Vortex preparation and application of amino acid-based ruthenium complex self-delivery assembly

Abstract

The invention discloses vortex preparation and application of an amino acid-based ruthenium complex self-delivery assembly, and belongs to the technical field of fine chemical engineering. According to the molecule, amino acid is directly coordinated to a ruthenium metal center in a normal-temperature stirring mode under a dark condition, and the amino acid-based ruthenium complex with near-infrared light activating coordinate bond breakage and singlet oxygen (ROS) releasing is constructed. The introduction of amino acid not only improves the water solubility of the complex, but also endows the complex with a self-assembly function, and a large number of nano assemblies can be prepared by using a multi-channel vortex mixer to meet the requirements of biological experiments and clinical transformation. The complex can realize a cascade targeting process from an endoplasmic reticulum to a cell nucleus, and gives full play to the advantages of photodynamic therapy (PDT) and photo-activated chemotherapy (PACT) of the ruthenium complex. The synthesis of the complex and the preparation method of the assembly are simple, the dark toxicity is low, the anti-tumor effect is remarkable, and the potential of clinical application is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of fine chemicals, and relates to the preparation of ruthenium complex nanomedicine materials, which can be used in PDT and PACT anti-tumor treatments. Through cascaded organelle targeting, it can effectively inhibit tumor growth and metastasis. Background Art

[0002] At present, cancer has become a global security issue threatening human life and health. According to data from the World Health Organization, nearly 10 million people die from cancer every year. Improving the treatment effect of tumors is the tireless pursuit goal of researchers and clinicians. Since cisplatin was approved for clinical cancer treatment in 1978, metal drugs have become an important research direction in tumor treatment. However, in clinical applications, cisplatin and its derivatives have been proven to have obvious side effects on cancer patients, and at the same time, their treatment effects are greatly reduced due to problems such as drug resistance and poor targeting.

[0003] In recent years, ruthenium polypyridyl complexes have received much attention from researchers due to their excellent photophysical and photochemical properties. Such complexes form a "caged" structure through coordination bonds, making them non-toxic in the dark. When irradiated with light of a specific wavelength in the tumor area, ligand dissociation occurs, generating singlet oxygen (ROS) while restoring the binding ability of the ruthenium center to biomolecules, thereby effectively killing tumor cells. However, ruthenium complexes have problems such as poor water solubility and low delivery efficiency. If precise targeted delivery in vivo can be achieved, the dosage can be reduced while maintaining anti-tumor efficiency. The most commonly used strategy at present is to polymer-encapsulate or modify ruthenium complexes. This strategy adds extra steps and has poor repeatability and complex structures, which easily cause batch-to-batch differences. At the same time, it is difficult to precisely control the specific structure of polymers, unable to finely regulate the functions of complexes, and there are also degradation problems themselves. Therefore, it is of great significance to design a simple and precisely structured ruthenium complex self-delivery targeted nano-drug. Summary of the Invention

[0004] In view of the above deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing a highly selective targeted self-delivery ruthenium complex assembly, aiming to solve problems such as poor delivery efficiency, complex preparation, and poor biocompatibility of ruthenium complexes.

[0005] The technical solution of the present invention is as follows: An amino acid-based ruthenium complex, and the complex has the following general structural formula:

[0006]

[0007] Wherein, R1 is a bipyridyl ligand, R2 is a terpyridyl ligand, and A1 is an amino acid ligand;

[0008] Z - is a halogen anion, nitrate anion, sulfate anion, hexafluorophosphate anion;

[0009] The complex is electrically neutral;

[0010] R1 is selected from the following structures:

[0011]

[0012] wherein X is hydrogen, a straight-chain or branched-chain alkane having 1 to 5 carbon atoms;

[0013] R2 is selected from the following structures:

[0014]

[0015] wherein X1 is hydrogen, trifluoromethyl, methoxy, isopropyl; X2 is hydrogen, trifluoromethyl, a straight-chain or branched-chain alkane having 1 to 5 carbon atoms;

[0016] A1 is selected from the following structures:

[0017]

[0018] wherein Y is sulfur or selenium.

[0019] Further, the X is hydrogen, methyl, ethyl or isopropyl.

[0020] Further, X2 is hydrogen, trifluoromethyl, methyl, ethyl or isopropyl.

[0021] Further, X is hydrogen, X1 is hydrogen, X2 is hydrogen, and Y is sulfur.

[0022] Further, the structure of the complex is:

[0023]

[0024]

[0025] A nanoparticle, wherein the nanoparticle comprises at least one of the above ruthenium complexes.

[0026] Further, the prepared nanoparticle is obtained by self-assembly in water and a multi-channel vortex mixer.

[0027] Further, the diameter of the nanoparticle is 90 - 150 nm.

