Chiral iridium complex nano assembly as well as preparation method and application thereof

Abstract

The invention discloses a chiral iridium complex nano assembly as well as a preparation method and application thereof. The chiral iridium complex nano assembly disclosed by the invention is obtained by carrying out ultrasonic treatment, dialysis and filtration on delta-IrBMS8-mPEG or lambda-IrBMS8-mPEG. The chiral iridium complex nano assembly disclosed by the invention can induce chirality-dependent ferroptosis in a photodynamic therapy (PDT) process and cooperate with blocking of immune checkpoints, so that an anti-tumor immune process is effectively activated, and an efficient synergistic treatment effect is achieved. The preparation method disclosed by the invention is simple and controllable in process, excellent in biological safety and suitable for anti-tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a preparation method of chiral iridium complex nano-assembly and application thereof. BACKGROUND

[0002] Metal drugs, as an important branch of anticancer drugs, play a unique role in the diagnosis and treatment of cancer. Cisplatin, a milestone in modern metal drugs, was approved by FDA for clinical tumor treatment in 1978. Due to the successful application of cisplatin in cancer treatment, the development of platinum drugs has been very rapid in the past few decades, and various cisplatin metal drugs such as carboplatin and oxaliplatin have been successively approved for marketing. At present, more than 50% of tumor patients rely on cisplatin and its derivative drugs for chemotherapy. However, the DNA damage caused by cisplatin metal drugs is easily repaired by the DNA repair system, thereby causing tumor cell drug resistance, and also lacking of targeting, easily causing systemic toxicity, and low bioavailability, which greatly limits their clinical efficacy.

[0003] In order to overcome the drug resistance and systemic toxicity of cisplatin metal drugs, researchers have carried out a lot of work. On the one hand, the structure-activity relationship of traditional cisplatin metal drugs is changed, and various types of platinum complexes are developed. On the other hand, the anti-tumor effect of other metal complexes is explored. At present, the European Union has approved a palladium porphyrin photosensitizer for the treatment of prostate cancer, and Ru photosensitizer TLD-1433 and Mn porphyrin photosensitizer BMX-001 are in the preclinical trial stage. Os, Rh, Au, Ir and other transition metal complexes also show strong anti-tumor effect. Among them, iridium complexes have the characteristics of coordination saturation, structural stability and adjustable configuration, and often also show high efficient catalytic properties, rich redox states and photoelectric properties, and have attracted widespread attention in the field of anti-tumor.

[0004] Iridium complexes are widely used in chemotherapy drugs, photosensitizers and tracer imaging due to their strong anti-cancer activity, unique mechanism of action and limited side effects, but have the shortcomings of easy racemization, unknown intracellular precise action target and poor targeting of biological macromolecules. Chirality is an important tool in modern drug development, and molecular recognition of chiral biological targets can provide a theoretical basis for the design of new active drugs. The chirality of metal complexes is an inherent factor of their biological activity, and chiral metal drugs are a rapidly developing field in current anti-cancer research. Inspired by the first clinically approved chiral anti-tumor metal drug oxaliplatin, chiral metal drugs have attracted more and more attention, and a number of works have confirmed that chiral metal complexes can specifically and selectively recognize and bind chiral DNA and enzyme molecules, and play the function of precise targeting of biological macromolecules. Therefore, it is crucial to construct metal iridium complexes with precise chiral structure for the development of precise drugs.

[0005] In addition, in order to overcome the low target accumulation level of traditional small molecule metal drugs, a nano-formulation delivery strategy is usually introduced to improve the efficacy of the chemotherapy drugs and reduce their toxicity. However, the combination of nano-formulations and metal drugs inevitably causes problems such as unclear metal drug-nano-carrier interaction mechanism, low controllability and poor repeatability. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a preparation method of a chiral iridium complex nano-assembly and its application. The present application utilizes the PD-L1 receptor targeting property of BMS8, combines the strong anticancer activity and limited side effects of iridium complexes, and solves the problems of systemic toxicity and easy tumor cell drug resistance of existing metal drugs such as cisplatin, which are difficult to provide effective treatment for solid tumors due to the lack of targeting property. The chiral iridium complex nano-assembly prepared by the present application has the advantages of high bioavailability, strong targeting property and small toxic and side effects.

[0007] The technical solutions of the present application are as follows:

[0008] A chiral iridium complex nano-assembly, wherein the chiral iridium complex nano-assembly is obtained by ultrasonic treatment, dialysis and filtration of Δ-IrBMS8-mPEG or Λ-IrBMS8-mPEG; the structures of Δ-IrBMS8-mPEG and Λ-IrBMS8-mPEG are as follows:

[0009]

[0010] The power of the ultrasonic treatment is 18 kHz; the flow rate of the solution added during the ultrasonic treatment is 2.4 mL / h; and the membrane molecular weight cut-off of the dialysis is 10 kDa.

