of mice in each group were within the normal range, and the ART group was slightly elevated, also within the normal range, indicating that the prepared AP / pGM-CSF / LNP (NPs) nano-preparation and AP / pGM-CSF / LNP combined with BMS-1 had good safety.

[0094] In the BUN biochemical index detection, the determination results of each group of normal saline (NaCl), ART intratumoral injection, ART-PRO™ intratumoral injection (AP), AP / pGM-CSF intratumoral injection, AP / pGM-CSF / LNP intratumoral injection (NPs), BMS-1 intravenous injection, and AP / pGM-CSF / LNP intratumoral injection combined with BMS-1 intravenous injection (APGLNPs) were 17.43±2.30 mg / dL, 24.86±3.71 mg / dL, 22.36±8.74 mg / dL, 16.59±2.03 mg / dL, 19.01±5.13 mg / dL, 18.08±5.78 mg / dL, and 19.16±5.52 mg / dL, respectively. The normal range of CREA in the blood of mice was 10.81-34.74 mg / dL. The BUN levels of mice in each group were within the normal range, and the ART and AP groups were slightly elevated, also within the normal range, indicating that the prepared AP / pGM-CSF / LNP (APGLNPs) nano-preparation and combined BMS-1 had good safety.

[0095] In the CREA biochemical index detection, the results of each group of normal saline (NaCl), ART intratumoral injection, ART-PRO™ intratumoral injection (AP), AP / pGM-CSF intratumoral injection, AP / pGM-CSF / LNP intratumoral injection (NPs), BMS-1 intravenous injection, and AP / pGM-CSF / LNP intratumoral injection combined with BMS-1 intravenous injection (APGLNPs) were 29.25 ± 6.58 μmol / L, 28.08 ± 4.99 μmol / L, 34.90 ± 14.62 μmol / L, 24.64 ± 4.52 μmol / L, 26.18 ± 3.52 μmol / L, 26.75 ± 12.37 μmol / L, and 30.31 ± 7.22 μmol / L, respectively. The normal range of CREA in the blood of mice was 10.91-85.09 μmol / L. The CREA levels of mice in each group were within the normal range, and the AP group was slightly elevated but also within the normal range, indicating that the prepared AP / pGM-CSF / LNP (APGLNPs) nanometer preparation and the combination of BMS-1 had good safety.

[0096] Example 8

[0097] Cell transfection of AP carrier and GM-CSF-pcDNA3.1(+) plasmid complex, combined ratio screening, the plasmid has negative charge, the cationic liposome has positive charge, the AP has positive charge, first we apply the condensation block experiment to find the suitable ratio of plasmid and cationic liposome combination, the results are shown in Figure 12.

[0098] Flow detection of transfection effect: CT26 cells were inoculated in 6-well plates one day in advance, 10 5 cells / well, and the next day the medium was aspirated from the cells and replaced with serum-free medium in an incubator for 30 minutes. GM-CSF-pcDNA3.1(+) plasmid, AP, and cationic liposome nanometer (LNP) complex were prepared, and the three were mixed in the proportions of 16 μg, 48 μg, and 48 μg, and 16 μg, 48 μg, and 64 μg, respectively, and incubated for 30 minutes to form a lipid nanometer (LNP) complex. The complex was added to the cell culture medium to be transfected, and after 24 hours of transfection, the medium was replaced with full medium containing 10% FBS for continued culture for 48 hours. Flow cytometry was used to detect the expression of GM-CSF plasmid in CT26 cells, and the results are shown in Figure 13.

[0099] Western blot detection of transfection effect: 48 hours after transfection, the cells were collected and washed twice with PBS, and the protein was extracted with RIPA protein lysis buffer (containing 1% PMSF). After lysis on ice for 30 minutes, centrifugation at 4°C and 12000 rpm for 10 minutes, the supernatant (cell protein) was collected. The extracted protein was quantified using a BCA protein quantification kit, and then protein loading buffer solution was added and heated at 100°C for 5 minutes to prepare the protein sample for WB detection.

[0100] Gel retardation experiment showed that the plasmid DNA could be completely combined according to the proportion used in the experiment (as shown in Figure 12). We used flow cytometry and Western blot to detect the ternary complex formed by GM-CSF-pcDNA3.1(+) plasmid, AP, and LNP to deliver GM-CSF plasmid DNA to CT26 cells, and the results are shown in Figure 13 (flow cytometry detection) and Figure 14 (western blot detection). Both proportions of AP / pGM-CSF / LNP can deliver GM-CSF plasmid to CT26 cells and express, and increasing the proportion of cationic LNP can increase the transfection efficiency.

[0101] Example 9 Transwell invasion experiment

[0102] CT26, HCT116 cell suspension 200 μl was added to the Transwell chamber (50000 cells / well). Matrigel gel was diluted with serum-free cell culture medium on ice, and after mixing, it was added to the chamber. 500 μl of medium containing 15% FBS was added to the lower chamber of the 24-well plate, and the chamber was placed in the culture plate. Different drugs were added to the upper chamber and serum-free medium as a control, and cultured for 24 hours. At the time point, the chamber was taken out, washed with PBS, and the cells and Matrigel in the upper chamber were gently wiped off with a cotton swab. After being fixed with 4% paraformaldehyde for 20 minutes, the chamber was dried, stained with 0.1% crystal violet for 30 minutes, washed with PBS for 3 times, and dried at 37°C. The culture solution in the hole was discarded, and the upper layer of non-migrated cells was gently wiped off with a cotton swab. The chamber was placed under a microscope, and 5 fields of view were randomly selected, photographed and counted. The results are shown in Figures 15A-B.