[0028] The application of the above amino acid-based ruthenium complex, wherein the amino acid-based ruthenium complex is used for preparing a drug for treating tumors, and further, for preparing a drug for tumor PDT or PACT.

[0029] The application of the above-mentioned nanoparticles, which are used for preparing drugs for treating tumors, and further for preparing drugs for tumor PDT or PACT.

[0030] A pharmaceutical composition comprising the above-mentioned ruthenium complex or the above-mentioned nanoparticles.

[0031] Compared with the prior art, the present invention has the following prominent advantages:

[0032] (1) Simple preparation, clear structure and easy to regulate: Under the conditions of normal temperature and light avoidance, the present invention smoothly introduces commercial amino acids into the ruthenium complex, endowing it with self-assembly function, optimizing the molecular design and reducing the complexity of the synthesis of nano-drugs; meanwhile, a large number of nano-assemblies can be prepared by simple vortex mixing to meet the needs of clinical transformation.

[0033] (2) Reducing molecular toxicity and improving delivery efficiency: Compared with traditional methods such as polymer modification or encapsulation, the present invention realizes self-assembly and targeted delivery only by using amino acids, and can avoid the toxicity caused by the introduction of non-biological molecules and the leakage of complexes.

[0034] (3) Specific targeting improves the therapeutic effect: The complex molecule designed by the present invention can achieve cascade targeting from the endoplasmic reticulum to the nucleus, giving full play to the characteristics of the ruthenium complex. First, it targets to the endoplasmic reticulum, generates ROS and releases ruthenium ligands under light irradiation. During this process, ROS can damage the endoplasmic reticulum and induce pyroptosis of cells, amplifying the PDT therapeutic effect. On the other hand, the released ruthenium ligands can smoothly enter the nucleus adjacent to the endoplasmic reticulum and act on DNA to play a PACT role.

[0035] This molecule directly coordinates amino acids to the ruthenium metal center by stirring at normal temperature in the dark, constructing an amino acid-based ruthenium complex that can be activated by near-infrared light to break the coordination bond and release singlet oxygen (ROS). Amino acids are naturally occurring molecules in living organisms, and their derivatives or analogs have low immunogenicity and toxicity. The introduction of amino acids can reduce the non-specific damage of drugs to normal cells. Some amino acids or their modified structures can be specifically taken up by cells enhanced by amino acid transporters overexpressed on the tumor surface, enhancing the enrichment of drugs in the tumor area. In addition, amino acids have good water solubility, and can self-assemble in aqueous solution by adjusting the intermolecular forces to form assemblies with different morphologies, playing an important role in the field of nanomedicine. Introducing amino acids into ruthenium complexes undoubtedly provides a new idea with better biocompatibility for the field of cancer treatment.

[0036] The introduction of amino acids not only improves the water solubility of the complex, but also endows it with self-assembly function. Placing it in a multi-channel vortex mixer can prepare a large number of nano-spheres with a diameter of about 100-140 nm, meeting the requirements of biological experiments. At the same time, the exposed carboxyl groups can change the overall charge of the complex through the protonation and deprotonation processes, thereby changing the intermolecular forces. When the pH gradually decreases, the carboxyl groups are gradually protonated, resulting in the charge of the complex changing from +1 to +2, increasing or decreasing the intermolecular repulsion, and then reducing the particle size of the nano-assembly. This feature can enhance the penetration depth of the nano-assembly in solid tumors and increase the cell uptake rate. Cell experiments show that the complex of the present invention can achieve the cascade targeting process from the endoplasmic reticulum to the nucleus, giving full play to the advantages of ruthenium complex photodynamic therapy (PDT) and photoactivated chemotherapy (PACT). After intravenous tail vein administration, the designed complex can effectively accumulate in mouse tumor tissues through the enhanced permeability and retention (EPR) effect, inhibit subcutaneous and orthotopic breast tumors in mice, and prevent lung metastasis. Therefore, the preparation method of the complex synthesis and assembly system in the present invention is simple, has low dark toxicity, and has a significant anti-tumor effect, showing the potential for clinical application. Brief Description of the Drawings

[0037] Figure 1 In which, a is the TEM image of compound 4a; b is the DLS particle size statistics of compound 4a in different solutions.

[0038] Figure 2 In which, a is the photolysis ultraviolet absorption diagram of compound 4a; b is the ultraviolet absorption diagram of ROS degrading DPBF.

[0039] Figure 3 It shows the enrichment of compound 4a in cells over time.

[0040] Figure 4 In which, a is the content of compound 4a in different organelles before light irradiation; b is the content of compound 4a in different organelles after light irradiation.

[0041] Figure 5 In which, a is the detection of adenosine triphosphate (ATP) secretion; b is the detection of lactate dehydrogenase release.

[0042] Figure 6 In which, a is the toxicity diagram of compound 4a at different concentrations on 4T1 cells under light or dark conditions; b is the toxicity diagram of compound 4a at different concentrations on MCF7 cells under light or dark conditions.