[0011] A preparation method of the chiral iridium complex nano-assembly, wherein the preparation method comprises the following steps:

[0012] Δ-IrBMS8 and Λ-IrBMS8 are subjected to a reduction reaction with NaBH4 to obtain dark red solids; under the catalysis of DMAP and DCC, the dark red solids are subjected to an acylation reaction with mPEG-COOH to obtain Δ-IrBMS8-mPEG and Λ-IrBMS8-mPEG; then Δ-IrBMS8-mPEG and Λ-IrBMS8-mPEG are subjected to ultrasonic treatment, dialysis and filtration, respectively, to obtain Δ-IrBMS8-NPs and Λ-IrBMS8-NPs nano-assemblies, respectively.

[0013] Further, the solvent of the reduction reaction is a mixture of DCM and MeOH in a volume ratio of 1:1, and the reducing agent is NaBH4; the molar ratio of the reducing agent to Δ-IrBMS8 or Λ-IrBMS8 is 2.5-3.5:1;

[0014] The solvent of the acylation reaction is DCM, the mass ratio of the mPEG-COOH to Δ-IrBMS8-NPs or Λ-IrBMS8-NPs is 1:0.2-0.3; the mass ratio of the mPEG-COOH to DMAP is 100:1; and the mass ratio of the DMAP to DCC is 1:9-12;

[0015] The power of the ultrasound is 18 kHz; the flow rate of the solution added during the ultrasound is 2.4 mL / h; and the molecular weight cut-off of the dialysis membrane is 10 kDa.

[0016] Further, the preparation method of Δ-IrBMS8 and Λ-IrBMS8 is as follows:

[0017]

[0018] The specific steps are as follows:

[0019] 4-(Isoquinoline-1-yl)benzaldehyde is used as the main ligand, and iridium chloride hydrate is used as the iridium source to perform a reaction, and then (R)-Fphox and triethylamine are added to perform a reaction to obtain chiral iridium complex precursors Δ-Irpiq-R and Λ-Irpiq-R;

[0020] Δ-Irpiq-R and Λ-Irpiq-R are subjected to coordination reactions with BMS8-NN, respectively, to obtain Δ-IrBMS8 and Λ-IrBMS8, respectively.

[0021] Further, the molar ratio of 4-(isoquinoline-1-yl)benzaldehyde to iridium chloride hydrate is 2.4-2.5:1; the molar ratio of iridium chloride hydrate to (R)-Fphox is 1:1.1-1.3; and the molar ratio of iridium chloride hydrate to triethylamine is 1:5-10;

[0022] The catalyst of the coordination reaction is trifluoroacetic acid, the temperature is 25°C, and the time is 2-4 h; and the molar ratio of the trifluoroacetic acid to Δ-Irpiq-R or Λ-Irpiq-R is 4-6:1;

[0023] When Δ-IrBMS8 is prepared, the molar ratio of Δ-Irpiq-R to BMS8-NN is 1:1.1-1.3;

[0024] When Λ-IrBMS8 is prepared, the molar ratio of Λ-Irpiq-R to BMS8-NN is 1:1.1-1.3.

[0025] Further, the preparation method of the (R)-Fphox is carried out according to the following scheme:

[0026]

[0027] The specific steps are as follows: 2-fluoro-6-hydroxybenzonitrile and D-phenylalaninol are dissolved in toluene, and a ring closure reaction is carried out under the catalysis of zinc chloride to obtain compound (R)-Fphox.

[0028] Further, the molar ratio of 2-fluoro-6-hydroxybenzonitrile to D-phenylalaninol is 1:1.3; the molar ratio of zinc chloride to 2-fluoro-6-hydroxybenzonitrile is 1:40; the temperature of the ring closure reaction is 130℃, and the time is 24h.

[0029] Further, the preparation method of the BMS8-NN is carried out according to the following scheme:

[0030]

[0031] The specific steps are as follows:

[0032] (2-methylbiphenyl-3-yl)methanol is used as a starting material, and a bromination reaction is carried out with PBr3 to obtain intermediate compound a, intermediate compound a is subjected to an etherification reaction with 3-bromo-4-hydroxybenzaldehyde to obtain intermediate compound b, and intermediate compound b is subjected to a reductive amination reaction with 2-piperidinecarboxylic acid and NaBH3CN to obtain intermediate compound c;

[0033] After 5-amino-1,10-phenanthroline is reacted with p-hydroxybenzaldehyde at 65℃ for 12h, NaBH4 is added to carry out an amidation reaction to obtain intermediate compound d;

[0034] Intermediate compound c is subjected to a condensation reaction with intermediate compound d to obtain the targeting ligand BMS8-NN of PD-L1.

[0035] Further, the molar ratio of PBr3 to (2-methylbiphenyl-3-yl)methanol is 1.1-1.3:1; the solvent of the bromination reaction is THF, the temperature is room temperature, and the time is 2-4h;

[0036] The molar ratio of 3-bromo-4-hydroxybenzaldehyde to intermediate compound a is 0.7-0.8:1; the solvent of the etherification reaction is acetone, the temperature is 55-65℃, and the time is 10-14h;

[0037] The molar ratio of 2-piperidinecarboxylic acid, NaBH3CN to intermediate compound b is 3:3:1, the solvent of the reductive amination reaction is DMF, the temperature is 75-85℃, and the time is 2-3h.