[0103] Example 10 Glutathione (GSH) concentration detection

[0104] Cells were plated in 6-well plates one day in advance, and on the next day, the cells were administered with the following drugs according to the experimental grouping: control, ART, AP, LNP, pGM-CSF / AP, AP / pGM-CSF / LNP, and AP / pGM-CSF / LNP+Fer-1. The drugs were co-cultured with the cells for 24 hours, and then the cells were collected and broken by a cell crusher to release the protein and glutathione (GSH). The cells were centrifuged at 10,000 g for 10 minutes, and the supernatant was mixed with the reagent in the kit at a ratio of 1:1. The mixture was centrifuged at 4,500 g for 10 minutes, and the supernatant was collected. The GSH level was then determined by the Ellman method. Reduced glutathione (GSH) can react with dithiothionitrobenzoic acid (DTNB) to produce thionitrobenzoic acid and glutathione disulfide. Nitrothiobenzoic acid is a yellow compound, and the content of reduced glutathione (GSH) can be determined by colorimetry at 405 nm. The protein content in the tissue and cells also needs to be detected, and the BCA protein quantification method was used for detection. The results are shown in FIG. 16A.

[0105] Example 11 Determination of Malondialdehyde (MDA)

[0106] Cells were plated in 6-well plates one day in advance, and on the next day, the cells were administered with the following drugs according to the experimental grouping: control, ART, AP, LNP, pGM-CSF / AP, AP / pGM-CSF / LNP, and AP / pGM-CSF / LNP+Fer-1. The drugs were co-cultured with the cells for 24 hours, and then the cells were collected and broken by a cell crusher to release the protein and glutathione (GSH). The cells were centrifuged at 10,000 g for 10 minutes, and the supernatant was mixed with the reagent in the kit at a ratio of 1:1. The mixture was centrifuged at 4,500 g for 10 minutes, and the supernatant was collected. The GSH level was then determined by the Ellman method. Reduced glutathione (GSH) can react with dithiothionitrobenzoic acid (DTNB) to produce thionitrobenzoic acid and glutathione disulfide. Nitrothiobenzoic acid is a yellow compound, and the content of reduced glutathione (GSH) can be determined by colorimetry at 405 nm. The protein content in the tissue and cells also needs to be detected, and the BCA protein quantification method was used for detection. The results are shown in FIG. 16A.

[0105] Example 11 Determination of Malondialdehyde (MDA)

[0106]

Claims

1. An artemisinin-hemojuvelin conjugate, characterized in that, The artemether-artemotil is obtained by chemical reaction such as amide bond coupling with protamine.

2. The artemisinin derivative-proteinate conjugate according to claim 1, characterized in that, The molar ratio of artemether-artemotil to protamine in the artemether-artemotil-protamine conjugate is preferably 1-10:

1.

3. The artemisinin derivative-proteinate conjugate of claim 1, wherein, The artemether-artemotil-protamine conjugate is prepared by the following method: (1) Artemether is used as raw material, dissolved in dimethylformamide (DMF) aqueous solution, carboxyl activator is added, and inorganic or organic acid is used to adjust the pH to 5.5; after uniform magnetic stirring at room temperature, sodium hydroxide (NaOH) solution is used to adjust the pH to 7-8, to obtain clear solution I; (2) Protamine sulfate is dispersed in an inorganic solution to obtain solution II; (3) Solution I is slowly dropped into solution II, and stirred at room temperature for 12-18 hours in the dark to obtain artemether-artemotil-protamine conjugate; or mixed in a microfluidic device.

4. The artemisinin derivative-protein conjugate of claim 3, wherein, The carboxyl activator is selected from 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS); the molar ratio of artemether, NHS and EDC·HCl is 1:1.5-3:1.5-3.

5. The artemether-artemotil-protamine conjugate of any one of claims 1-4 as a carrier for delivering nucleic acid, wherein the nucleic acid is selected from deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), preferably plasmid DNA (pDNA), messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA (miRNA), antisense oligonucleotide (ASO), CRISPR-Cas system components (such as sgRNA, Cas9 mRNA or protein), circular RNA, ribozyme, further preferably plasmid DNA or a complex of plasmid DNA and lipid particles.

6. Use according to claim 5, characterized in that The nucleic acid is plasmid DNA or a complex of plasmid DNA and cationic lipid nanoparticles for inhibiting tumor growth and / or metastasis and regulating tumor immune microenvironment.

7. Use according to claim 5, characterized in that The nucleic acid is a plasmid containing granulocyte-macrophage colony-stimulating factor gene or a complex of a plasmid containing granulocyte-macrophage colony-stimulating factor gene and cationic lipid nanoparticles.

8. A nanoparticle having an iron-deatli-inducing function and delivering a nucleic acid nanocomplex, characterized by The nucleic acid nano-complex is a stable nanoparticle formed by electrostatic interaction between the artemether-artemotil-protamine conjugate of any one of claims 1-4 and plasmid DNA; or the nucleic acid nano-complex is a stable nanoparticle formed by electrostatic interaction between the artemether-artemotil-protamine conjugate of any one of claims 1-4, plasmid DNA and cationic lipid particles, with a particle size of 10-500 nm and a Zeta potential of +2 mV to +45 mV.

9. Use of the artemether-artemotil-protamine conjugate of any one of claims 1-4 or the nano-complex having the function of inducing ferroptosis and delivering nucleic acid of claim 8 in the preparation of an anti-tumor drug or an anti-tumor drug in combination with a PD-1 / PD-L1 inhibitor.

10. A pharmaceutical composition, characterized by The artemether-protamine conjugate according to any one of claims 1 to 4 or the nanoparticle complex having the function of inducing ferroptosis and delivering nucleic acid according to claim 8.

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