[0043] Figure 7 It is the in vivo fluorescence imaging diagram of mice after intravenous tail vein injection of the complex.

[0044] Figure 8In a, it is the content diagram of Ru element in tumor tissues and main organs after tail vein injection; in b, it is the relative tumor volume change diagram of different experimental groups during the treatment period.

[0045] Figure 9 In a, it is the TEM diagram of compound 4b; in b, it is the TEM diagram of compound 4c.

[0046] Figure 10 In a, it is the DLS particle size statistical diagram of compound 4b in aqueous solution; in b, it is the DLS particle size statistical diagram of compound 4c in aqueous solution.

[0047] Figure 11 In a, it is the toxicity diagram of compound 4b to 4T1 cells under light or dark conditions; in b, it is the toxicity diagram of compound 4c to 4T1 cells under light or dark conditions.

[0048] Figure 12 In a, it is the structural formula of compound 5; in b, it is the structural formula of compound 6.

[0049] Figure 13 In a, it is the toxicity diagram of compound 5 to 4T1 cells under light or dark conditions; in b, it is the toxicity diagram of compound 6 to 4T1 cells under light or dark conditions. Detailed implementation manners

[0050] The present invention provides a preparation strategy and application of an amino acid-based ruthenium complex self-delivery assembly. To make the invention purpose and technical solution of the present invention clearer and more definite, the following further explains the present invention in detail.

[0051] An amino acid-based ruthenium complex, characterized in that it has the following structure I, wherein R1 is a bipyridine type, R2 is a terpyridine type, and A1 is an amino acid type.

[0052]

[0053] Structural formula I

[0054] Some specific amino acid-based ruthenium complexes, R1 has the following structure, wherein X is a straight-chain alkane with 1-5 carbon atoms, a benzene ring, or an isopropyl group.

[0055]

[0056] Some specific amino acid-based ruthenium complexes, R2 has the following structure, wherein X1 is a trifluoromethyl group, a methoxy group, or an isopropyl group; X2 is a methyl group, an ethyl group, an isopropyl group, or a trifluoromethyl group.

[0057]

[0058] Some specific ruthenium complexes with amino acid groups, where A1 has the following structure, and Y is sulfur (S) or selenium (Se).

[0059]

[0060] A self-assembled nanoparticle comprising at least one of the complex small molecules described by Structural Formula I.

[0061] A nanoparticle, the preparation method of which is to prepare it by self-assembly in water and a multi-channel vortex mixer.

[0062] The diameter of the nanoparticle is 90 - 150 nm.

[0063] A specific ruthenium complex with short peptide groups has the following structure:

[0064] The present invention provides a synthesis method for a specific ruthenium complex with short peptide groups, and the specific steps are as follows:

[0065]

[0066] (1) Synthesis of Compound 1

[0067] Dissolve RuCl3 and terpyridine in a certain amount of ethanol solution, heat the mixture under stirring for a certain time and then cool it to room temperature, filter and collect the precipitate, wash it three times with ethanol and diethyl ether respectively, and finally dry it under vacuum to obtain a brown powder (0.70 g, 80%).

[0068] (2) Synthesis of Compound 2

[0069] Dissolve Compound 1 and 2,2'-biquinoline in 50 mL of 75% ethanol, bubble nitrogen for 10 min and then reflux overnight in the dark, remove the solvent under reduced pressure, and purify the crude product by silica gel column chromatography to obtain a purple powder (0.48 g, 61%).

[0070] (3) Synthesis of Compound 3

[0071] Dissolve Compound 2 and AgPF6 in 8 mL of aqueous solution, reflux in the dark for 12 h, cool to room temperature and precipitate with saturated KPF6 solution to obtain Compound 3.

[0072] (4) Synthesis of Compound 4a

[0073] Add Compound 3 and commercial Fmoc-methionine to acetone solution, stir in the dark at room temperature for 8 h, add diethyl ether to precipitate in the reaction solution to obtain Compound 4a.

[0074] A specific method for preparing nanoparticles. Prepare a DMSO stock solution of 3 mmol of compound 4a, and inject it into channel 1 of a vortex mixer at a flow rate of 3 mL / min. Inject ultrapure water into the remaining three channels at flow rates of 12, 3, and 12 mL / min respectively, and a large number of assemblies can be obtained.

[0075] The above-mentioned amino acid-based ruthenium complex self-assembled nanoparticles can be applied in the field of anti-tumor.

[0076] The present invention also provides amino acids with different protecting groups for different ruthenium complexes, and by regulating the amino acid protecting groups, the self-assembly of the complexes can be effectively promoted.

[0077] The molecules of the present invention have good tumor targeting ability, can aggregate in the tumor area through EPR, reduce the nanoparticle size by means of the protonation effect of the amino acid carboxyl group, and can further promote the uptake of tumor cells.