[0038] The molar ratio of the 5-amino-1,10-phenanthroline to p-hydroxybenzaldehyde is 1:1.1~1.3; the solvent of the amidation reaction is anhydrous ethanol; the temperature of the amidation reaction is room temperature, and the time is 2-3h;

[0039] The molar ratio of the intermediate compound c and the intermediate compound d is 1:1~1.5, the solvent of the condensation reaction is DMF, the condensing agent is HATU, the temperature is room temperature, and the time is 11-13h.

[0040] An anti-tumor drug, comprising the chiral iridium complex nano-assembly of claim 1 or the chiral iridium complex nano-assembly prepared by the preparation method of any one of claims 2-9.

[0041] The present application has the beneficial technical effects that:

[0042] The present application assembles the chiral metal complex by self-assembly, and the nano-particles after assembly have the same optical chirality as the complex, and the nano-particles with two chiralities have different anti-tumor activities, thus avoiding the problems of low controllability and poor repeatability of ordinary nano-drugs, improving the bioavailability and tumor enrichment rate of the chiral metal drugs, and further activating and enhancing the immune regulation function of the chiral iridium complex nano-assembly.

[0043] The present application first introduces the BMS8 small molecule structure of the PD-L1 receptor target as a ligand group, which theoretically helps to improve the targeting of the complex to tumor cells and reduce the toxic side effects of the drug; secondly, the chirality of the ring metal iridium structure can provide chiral-dependent anti-tumor effect regulation, and the photoelectric properties of the iridium complex help to introduce optical auxiliary treatment, and the covalent modification of the amphiphilic PEG long chain can make the metal chiral complex self-assemble into a nano-assembly, change the mode of drug delivery, and improve the bioavailability. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 It is a structural schematic diagram of the chiral iridium complex nano-assembly Δ-IrBMS8-NPs and Λ-IrBMS8-NPs of the present application.

[0045] Figure 2 It is a TEM and SEM morphology diagram of the chiral iridium complex nano-assembly prepared by the present application.

[0046] Figure 3 It is a CD spectrum comparison diagram of Δ-IrBMS8-NPs and Λ-IrBMS8-NPs.

[0047] Figure 4Dynamic light scattering plot of Δ-IrBMS8-NPs and Λ-IrBMS8-NPs.

[0048] Figure 5 Intracellular ROS fluorescence imaging plot induced by Δ-IrBMS8-NPs.

[0049] Figure 6 Tumor volume change plot of tumor-bearing mice.

[0050] Figure 7 Flow cytometry plot of splenic effector CD8+ T cells. DETAILED DESCRIPTION

[0051] The present application will be described in detail below with reference to the accompanying drawings and examples.

[0052] The mPEG-COOH (2000 Da) used in the following examples of the present application was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., with the product number of BD633654.

[0053] The experimental animals used in the following examples of the present application were CT26 tumor-bearing mice, which were constructed as follows: 6-8 week old female BALB / c mice were purchased, and after adaptive culture at 25℃ for 7 days, 1.0×10 6 CT26 (100 μL PBS) cells to obtain CT26 tumor-bearing mice.

[0054] Example 1

[0055] Synthesis of ligand BMS8-NN, the synthetic route of which is as follows:

[0056]

[0057] The specific synthesis method is as follows:

[0058] S1-1: Synthesis of compound a: (2-methylbiphenyl-3-yl)methanol (3.37 g, 17 mmol) was dissolved in THF (40 mL), PBr3 (1.75 mL, 18.7 mmol) was added dropwise at 0℃, and the reaction was carried out at room temperature for 3 h. Column chromatography (PE) gave a light yellow solid (compound a, 4.43 mmol, 2.38 g, yield 99%, 1 H NMR δ 4.79 ppm).

[0059] S1-2: Synthesis of compound b: Compound a (1.38 g, 4 mmol) was dissolved in acetone (15 mL) with 3-bromo-4-hydroxybenzaldehyde (603 mg, 3.0 mmol), K2CO3(2.2 g, 15.9 mmol) and reacted at 60 °C for 12 h. Column chromatography (PE:EA = 50:1) gave white solid (compound b, 400 mg, 1.05 mmol, yield 35%, 1H NMR δ 9.88 ppm). 1 H NMR δ 9.88 ppm).

[0060] S1-3: Synthesis of BMS8 (compound c): Compound b (380 mg, 1 mmol) was dissolved in DMF (1 mL) with 2-piperidinecarboxylic acid (387 mg, 3.0 mmol), NaBH3CN (189 mg, 3.0 mmol) and 0.1 mL glacial acetic acid was added at 80 °C for 2 h. Column chromatography (EA:MeOH = 30:1) gave white solid BMS8 (compound c, 414 mg, 0.84 mol, yield 84%).