[0078] Among them, the enrichment process of the complex molecule in the cell is as follows: first, it is enriched on the endoplasmic reticulum, dissociates after being irradiated with light of a specific wavelength, then cascades and targets into the cell nucleus, forms an assembly with DNA and destroys DNA.

[0079] Among them, the complex molecule will generate ROS during the light irradiation process to destroy the endoplasmic reticulum, cause pyroptosis of the cell, and further promote cell death.

[0080] Among them, the complex molecule can effectively target and aggregate in the tumor tissue of mice, inhibit subcutaneous and orthotopic mammary tumors in mice, and prevent lung metastasis.

[0081] It should be clear that the examples of the specific implementation manners described here are only for further explaining and illustrating the present invention, rather than limiting the present invention.

[0082] Example 1: Preparation of an amino acid-based ruthenium complex and its nanoassembly

[0083]

[0084] (1) Synthesis of compound 1

[0085] Dissolve RuCl3 (0.52 g, 2.0 mmol) and terpyridine (tpy, 0.56 g, 2.0 mmol) in 150 mL of ethanol. Under stirring conditions, heat the mixture to 85 °C, keep it for 4 h and then cool it to room temperature. Filter and collect the precipitate, wash it with ethanol and ether respectively, and finally dry it under vacuum to obtain a brown powder (0.70 g, 80%), which is directly used for the next reaction.

[0086] (2) Synthesis of compound 2

[0087] Compound 1 (0.35 g, 0.8 mmol) and 2,2'-biquinoline (biq, 0.2 g, 0.8 mmol) were dissolved in 50 mL of 75% ethanol. After bubbling with nitrogen for 10 min, the mixture was refluxed for 24 h in the dark. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a purple powder (0.48 g, 61%).

[0088] 1 H NMR (500 MHz, CD3OD) δ (ppm): 9.66 (d, J = 8.6 Hz, 1H), 8.99 (d, J = 8.8 Hz, 1H), 8.93 (d, J = 8.8 Hz, 1H), 8.68 (d, J = 8.8 Hz, 1H), 8.65 (d, 8.3 Hz, 2H), 8.49 (d, J = 7.9 Hz, 2H), 8.32 (d, J = 8.7 Hz, 1H), 8.25 (d, J = 8.8 Hz, 1H), 8.19 (t, J = 8.3 Hz, 1H), 7.96 - 7.83 (m, 6H), 7.81 (d, J = 8.2 Hz, 1H), 7.44 (t, J = 8.8 Hz, 1H), 7.31 (t, J = 6.9 Hz, 2H), 7.20 (t, J = 8.8 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H). ESI-MS: (m / z): calculated for: 626.07, measured: 626.32.

[0089] (3) Synthesis of Compound 3

[0090] Compound 2 (28 mg, 0.049 mmol) and AgPF6 (28 mg, 0.11 mmol) were dissolved in 8 mL of aqueous solution. The mixture was refluxed for 12 h in the dark, cooled to room temperature, and precipitated with saturated KPF6 solution to obtain Compound 3.

[0091] 11H NMR (500 MHz, D2O) δ (ppm): 8.89 (d, J = 8.7 Hz, 1H), 8.78 (d, J = 8.7 Hz, 1H), 8.65 - 8.55 (m, 3H), 8.40 - 8.32 (m, 4H), 8.27 (t, J = 7.8 Hz, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.96 - 7.84 (m, 4H), 7.57 (d, J = 5.3 Hz, 2H), 7.52 (d, J = 7.9 Hz, 1H), 7.27 (t, J = 8.7 Hz, 1H), 7.20 - 7.12 (m, 3H), 6.65 (d, J = 8.7 Hz, 1H). ESI-MS: (m / z): calculated for: 609.11, measured: 609.23.

[0092] (4) Synthesis of Compound 4a

[0093]

[0094] Compound 3 and commercially available Fmoc-methionine were added to an acetone solution in a molar mass ratio of 1:2. After stirring at room temperature in the dark for 8 h, diethyl ether was added to the reaction solution for precipitation to obtain pure compound 4a.

[0095] 1 1H NMR (500 MHz, DMSO-d6) δ 12.67 (s, 1H), 9.27 - 9.05 (m, 2H), 8.96 - 8.76 (m, 3H), 8.68 - 8.40 (m, 5H), 8.13 - 7.83 (m, 9H), 7.70 (d, J = 9.3 Hz, 3H), 7.50 - 7.21 (m, 9H), 6.38 (d, J = 8.9 Hz, 1H), 4.39 - 4.07 (m, 4H), 2.07 - 1.72 (m, 3H), 1.48 (t, J = 64.4 Hz, 4H). ESI-MS: (m / z): calculated for: 962.22, measured: 962.35.