[0061] S1-4: Synthesis of compound d: 5-Amino-1,10-phenanthroline (390 mg, 2 mmol) was dissolved in anhydrous ethanol (10 mL) with p-hydroxybenzaldehyde (256 mg, 2.1 mmol) and heated to 65 °C under N2protection with dropwise addition of 90 μL glacial acetic acid for 12 h. Vacuum concentration and filtration gave yellow solid. The yellow solid was dissolved in ethanol / dichloromethane (20 mL 1:1, v / v) and NaBH4(757 mg, 20 mmol) was added slowly and reacted at room temperature for 2 h. The reaction was quenched with water and extracted with DCM (20 mL x 3). The organic phase was combined and washed with brine, dried over anhydrous Na2SO4, and concentrated under vacuum to give yellow solid, which was intermediate compound d, and used directly in the next step.

[0062] S1-5: Synthesis of ligand BMS8-NN: Compound c (247 mg, 0.5 mmol) was dissolved in DMF (2 mL) with phenanthroline derivative (compound d, 150 mg, 0.5 mmol) and condensed at 25 °C for 12 h under N2protection with HATU / DIPEA (HATU, 285 mg, 0.75 mmol; DIPEA, 325 mg, 2.5 mmol) as catalyst. Column chromatography (DCM:MeOH:H2O = 3:0.5:0.1) gave yellow solid (BMS8-NN, 318 mg, 0.41 mmol, yield 82%, ESI-MS m / z 781.32).

[0063] Example 2

[0064] Synthesis of Δ-IrBMS8 and Λ-IrBMS8, the specific synthesis method is as follows:

[0065] S2-1: Synthesis of (R)-Fphox, the synthetic route is as follows:

[0066]

[0067] The specific synthesis method is: 2-fluoro-6-hydroxybenzonitrile (2.74 g, 20 mmol), D-phenylalaninol (3.56 g, 26 mmol), ZnCl2(68 mg, 0.5 mmol) and 20 mL of toluene are added to a 50 mL schlenk tube, and the reaction is stirred at 130°C under N2atmosphere for 24 h. After the reaction is cooled, it is quenched with H2O, extracted with EA, the organic phases are combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and rotary evaporated. The crude product is purified by column chromatography (eluent: PE:EA = 10:1, v / v) to obtain a white solid with a yield of 70% (3.6 g, 14 mmol).

[0068] S2-2: Synthesis of Δ-Irpiq-R and Λ-Irpiq-R, the synthetic route is as follows:

[0069]

[0070]

[0071] The specific synthesis method is: 4-(isoquinolin-1-yl)benzaldehyde is used as the main ligand, and iridium chloride hydrate is used as the iridium source. 4-(Isoquinolin-1-yl)benzaldehyde (583 mg, 2.5 mmol) and iridium source (356 mg, 1 mmol) are dissolved in a mixed solution of ethylene glycol ethyl ether and water (10 mL, 3:1, v / v) under N2protection, and the reaction is carried out at 130°C for 24 h. After the reaction is completed, the reaction mixture is filtered and washed, and then dried under vacuum to obtain a black solid (which is directly used for the next step reaction). The black solid and (R)-Fphox (310 mg, 1.2 mmol) are dissolved in anhydrous ethanol (40 mL), and triethylamine (1 g, 9.9 mmol) is added under N2protection. The reaction is refluxed for 12 h, and then column chromatography is used to separate the corresponding chiral iridium complex precursors Δ-Irpiq-R and Λ-Irpiq-R.

[0072] S2-3: Synthesis of Δ-IrBMS8: Δ-Irpiq-R (47.5 mg, 0.05 mmol) was dissolved in DCM (20 mL) with BMS8-NN (47 mg, 0.06 mmol), trifluoroacetic acid (20 μL, 0.27 mmol) was added, and the reaction was allowed to proceed at room temperature for 2 h. Column chromatography (DCM:MeOH = 60:1) gave dark red solid (Δ-IrBMS8, 64 mg, 0.04 mmol, yield 88%, HRMS [M-Cl-]+: 1433.3510).

[0073] S2-4: Synthesis of Λ-IrBMS8: Λ-Irpiq-R (47.5 mg, 0.05 mmol) was dissolved in DCM (20 mL) with BMS8-NN (47 mg, 0.06 mmol), trifluoroacetic acid (20 μL) was added, and the reaction was allowed to proceed at room temperature for 2 h. Column chromatography (DCM:MeOH = 60:1) gave dark red solid (Λ-IrBMS8, 64 mg, 0.04 mmol, yield 88%, HRMS [M-Cl-]+: 1433.3510).

[0074] Example 3

[0075] The preparation of chiral iridium complex nanoassemblies is as follows:

[0076] S3-1: PEG modification of Δ-IrBMS8: Δ-IrBMS8 (29.4 mg, 0.02 mmol) was dissolved in 10 mL of DCM / MeOH (V DCM / V MeOH =1:1) and NaBH4(2.5 mg, 0.066 mmol) was added while stirring. The reduction reaction was allowed to proceed for 1 h. After the reaction was completed, the reaction solution was spin-dried, water and EA were added to extract the combined organic phase, anhydrous Na2SO4was added for drying, filtered, and spin-dried to obtain dark red solid (29.4 mg, 0.02 mmol). The dark red solid (29.4 mg, 0.02 mmol), mPEG-COOH (2000 Da, 100 mg), and DMAP (1 mg) were dissolved in DCM (2 mL), and a DCC (10.3 mg, 50 μmol) solution in DCM was added dropwise to catalyze the condensation reaction. The condensation reaction was allowed to proceed at room temperature for 48 h, and column chromatography gave Δ-IrBMS8-mPEG (31 mg, yield 45%, HRMS [M-Cl-]+: 1433.3510). 1 H NMR δ 3.50 ppm broad peak; the molecular structure is shown in Figure 1

[0077] ​S3-2: Preparation of Δ-IrBMS8-NPs nanoassemblies: Δ-IrBMS8-mPEG was dissolved in DMSO at a final concentration of 5 mg / mL, and sonicated using a Biosafer 650-92 ultrasonicator at 18 kHz, 2.4 mL / h flow rate. The sonication conditions were selected to obtain Δ-IrBMS8-NPs nanoassemblies with a diameter of 160 nm. The Δ-IrBMS8-NPs nanoassemblies were dialyzed (dialysis cutoff 10 kDa) against deionized water at a constant rate using a peristaltic pump, and then filtered through a 0.45 μm filter membrane. The Δ-IrBMS8-NPs nanoassemblies were characterized by SEM and TEM, and the results are shown in FIG. 3A and FIG. 3B, respectively. The SEM and TEM results show spherical nanoparticles (diameter 160.8 ± 12.3 nm, PDI 0.15). The CD spectrum of the Δ-IrBMS8-NPs nanoassemblies is shown in FIG. 3C, and the DLS results are shown in FIG. 3D. The DLS and CD spectrum confirm the chirality retention. S3-2: Preparation of Δ-IrBMS8-NPs nanoassemblies: Δ-IrBMS8-mPEG was dissolved in DMSO at a final concentration of 5 mg / mL, and sonicated using a Biosafer 650-92 ultrasonicator at 18 kHz, 2.4 mL / h flow rate. The sonication conditions were selected to obtain Δ-IrBMS8-NPs nanoassemblies with a diameter of 160 nm. The Δ-IrBMS8-NPs nanoassemblies were dialyzed (dialysis cutoff 10 kDa) against deionized water at a constant rate using a peristaltic pump, and then filtered through a 0.45 μm filter membrane. The Δ-IrBMS8-NPs nanoassemblies were characterized by SEM and TEM, and the results are shown in FIG. 3A and FIG. 3B, respectively. The SEM and TEM results show spherical nanoparticles (diameter 160.8 ± 12.3 nm, PDI 0.15). The CD spectrum of the Δ-IrBMS8-NPs nanoassemblies is shown in FIG. 3C, and the DLS results are shown in FIG. 3D. The DLS and CD spectrum confirm the chirality retention. Figure 2 S3-2: Preparation of Δ-IrBMS8-NPs nanoassemblies: Δ-IrBMS8-mPEG was dissolved in DMSO at a final concentration of 5 mg / mL, and sonicated using a Biosafer 650-92 ultrasonicator at 18 kHz, 2.4 mL / h flow rate. The sonication conditions were selected to obtain Δ-IrBMS8-NPs nanoassemblies with a diameter of 160 nm. The Δ-IrBMS8-NPs nanoassemblies were dialyzed (dialysis cutoff 10 kDa) against deionized water at a constant rate using a peristaltic pump, and then filtered through a 0.45 μm filter membrane. The Δ-IrBMS8-NPs nanoassemblies were characterized by SEM and TEM, and the results are shown in FIG. 3A and FIG. 3B, respectively. The SEM and TEM results show spherical nanoparticles (diameter 160.8 ± 12.3 nm, PDI 0.15). The CD spectrum of the Δ-IrBMS8-NPs nanoassemblies is shown in FIG. 3C, and the DLS results are shown in FIG. 3D. The DLS and CD spectrum confirm the chirality retention. Figure 3 S3-2: Preparation of Δ-IrBMS8-NPs nanoassemblies: Δ-IrBMS8-mPEG was dissolved in DMSO at a final concentration of 5 mg / mL, and sonicated using a Biosafer 650-92 ultrasonicator at 18 kHz, 2.4 mL / h flow rate. The sonication conditions were selected to obtain Δ-IrBMS8-NPs nanoassemblies with a diameter of 160 nm. The Δ-IrBMS8-NPs nanoassemblies were dialyzed (dialysis cutoff 10 kDa) against deionized water at a constant rate using a peristaltic pump, and then filtered through a 0.45 μm filter membrane. The Δ-IrBMS8-NPs nanoassemblies were characterized by SEM and TEM, and the results are shown in FIG. 3A and FIG. 3B, respectively. The SEM and TEM results show spherical nanoparticles (diameter 160.8 ± 12.3 nm, PDI 0.15). The CD spectrum of the Δ-IrBMS8-NPs nanoassemblies is shown in FIG. 3C, and the DLS results are shown in FIG. 3D. The DLS and CD spectrum confirm the chirality retention. Figure 4 S3-2: Preparation of Δ-IrBMS8-NPs nanoassemblies: Δ-IrBMS8-mPEG was dissolved in DMSO at a final concentration of 5 mg / mL, and sonicated using a Biosafer 650-92 ultrasonicator at 18 kHz, 2.4 mL / h flow rate. The sonication conditions were selected to obtain Δ-IrBMS8-NPs nanoassemblies with a diameter of 160 nm. The Δ-IrBMS8-NPs nanoassemblies were dialyzed (dialysis cutoff 10 kDa) against deionized water at a constant rate using a peristaltic pump, and then filtered through a 0.45 μm filter membrane. The Δ-IrBMS8-NPs nanoassemblies were characterized by SEM and TEM, and the results are shown in FIG. 3A and FIG. 3B, respectively. The SEM and TEM results show spherical nanoparticles (diameter 160.8 ± 12.3 nm, PDI 0.15). The CD spectrum of the Δ-IrBMS8-NPs nanoassemblies is shown in FIG. 3C, and the DLS results are shown in FIG. 3D. The DLS and CD spectrum confirm the chirality retention.