[0096]

[0097] Compound 3 and commercial Cbz-methionine were added to an acetone solution in a molar mass ratio of 1:2. After stirring at room temperature and in the dark for 8 h, ether was added to the reaction solution for precipitation to obtain pure compound 4b.

[0098] 1 H NMR (500 MHz, DMSO-d6) δ 9.20-9.09 (m, 2H), 8.94-8.80 (m, 3H),8.76-8.39 (m, 7H), 8.16-7.85 (m, 8H), 7.56-7.19 (m, 11H), 6.40 (d, J = 8.9Hz, 1H), 5.02 (q, J = 12.3 Hz, 2H), 1.88 (d, J = 144.6 Hz, 2H), 1.69-1.27 (m,4H). ESI-MS: (m / z): calculated for: 874.31, measured: 874.25.

[0099]

[0100] Compound 3 and commercial Boc-methionine were added to an acetone solution in a molar mass ratio of 1:2. After stirring at room temperature and in the dark for 8 h, ether was added to the reaction solution for precipitation to obtain pure compound 4c.

[0101] 1 H NMR (500 MHz, DMSO-d6) δ 9.20-9.11 (m, 2H), 8.88 (dt, J = 26.4,8.7 Hz, 3H), 8.68 (dd, J = 26.5, 8.3 Hz, 3H), 8.58-8.45 (m, 4H), 8.22-7.79(m, 8H), 7.50 (dp, J = 37.8, 7.5 Hz, 3H), 7.24 (q, J = 10.0, 9.1 Hz, 1H),6.78 (d, J = 8.2 Hz, 1H), 6.39 (d, J = 8.9 Hz, 1H), 1.49 (d, J = 29.8 Hz,2H), 1.29 (d, J = 57.5 Hz, 12H). ESI-MS: (m / z): calculated for: 840.29,measured: 840.23.

[0102] Example 2: Preparation method of self-assembled nanoparticles

[0103] In some specific embodiments, an amino acid-based ruthenium complex is added to an aqueous solution to prepare a solution of 50-200 μmol, for example, solutions of 50, 100, 150, and 200 μmol are prepared; then it is left standing at room temperature to obtain an assembly with uniform size.

[0104] The amino acid-based ruthenium complex 4a is added to an aqueous solution to prepare a 100 μmol solution, and it is left standing at room temperature for 2 h to obtain an assembly with uniform size, that is, an aqueous solution of complex 4a nanoparticles is obtained.

[0105] The amino acid-based ruthenium complex 4a is prepared as a 3 mmol DMSO stock solution and injected into channel 1 of a vortex mixer at a flow rate of 3 mL / min, and ultrapure water is injected into the remaining three channels at flow rates of 12, 3, and 12 mL / min, respectively, to obtain a large number of assemblies with uniform size.

[0106] The amino acid-based ruthenium complexes 4b and 4c are also prepared by the above method to obtain the corresponding aqueous solutions of nanoparticles.

[0107] Example 3: TEM characterization of self-assembled nanoparticles and DLS test of stability in different solutions

[0108] The aqueous solution of complex 4a nanoparticles prepared in Example 2 is dropped onto the surface of a copper grid. After drying, a JEOL JEM-2100F transmission electron microscope (working voltage: 200 kV) is used to characterize its assembled morphology, as Figure 1 shown in a. It can be seen that the size of its assembly is uniform, about 140 nm, which can meet the EPR effect and enable the assembly to be effectively enriched in the tumor area. Then, a Zetasizer Nano-ZS90 is used to perform dynamic light scattering (DLS) experiments on the particle size of the nano-assembly in different solutions, indicating that its particle size is the same as that in the aqueous solution. The obtained hydrodynamic diameter is 135 nm, and the PDI is 0.132, indicating that the nano-assembly has good dispersibility and stability in different solution systems and can stably exist during the in vivo circulation process.

[0109] Example 4: Photoresponsive study of amino acid-based ruthenium complexes

[0110] The photoresponsive performance of the complex is detected by the ultraviolet-visible absorption spectrum of the complex aqueous solution under irradiation with light of a specific wavelength. 20 μM of the amino acid-based ruthenium complex 4a is added to water, 2 mL is taken and added to a cuvette, and then the absorption spectrum of the solution is measured every 2 min under irradiation with 660 nm near-infrared light to observe the change of the absorption peak. As Figure 2As shown in Fig. a, after 12 min of irradiation, the absorption peak of the complex gradually shifted from 525 nm to 550 nm, indicating that the amino acid in the complex had completely dissociated and the complex had a fast light responsiveness.