[0078] S3-3: PEG modification of Δ-IrBMS8: Δ-IrBMS8 (29.4 mg, 0.02 mmol) was dissolved in 10 mL of DCM / MeOH (V DCM MeOH = 1:1) with stirring, and NaBH4(2.5 mg, 0.066 mmol) was added. The reduction reaction was continued for 1 h with stirring, and the reaction was completed. The reaction solution was then dried by rotary evaporation, and water and EA were added to extract the combined organic phase. The organic phase was dried over anhydrous Na2SO4, filtered, and dried by rotary evaporation to obtain a dark red solid (29.4 mg, 0.02 mmol). The dark red solid (29.4 mg, 0.02 mmol), mPEG-COOH (2000 Da, 100 mg), and DMAP (1 mg) were dissolved in DCM (2 mL), and a DCC (10.3 mg, 50 μmol) solution in DCM was added dropwise to catalyze the condensation reaction. The condensation reaction was continued for 48 h at room temperature, and Λ-IrBMS8-mPEG (31 mg, 45% yield, 95% purity) was obtained by column chromatography. 1 H NMR δ 3.50 ppm broad peak; molecular structure shown in FIG. 3E. Figure 1

[0079] S3-4: Preparation of Δ-IrBMS8-NPs nanoassemblies: Δ-IrBMS8-mPEG was dissolved in DMSO at a final concentration of 5 mg / mL, and sonicated using a Biosafer 650-92 ultrasonicator at 18 kHz, 2.4 mL / h flow rate. The sonication conditions were selected to obtain Δ-IrBMS8-NPs nanoassemblies with a diameter of 160 nm. The Δ-IrBMS8-NPs nanoassemblies were dialyzed (dialysis cutoff 10 kDa) against deionized water at a constant rate using a peristaltic pump, and then filtered through a 0.45 μm filter membrane. The Δ-IrBMS8-NPs nanoassemblies were characterized by SEM and TEM, and the results are shown in FIG. 3A and FIG. 3B, respectively. The SEM and TEM results show spherical nanoparticles (diameter 160.8 ± 12.3 nm, PDI 0.15). The CD spectrum of the Δ-IrBMS8-NPs nanoassemblies is shown in FIG. 3C, and the DLS results are shown in FIG. 3D. The DLS and CD spectrum confirm the chirality retention. ​​Procedure, DMSO solution of Δ-IrBMS8-mPEG was added to 5 mL of deionized water at a constant rate for dialysis (dialysis molecular weight cutoff is only 10 kDa), and after dialysis, it was filtered through a 0.45 μm filter membrane to obtain Δ-IrBMS8-NPs nanassemblies. The SEM and TEM results of Δ-IrBMS8-NPs nanassemblies are shown in FIG. 1A and FIG. IB, respectively, showing spherical nanoparticles (particle size 227.4 ± 13.7 nm, PDI 0.15). Figure 2 The CD spectrum of Δ-IrBMS8-NPs nanassemblies is shown in FIG. 2A, and the DLS result is shown in FIG. 2B. DLS and CD spectrum confirmed the chirality retention. Figure 3 Figure 4

[0080] Example 4

[0081] Detection of Anti-tumor Effect of Chiral Iridium Complexes

[0082] (1) Effect of Chiral Iridium Complexes on Intracellular ROS Level

[0083] Mouse colorectal cancer cells CT26 (purchased from American Type Culture Collection (ATCC)) were seeded at 1.0 x 10 5 cells / well in 35 mm glass-bottom confocal dishes and incubated for 12 hours. Then 0.2 μM BMS8, Δ-IrBMS8, Λ-IrBMS8, Δ-IrBMS8-NPs, Λ-IrBMS8-NPs were added for co-incubation. After 12 hours, cells were irradiated at 450 nm light (30 mW / cm2) for 10 minutes, followed by further incubation. After 12 hours, the old medium was removed, washed with serum-free DMEM medium for 3 times, and stained by adding PBS buffer containing 5 μM SOSG (S36002, Thermo Fisher Scientific) / MitoSOX Red (M36008, Thermo Fisher Scientific). After incubation at 37 °C, 5% CO2for 30 minutes, the medium was discarded, carefully washed with serum-free DMEM medium for 2 times, and imaged by confocal microscopy (GFP channel) / (Rhodamine channel). The intracellular ROS level was observed, and the results are shown in FIG. 3A and FIG. 3B. Figure 5 Figure 5 As can be seen from the results, the fluorescence intensity of the cells treated with the two chiral nanoparticles is higher, indicating that the intracellular ROS level is significantly increased, and the two fluorescence indicates that type I and type II mechanisms are combined in the PDT process, in which O2 ·- and 1 ​​​O2 accumulation was quite noticeable. We further analyzed the O2... ·- and 1 The mean fluorescence intensity of O2 imaging indicates that Δ-IrBMS8-NPs induce more O2 in CT26 cells. ·- and 1 O2 accumulation is consistent with phototoxicity results.

[0084] Seven-week-old CT26 tumor-bearing mice were used as experimental subjects, and experimental and control groups were set up according to Table 1:

[0085] Table 1. Treatment methods for CT26 tumor-bearing mice

[0086]

[0087] Mice in each group were treated according to the treatment methods in Table 1. After 15 days of treatment, experimental observations were conducted on the mice's tissues and organs.

[0088] (2) Effect of chiral iridium complexes on tumor volume

[0089] Tumors were collected from each group of mice, and changes in tumor volume were observed (tumor size was measured using calipers). The results are as follows: Figure 6 As shown. From Figure 6 The results showed that the Δ-IrBMS8-NPs+light group exhibited the best tumor suppression effect.

[0090] (3) Effects of chiral iridium complexes on the proportion of CD8+ T cells in the spleen

[0091] Spleens were harvested from mice in each group, and the changes in the proportion of CD8+ T cells in the spleen were detected by flow cytometry. The results are as follows: Figure 7 As shown. From Figure 7 The results showed that treatment with BMS8, Δ-IrBMS8-NPs, Δ-IrBMS8-NPs+light, and Δ-IrBMS8-NPs+light induced CD4+ in the spleen of mice. + The number of T cells increased significantly, and CD8+ levels in the spleen of mice were significantly increased after treatment with Δ-IrBMS8-NPs, Δ-IrBMS8-NPs+light, and Δ-IrBMS8-NPs+light. + The number of T cells was significantly increased. Overall, CD4+ levels in the spleen of mice treated with Δ-IrBMS8-NPs+light were significantly increased. + T cells and CD8 + T cells were significantly increased, and compared with the Δ-IrBMS8-NPs+light treatment group, the Δ-IrBMS8-NPs+light treatment group activated more cytotoxic T cells.

[0092] Therefore, the chiral complex monomers Δ-IrBMS8 and Λ-IrBMS8 and the chiral iridium complex nanoassemblies Δ-IrBMS8-NPs and Λ-IrBMS8-NPs prepared by the application are all suitable for anti-tumor immunotherapy, but the nanoassemblies Δ-IrBMS8-NPs have a better effect of activating immunity, especially under light conditions.

[0093] The above merely describes preferred embodiments of the present application, and the present application is not limited to the above embodiments. It can be understood that other improvements and changes directly derived or thought of by those skilled in the art without departing from the spirit and concept of the present application shall be considered to be within the protection scope of the present application.

Claims

1. A chiral iridium complex nanoassembly, characterized in that, The chiral iridium complex nanoassemblies are obtained by sonication, dialysis, and filtration of Δ-IrBMS8-mPEG or Λ-IrBMS8-mPEG; the structures of Δ-IrBMS8-mPEG and Λ-IrBMS8-mPEG are as follows: The ultrasonic power is 18 kHz; the flow rate of the solution added during ultrasonication is 2.4 mL / h; and the molecular weight cutoff of the dialysis membrane is 10 kDa.

2. A method for preparing the chiral iridium complex nanoassembly according to claim 1, characterized in that, The preparation method includes the following steps: Δ-IrBMS8 and Λ-IrBMS8 were reduced with NaBH4 to obtain dark red solids. Under the catalysis of DMAP and DCC, the dark red solids were acylated with mPEG-COOH to obtain Δ-IrBMS8-mPEG and Λ-IrBMS8-mPEG. Then, Δ-IrBMS8-mPEG and Λ-IrBMS8-mPEG were subjected to sonication, dialysis and filtration to obtain Δ-IrBMS8-NPs and Λ-IrBMS8-NPs nanoassemblies, respectively.