[0111] Example 5: Test on the ROS generation ability of amino acid-based ruthenium complexes

[0112] During this process, 1,3-diphenylbenzofuran (DPBF) was selected as the singlet oxygen trapping reagent, and the ROS generation ability of the complex was evaluated by the mechanism that ROS can rapidly oxidize DPBF, resulting in the attenuation of its maximum absorption peak (411 nm). As Figure 2 shown in Fig. b, the amino acid-based ruthenium complex 4a designed in the present invention can rapidly oxidize DPBF at a concentration of 3 μmol, indicating that it has a strong ROS generation ability.

[0113] Example 6: Cellular uptake of amino acid-based ruthenium complex self-assemblies

[0114] The cells used in this experiment were MCF-7 cells (mouse breast cancer cells) and 4T1 cells (mouse breast cancer cells). The above cells were inoculated into DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture), and then cultured in a cell incubator at 37 °C and 5% CO2. When the cells grew to the logarithmic growth phase, the cells were digested with trypsin containing EDTA and then transferred to a 96-well plate or a confocal culture dish for subsequent cell experiments.

[0115] To determine the optimal time for cell therapy, it is necessary to determine the maximum enrichment time of the assemblies in cells through a cellular uptake experiment. In this experiment, Cy 5 was encapsulated in the assemblies, and its fluorescence was used to judge the optimal enrichment time of the complex assemblies. Human breast cancer cells MCF-7 were used for subsequent experiments. First, MCF-7 cells were cultured in a 35 mm confocal culture dish and incubated in a cell incubator at 37 °C for 24 h. When the cells were completely adherent, they could be used for the cellular uptake experiment. At this time, an aqueous solution of complex 4a nanoparticles with a concentration of 20 μM was incubated with MCF-7 cells for different times (0.5 h, 1 h, 4 h, 6 h, 8 h), and an FV-3000 laser confocal scanning microscope (Olympus) was used to observe the fluorescence changes in cells at different times. The maximum enrichment time of the nanoparticles in cells was judged by the fluorescence intensity at different times. Before imaging, it was washed 3 times with phosphate buffer saline (PBS).

[0116] As Figure 3As shown, weak fluorescence appeared in the cells 0.5 h after the addition of the assembly. As time increased, the intracellular fluorescence signal gradually enhanced, reaching the maximum at 4 h and then gradually weakening, indicating that the cells had enriched the most nanoparticles at 4 h. Therefore, in subsequent cytotoxicity experiments, the complex assembly was incubated with the cells for 4 h and then irradiated.

[0117] Example 7: Targeting of amino acid-based ruthenium complex assemblies to cell organelles

[0118] First, MCF-7 cells were seeded in a 9×9 cm culture dish. After the cell density reached 90%, 20 μmol of the aqueous solution of complex 4a nanoparticles was added thereto. After incubation for 4 h, it was divided into a light irradiation group and a dark group. The light irradiation group was irradiated under a 660 nm near-infrared lamp for 15 min, and then placed in an incubator for continued incubation for 1 h. The obtained cells were subjected to extraction and digestion of various cell organelles, and the ruthenium element content was measured by ICP-MS to evaluate the targeting of different cell organelles. As Figure 4 shown, a is the content of compound 4a in different cell organelles before light irradiation; b is the content of compound 4a in different cell organelles after light irradiation; before light irradiation, the assembly was highly enriched in the endoplasmic reticulum, and after light irradiation, the ruthenium element content in the nucleus increased sharply, indicating that the designed complex assembly has the function of photo-responsive cascade targeting, can fully exert the PDT and PACT functions of the ruthenium complex, and has a better killing effect on cancer cells.

[0119] Example 8: Release of pyroptosis factors mediated by amino acid-based ruthenium complex assemblies

[0120] (1) Detection of adenosine triphosphate (ATP) secretion.

[0121] The cells were seeded in a 24-well plate at a density of 1×10 5 / cm 2 and cultured until the cell density grew to 90%. The cells were divided into 4 groups, and each group was set with three parallel experiments. After adding 20 μmol of the aqueous solution of complex 4a nanoparticles for 4 h, the light irradiation group was irradiated for 15 min, and then incubated in an incubator for 1 h. The culture medium was aspirated and added to a black-bottomed 96-well plate. After adding the ATP detection working solution to each well, it was left standing for 3 - 5 min, and the chemiluminescence was measured by an enzyme-linked immunosorbent assay reader. It can be seen that the ATP release amount increased significantly after light irradiation ( Figure 5 a in), indicating that the complex assembly has the characteristic of inducing cell pyroptosis.