3. The preparation method according to claim 2, characterized in that, The solvent for the reduction reaction is a mixture of DCM and MeOH in a volume ratio of 1:1, and the reducing agent is NaBH4; the molar ratio of the reducing agent to Δ-IrBMS8 or Λ-IrBMS8 is 2.5-3.5:

1. The solvent for the acylation reaction is DCM; the mass ratio of mPEG-COOH to Δ-IrBMS8-NPs or Λ-IrBMS8-NPs is 1:0.2-0.3; the mass ratio of mPEG-COOH to DMAP is 100:1; and the mass ratio of DMAP to DCC is 1:9-12. The ultrasonic power is 18 kHz; the flow rate of the solution added during ultrasonication is 2.4 mL / h; and the molecular weight cutoff of the dialysis membrane is 10 kDa.

4. The preparation method according to claim 2, characterized in that, The preparation methods of Δ-IrBMS8 and Λ-IrBMS8 are carried out according to the following process: The specific steps are as follows: Using 4-(isoquinoline-1-yl)benzaldehyde as the main compound and hydrated iridium chloride as the iridium source, the reaction was carried out, and (R)-Fphox and triethylamine were added to react and obtain the cyclic metal iridium chiral complex precursors Δ-Irpiq-R and Λ-Irpiq-R. Δ-Irpiq-R and Λ-Irpiq-R were subjected to coordination reactions with BMS8-NN to obtain Δ-IrBMS8 and Λ-IrBMS8, respectively.

5. The preparation method according to claim 4, characterized in that, The molar ratio of 4-(isoquinoline-1-yl)benzaldehyde to hydrated iridium chloride is 2.4-2.5:1; the molar ratio of hydrated iridium chloride to (R)-Fphox is 1:1.1-1.3; and the molar ratio of hydrated iridium chloride to triethylamine is 1:5-10. The catalyst for the coordination reaction is trifluoroacetic acid, the temperature is 25°C, and the time is 2-4 h; the molar ratio of trifluoroacetic acid to Δ-Irpiq-R or Λ-Irpiq-R is 4-6:

1. When preparing Δ-IrBMS8, the molar ratio of Δ-Irpiq-R to BMS8-NN is 1:1.1-1.3; When preparing Λ-IrBMS8, the molar ratio of Λ-Irpiq-R to BMS8-NN is 1:1.1-1.

3.

6. The preparation method according to claim 4, characterized in that, The preparation method of (R)-Fphox is carried out according to the following process: The specific steps are as follows: 2-fluoro-6-hydroxybenzyl nitrile and D-phenylpropanol are dissolved in toluene and subjected to a cyclization reaction catalyzed by zinc chloride to obtain compound (R)-Fphox.

7. The preparation method according to claim 6, characterized in that, The molar ratio of 2-fluoro-6-hydroxybenzyl nitrile to D-phenylpropanol is 1:1.3; the molar ratio of zinc chloride to 2-fluoro-6-hydroxybenzyl nitrile is 1:40; the cyclization reaction is carried out at a temperature of 130°C for 24 hours.

8. The preparation method according to claim 4, characterized in that, The BMS8-NN is prepared according to the following procedure: The specific steps are as follows: Starting with (2-methylbiphenyl-3-yl)methanol, it undergoes a bromination reaction with PBr3 to obtain intermediate compound a. Intermediate compound a undergoes an etherification reaction with 3-bromo-4-hydroxybenzaldehyde to obtain intermediate compound b. Intermediate compound b undergoes a reductive amination reaction with 2-piperidinecarboxylic acid and NaBH3CN to obtain intermediate compound c. After reacting 5-amino-1,10-o-phenanthroline with p-hydroxybenzaldehyde at 65°C for 12 h, NaBH4 was added to carry out an amidation reaction to obtain intermediate compound d. The intermediate compound c and intermediate compound d were subjected to a condensation reaction to obtain the PD-L1 targeting ligand BMS8-NN.

9. The preparation method according to claim 6, characterized in that, The molar ratio of PBr3 to (2-methylbiphenyl-3-yl)methanol is 1.1 to 1.3:1; the solvent for the bromination reaction is THF, the temperature is room temperature, and the time is 2-4 hours. The molar ratio of 3-bromo-4-hydroxybenzaldehyde to intermediate compound a is 0.7–0.8:1; the solvent for the etherification reaction is acetone, the temperature is 55–65°C, and the time is 10–14 h. The molar ratio of 2-piperidinecarboxylic acid, NaBH3CN, and intermediate compound b is 3:3:

1. The solvent for the reductive amination reaction is DMF, the temperature is 75-85℃, and the time is 2-3h. The molar ratio of 5-amino-1,10-o-phenanthroline to p-hydroxybenzaldehyde is 1:1.1-1.3; the solvent for the amidation reaction is anhydrous ethanol; the amidation reaction is carried out at room temperature for 2-3 hours. The molar ratio of intermediate compound c to intermediate compound d is 1:1 to 1.

5. The solvent for the condensation reaction is DMF, the condensing agent is HATU, the temperature is room temperature, and the time is 11-13 hours.

10. An antitumor drug, characterized in that, The antitumor drug comprises the chiral iridium complex nanoassembly of claim 1 or the chiral iridium complex nanoassembly prepared by any one of claims 2 to 9.

Images