[0122] (2) Detection of lactate dehydrogenase release

[0123] Cells were seeded into 96-well cell culture plates, and the experiment was performed when the cell density reached 80–90%. Each culture well was divided into the following groups: a well containing culture medium without cells (background blank control well), a well containing control cells without drug treatment (sample control well), a well containing cells without drug treatment for subsequent lysis (sample maximum enzyme activity control well), and a well containing cells treated with 20 μmol of the complex 4a nanoparticle aqueous solution (20 μmol). After incubation for 4 h, the well was illuminated, the culture medium was aspirated, and the cells were washed 2-3 times with PBS solution. Fresh culture medium (serum-free culture medium) was replaced, and the culture was continued as usual. One hour before the scheduled detection time point, the cell culture plate was removed from the cell culture incubator, and the LDH release reagent provided by the kit was added to the "sample maximum enzyme activity control well" in an amount of 10% of the original culture medium volume. After adding the LDH release reagent, the well was repeatedly pipetted several times to mix, and then the well was continued to be incubated in the cell culture incubator. After the scheduled time, the cell culture plate was centrifuged at 400 g for 5 min using a multi-well plate centrifuge. Take 60 μl of the supernatant from each well and add it to the corresponding well of a new 96-well plate, and then perform sample measurement. The lactate dehydrogenase detection solution is composed of lactic acid, INT solution, and enzyme solution. The proportions are based on the instructions of the kit. Add 60 μL of lactate dehydrogenase detection solution to each well, mix well, and incubate at room temperature in the dark for 30 min. Then measure the absorbance at 490 nm, and use 600 nm as the reference wavelength for dual-wavelength measurement.

[0124]

[0125] OD 490 and OD 600 Represents the absorbance of the sample well at 490 nm and 600 nm; OD con and OD max Refers to the absorbance of the sample control well and the sample maximum enzyme activity control well. Figure 5 In b, 4a can induce a large amount of LDH release under light, showing the ability to induce cell apoptosis.

[0126] Example 9: Cytotoxicity Evaluation of Amino Acid-Based Ruthenium Complex Assemblies

[0127] The cytotoxicity of the complex assembly was tested by MTT assay. MCF-7 cells and 4T1 cells were cultured in 96-well plates (about 1×10 4Cells (add 100 μL of DMEM medium thereto). When the cells adhered and reached a density of 90%, an aqueous solution of complex 4a nanoparticles with different concentrations was added thereto and incubated for 4 h, and then irradiated with 660 nm (30 mW) for 15 min. At the same time, another control group with the same experimental conditions but without light irradiation was also prepared for the study of dark toxicity of the cells. After incubating again at 37 °C for 12 h, 100 μL of DMEM solution with 0.5 mg / mL MTT was added to each well and incubated for another 4 h. Subsequently, the medium in the 96-well plate was removed, and 100 μL of DMSO was added to each well to dissolve the generated formazan crystals. A Bio-Rad microplate reader was used to measure the absorbance of the solution at 490 nm and calculate the cell viability:

[0128]

[0129] Among them, the experimental group represents the cell culture group treated with different concentrations of complex nanoassemblies; the blank group represents the cell culture group with only medium added: the control group represents the cell culture group without complex nanoassemblies added: OD is the absorbance value measured at 490 nm of the DMSO solution dissolving formazan crystals. Each experiment was repeated 4 times in parallel.

[0130] As Figure 6 shown, under dark conditions, even at high concentrations of amino acid-based ruthenium complexes, only weak toxicity can be produced to both types of cells, while after irradiation with 660 nm laser, a great mortality rate is shown in both types of cells, which effectively proves that the amino acid-based ruthenium complex can effectively kill cancer cells after irradiation with 600 nm light for 15 min, has good phototoxicity, and is safe under dark conditions.

[0131] Example 10: Study on the anti-tumor performance of amino acid-based ruthenium complex assemblies in vivo

[0132] Under the "Guide for the Care and Use of Laboratory Animals", 4T1 cells were subcutaneously injected into the axilla of the front limb of mice with a cell suspension of 5*10 6 / mL. When the tumor volume was about 100 mm 3 . The mice were randomly grouped for experiments, where V 肿瘤 = long diameter × short diameter 2 ×1 / 2.

[0133] (1) Optimal enrichment time and distribution of amino acid-based ruthenium complex in mice

[0134] 100 μmol of the aqueous solution of complex 4a was injected into mice via the tail vein. At regular intervals, a small animal imager was used to monitor the distribution of the complex in vivo. Through the fluorescence signal, the targeting ability of the complex and the optimal enrichment time in the tumor region could be preliminarily judged. Thereafter, three more mice were selected and injected with 100 μmol of the aqueous solution of complex 4a. When the optimal enrichment time was reached, the mice were sacrificed by cervical dislocation. The tumor tissues and main organ tissues were collected for ICP-MS testing to detect the content of Ru element in each tissue and quantitatively analyze the targeting ability of the complex.

[0135] It can be seen from Figure 7 that after intravenous injection, the fluorescence signal in the tumor region showed a trend of increasing first and then decreasing, reaching the maximum at 18 h, which was its optimal enrichment time, and then gradually decreasing, and basically disappearing until the third day, indicating that the complex could stay in the tumor tissue for a long time and meet the needs of multiple treatments. To further verify the targeting ability of the amino acid-based ruthenium complex to tumor tissues in mice, the content of Ru element in different tissues and organs was measured by ICP-MS. It was found that the complex was highly enriched in the tumor tissue ( Figure 8 in a), showing good selectivity.

[0136] (2) In vivo antitumor performance of amino acid-based ruthenium complex

[0137] To evaluate the in vivo antitumor effect of the amino acid-based ruthenium complex, mice were randomly divided into four groups: PBS, PBS + light, 4a, and 4a + light. After intravenous injection, at the optimal enrichment time, the tumor sites of the mice were irradiated for 20 min. The body weight and tumor volume of the mice were monitored every two days. After the treatment cycle, the mice were sacrificed by cervical dislocation, and the tumor tissues and main organ tissues were taken for H&E staining analysis. It can be seen that after treatment with 4a, the tumors of the mice hardly grew significantly, and the tumor inhibition rate reached 75%. However, the tumor growth of other control groups was not affected, indicating that the complex could effectively inhibit the growth of solid tumors under near-infrared light irradiation by intravenous injection ( Figure 8 in b).

[0138] Example 11: Study on the antitumor performance of amino acid-based ruthenium complexes 4b and 4c

[0139] First, the assembly performance of amino acid-based ruthenium complexes 4b and 4c was studied. It was found that they also had certain assembly performance, but relatively lower than that of 4a. TEM testing observed that 4b formed irregular assemblies with a size of 20 nm, while 4c formed assemblies with a size of less than 10 nm ( Figure 9 ). At the same time, DLS particle size testing found that the stability of the assemblies gradually decreased starting from 4b ( Figure 10 ). However, cell experiments found that 4b and 4c also had the effect of killing cells (Figure 11 ).

[0140] Example 12: Study on the Antitumor Properties of Derivatives of Amino Ruthenium Complexes

[0141]

[0142] In addition, two other derivatives, Compound 5 and Compound 6 ( Figure 12 ) were synthesized. The synthesis steps were the same as those of 4a, and only different ligands were replaced. The molecular structures of the final compounds were correctly characterized by mass spectrometry (Compound 5: ESI-MS: (m / z): calculated for: 862.19, measured: 862.32; Compound 5: ESI-MS: (m / z): calculated for: 962.22, measured: 962.21). Subsequently, their antitumor activities at the cellular level were studied, and it was found that both of them had antitumor activities. The minimum inhibitory concentration of Compound 5 was higher than that of Compound 4a, but it showed obvious anticancer cell proliferation effect, and only 55 μM was required to effectively kill 4T1 tumor cells under light conditions ( Figure 13 a). The minimum inhibitory concentration of Compound 6 for antitumor was slightly less than that of Compound 4a, showing good antitumor activity ( Figure 13 b). Therefore, the designed compounds all have the potential to be used as cancer drugs.

Claims

1. An amino acid-based ruthenium complex, characterized in that, The complex has the following general structural formula: ； Wherein, R1 is a bipyridine ligand, R2 is a terpyridine ligand, and A1 is an amino acid ligand; Z - is a halogen anion, nitrate anion, sulfate anion, hexafluorophosphate anion; The complex is electrically neutral; R1 is selected from the following structures: ； Wherein, X is hydrogen, a straight-chain or branched-chain alkane with 1-5 carbon atoms; R2 is selected from the following structures: ； Wherein, X1 is hydrogen, trifluoromethyl, methoxy, isopropyl; X2 is hydrogen, trifluoromethyl, a straight-chain or branched-chain alkane with 1-5 carbon atoms; A1 is selected from the following structures: ； Wherein, Y is sulfur or selenium.

2. The ruthenium complex according to claim 1, wherein: The X is hydrogen, methyl, ethyl or isopropyl.

3. The ruthenium complex according to claim 2, wherein: X2 is hydrogen, trifluoromethyl, methyl, ethyl or isopropyl.

4. The ruthenium complex according to claim 3, characterized in that: X is hydrogen, X1 is hydrogen, X2 is hydrogen, and Y is sulfur.

5. A nanoparticle, characterized in that, The nanoparticles include at least one of the ruthenium complexes described in claims 1-4.

6. A nanoparticle according to claim 5, wherein, The prepared nanoparticles are obtained by self-assembly in water or by a multi-channel vortex mixer.

7. A nanoparticle according to claim 6, characterized in that, The diameter of the particles is 90-150 nm.

8. Use of the amino acid-based ruthenium complex according to any one of claims 1 to 4, characterized in that: The amino acid-based ruthenium complex is used in the preparation of a drug for treating tumors.

9. Use of the nanoparticles according to any one of claims 5-6, characterized in that, The nanoparticles are used in the preparation of a drug for treating tumors.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition includes any one of the ruthenium complexes described in claims 1-4, or includes any one of the nanoparticles described in claims 5-6.

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